Aabs 3.1 (2016) by PaGe

eISSN: 2349-6991 pISSN: 2455-0396

Annals of Applied BioSciences An Interna onal, Open access, Indexed, Peer-reviewed Journal

AABS

Vol. 3, Issue 1, January-March 2016

Editor-in-Chief Dr Shelly Sehgal Dr Dip Agrawal

PaciďŹ c Group of eJournals

Annals of Applied Bio-Sciences Editors-in-Chief

Dr Shelly Sehgal Specialist Pathologist, Dept. of Pathology, SDN Hospital, Delhi, India Dr Dipti Agrawal Dept. of Gynecology and Obstetrics, SDN Hospital, Delhi, India

Co-Editors-in-Chief

Dr Sompal Singh Specialist Pathologist, Dept. of Pathology, N D M C Medical College & Hindu Rao Hospital, Delhi, India Dr Bhupendra Pushkar Asst. Professor, Dept. of Biotechnology, University of Mumbai, India

Associate Editors

Dr Vijaya Bhaskar Varadarajalu Chennai, Tamil Nadu, India Dr B K Guha Associate Prof. Anatomy, NSCB Medical College, Jabalpur, India Dr Hariom Gupta Associate Prof. Medicine SSMC and SGMH, Rewa. (MP), India Dr Shashikant Agarwal Associate Prof. Physiology, JMC, Jhalawar, India

Editorial Board Members

Dr Francesco Merolla Advanced Biomedical Sciences, University of Naples â&#x20AC;&#x153;Federico IIâ&#x20AC;? (ITALY), Italy Dr Chandrassegar Saravanan Novartis Institutes for Biomedical Research, Technology Square, Cambridge, MA Dr Nandhakumar Balakrishnan College of Veterinary Medicine North Carolina State University, Raleigh

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India) Website: www.pacificejournals.com www.pacificejournals.com/aabs E-mail: contactus.aabs@pacificejournals.com

Prof. M H Fulekar Professor & Dean, School of Environment and Sustainable Development, Central University Gujarat,, India Dr Igor Iuco Castro-Silva Professor, ClinicaOdontologica, Faculdade de Ciencias do Tocantins, Brazil Kapil Agarwal Engineer, Nagoya City, Aichi, Japan Dr Devesh Palharya Consultant Pathologist, Bhopal, India Dr D A Bhiwgade Dept. of Biotechnology & Bioinformatics, Padmashree Dr. D. Y. Patil University, Navi Mumbai,, India Dr Arpana Haritwal Consultant, Obs. &Gynaecology, Saket City Hospital, New Delhi, India Dr Radhika P Kamdar Emory University School of Medicine, Georgia,, United States Dr Saba Hasan Asst. Prof. Amity University, Lucknow, India Dr Manav Kapoor Assistant Professor Neuroscience, Icahn School of Medicine, Mount Sinai, New York, NY, 10029

Managing Editor

Dr Prashant Goyal Director-Laboratory,Accuprobe Healthcare and Diagnostics, Delhi, India

Annals of Applied Bio-Sciences Annals of Applied Bio-Sciences (AABS) is an international, fast double-blind peer-reviewed, indexed, open access, online and print multidisciplinary journal with high impact factor (2.9254) and IC value (56.90), published by ‘Pacific group of e-Journals’ (PaGe), an ISO 9001:2008 Certified academic publishing house, with a quick post-acceptance online publication.

AABS is a multidisciplinary journal in the all fields of sciences including Medicine(Anatomy, Anesthesia, Cardiology, Community Medicine, Dermatology, Internal Medicine, Neonatology, Oncology, Orthopedics, Pediatrics, Physiology, Plastic Surgery, Psychiatry, Radiology, Surgery, ENT, Nutrition, Anatomy, Forensic , Genetics, Obstetrics And Gynecology, Ophthalmology, Pediatric Surgery, Nephrology, Urology, Neurology etc.) and Dentistry, Nursing and Health Professions, Biological sciences, Biotechnology, Molecular biology, Cell biology, Genetic engineering, Biochemistry, Pharmacology, Pharmaceutical Science, Toxicology, Immunology, Microbiology, Virology, Veterinary Medicine, Veterinary Science, Agricultural, Environmental science, Ecology and Ecotoxicology, Biophysics, Biomedical sciences, Computational biology, Biological anthropology and Biological education etc.

Publication Frequency

Quarterly with continuous publication. Articles are published online in the current volume of the Journal as soon as they are accepted. However, the issue is published in printed form only at the end of the relevant quarter.

Disclaimer

1. Thoughts, Language and examples in published research papers are entirely of Authors of Research Papers. It is not necessary that both Editor and Editorial Board are satisfied by the research paper. The responsibility of the matter of the research paper/ article is entirely of author. Scientific knowledge is ever changing. As new research and experiences broaden our knowledge, old concepts may be required to be looked into afresh. The Authors and editors of the material herein have consulted sources believed to be reliable in their efforts to provide information that is complete and in accord with the standards accepted at the time of publication. However, in view of the possibility of human error by the authors, editors, or publisher, nor any other who has been involved in the preparation of the work warrants that the information contained herein in every aspect accurate or complete, and they are not responsible for any errors or omissions or the results obtained from the use of such information. Readers are advised to confirm the information contained herein with other sources. 2.

•

Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work’s authorship and initial publication in this journal. Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal’s published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.

Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.

In any condition and due to any reason, if any Educational/Research Institution (College or University) denies accepting the research paper published in AABS, it will not be the responsibility of the Editor, Publisher or the Management. Only the First author is entitled to receive the print copies of all co-authors.

All the legal disputes related to PaGe are subject to Delhi Jurisdiction only

Table of Contents Review Articles Management Modalities for Necrotising Enterocolitis: Targeting Toll Like Receptors Prasanta Kumar Tripathy, Rachita Nanda

Urine Cytology: A Review Archana S Bhat, Manjunath J, Manjunath Shettty

R1-5

R6-10

Original Article Diagnosis of Endophthalmitis with amplification of syndrome specific signature

A1-4

A Case Series of Retinochoroidal Toxoplasmosis from Western India: Guide to Salient Features, Diagnosis and Treatment Deepen Surendra Sheth, Pandurang Kulkarni, Sundaram Natarajan

A5-11

genes by Syndrome Evaluation System: A Retrospective Analysis Anoop Sivaraman, Pandurang Kulkarni, Sundaram Natarajan, Rishi Bhardwaj

Positive feedback between climate change, forest pests and the carbon cycle Anne I. M. Arnold, Annett Reinhardt, Ignacy Korczynski, Maren Gr端ning, Carsten Thies Assessment of the health related quality of life in patients suffering from hypertension and diabetes mellitus: A cross sectional study Ishtpreet Mann, Navtej Singh, Manisha ., Ashwani K Gupta, Prithpal S Matreja Radiological and Biochemical aspect of Alcoholic Liver Disease: A Prospective study Siddharth Bhargava, Navtej Singh, Prithpal S Matreja, Ashwani K Gupta, Arshdeep Singh Etiological and clinical spectrum of pancytopenia based on bone marrow examination and case records: A retrospective study Prashanth B Gandhi, Mohammed Afraz Pasha, Tharanath Shankar, MangalaGouri

A12-15 A16-21 A22-26 A27-32

Prevalence of molar pregnancy (a three year retrospective study) in a tertiary care ho Jangbhadur Singh, Shaveta Sharma, KirpalKour, Shazia Bashir

A33-36

Effect of Tetracycline therapy on Microfilaraemia and Antigenemia in Raipur city, Chhattisgarh state, India Santosh Kumar Agrawal

A37-41

Quantification of oligo-elements and heavy metals in the fruits (pods and seeds) of the introduced tree Gleditsiatriacanthos L. in Algeria Benhamiche Samia, Benhassaini Hachemi, Chabane Kheira, Romane Abderrahmane, Arjouni Moulay Youssef

A42-48

A49-53 Pros and Cons of Different Methods of Leucoreduction and Its Scope of Implementation in the Cost Constrained Settings Ankita Anand Katara, Abhishek Vinod Pandey, Jagdish D Dalsania, Rohit R Bhalara, Milan Purohit Role of Complements and Immunoglobulins in Type 1 Diabetes Mellitus Shailja Singh, Usha Singh, N K Agrawal, R G Singh Clinical Correlation of coagulopathy in vivax malaria Reshma Gopal Kini, Veronica Lobo, Raphael Lyngdoh, Vedasree Reddy Diagnostic role of IL-6 in Neonatal sepsis Neeraj Kumar, Manoj Kumar Singh, Rajeshwar Dayal, Shikha Gupta, Ruchika Garg Evaluation of various causes of thrombocytopenia in patients attending Hamidia Hospital, Bhopal Maneesh Sulya, Reeni Malik, Kamlesh Patel Clinical Profile of Swine Flu in Children at a tertiary care center Manoj Kumar Singh, Pankaj Kumar, Rajesh Kumar, Shikha Gupta, Prabhat Agrawal A retrospective study to evaluate tuberculosis burden by FNAC of cervical lymphnode from the patients coming to Hamidia Hospital. Maneesh Sulya, Sonal Gupta, Kamlesh Patel

A54-59 A60-66

A67-71 A72-74 A75-78 A79-81

Case Reports

Seroepidemiology of HIV infection in pregnant Women at a Referral Centre of North India Ruchika Garg, Shikha Singh, Saroj Singh, Indira Sarin, Manisha Pathak

A82-87

Clinical Spectrum of non-Hodgkin lymphoma: A Hospital based study of 410 cases. Vijayashree Shivappa Neeravari, Doddappa Mallappa Bannigidad, Anirudha Vasantacharya Kushtagi, Lakshmi Rao

A88-93

Discovery of novel gene biomarker for Acute Myeloid Leukemia through differential gene expression analysis Sweta Kumari Tripathi, Himanshu A Pandya

A94-100

Evaluation of glycosylated hemoglobin(HbA1c) levels in hypothyroid and hyperthyroid patients. Sumit Sohal, Aanchal Wats, ChittaranjanVij

A101-107

Diagnostic role of fine needle aspiration cytology in thyroiditis along with Thyroid hormone assay. Prahlad C Agrawal, Reena Naik, Kishori Moni Panda

A108-112

Role of Bronchoscopy in Diagnosing Sputum Smear Negative Pulmonary Tuberculosis Prashant Prakash, Pooja Agarwal, Prabhat Agarwal, Durga Prasad Singh, Ashutosh Gupta

A113-117

Diagnosis of primary abdominal wall endometriosis on cytology: An unusual presentation Ankit Kaushik, Anamika Jaiswal

C1-3

A Diagnostic Dilemma of Nasal Meningo-encephalocele: a case report Ritu Sharma, Vivek Gupta, Gaurav Kumar

C4-7

Peutz-Jegherâ&#x20AC;&#x2122;s syndrome: A diagnosis clinched on histopathology Somshamkar Chowdhury, Neha Tyagi, Leelawathi Dawson, Ashish K Mandal Ruptured Ovarian Pregnancy in a Young Primigravida Beant Singh, Balwinder Kaur, Parneet Kaur, Ramiti Gupta Pure mucinous carcinoma of male breast: case report of a rare histological subtype of male breast carcinoma Riju Rani Deka, Shiraj Ahmed, Amitabh Handique Early Gastric carcinoma: A rare variant of a common entity. Monal Trisal, Kangana Sengar, Sumedha Kotwal, Sanjay Deb, Ramesh Dawar

C8-12

C13-15 C16-20 C21-24

Review Article

Management Modalities for Necrotising Enterocolitis: Targeting Toll Like Receptors Prasanta Kumar Tripathy1, Rachita Nanda2 Department of Pediatric Surgery, Sardar Valhabhai Patel Post Graduate Institute of Pediatrics, Cuttack, India Department of Biochemistry, AIIMS, Raipur, Chhattisgarh, India. Keywords: Necrotizing Enterocolitis, Toll Like Receptor, Heat Shock Protein, Epidermal Growth Factor, Probiotics, Prebiotics

ABSTRACT

In spite of advances in the management of newborn infants, Necrotizing Enterocolitis(NEC) continues to be a complicated disease all over the world. Gradual development in the field of NEC discovered â&#x20AC;&#x2DC;Toll-like receptor 4â&#x20AC;&#x2122;(TLR4), which is a bacterial receptor having role in pathogenesis of the disease. TLR4 expression is increased in bowel mucosa of NEC babies and TLR4 activation causes mucosal injury and enterocyte apoptosis. Breast feeding is considered to decrease the risk of necrotizing enterocolitis. But, we neither have a definite molecule for the treatment of the disease nor for its prevention. CpG DNA which is present in the probiotic preparations and muramyl-di-peptide(MDP), inhibit TLR4 signaling may decrease NEC severity. Epidermal growth factors(EGF) present in amniotic fluid also reduce TLR4 signaling. Heparin binding EGF( HB-EGF) and Heat shock protein(Hsp 70) are possible approaches for management of NEC because of their action on TLR4. These may be considered as future management approach for this common and compromising disease of the newborn.

*Corresponding author: Dr. Rachita Nanda, Department of Biochemistry, AIIMS, Raipur, Chhattisgarh, India. PIN-492099 Mobile -+918518881763 email: dr.rachitananda@aiimsraipur.edu.in

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Review Article

R-2

INTRODUCTION Necrotizing Enterocolitis is one of the commonest medical and surgical emergencies in newborn. It is associated with high morbidity and mortality in neonatal intensive care units. Majority of cases are managed by medicines and supportive neonatal care, but surgical intervention is frequently required. In spite of improvement in care of newborns, availability of well equipped intensive care units and refinement of surgical practice, the prognosis is not good and it is poor when surgical intervention is required. Two common association with this disease are prematurity and feeding [1]. The disease starts from intestinal epithelium with inflammatory necrosis followed by systemic sepsis. Gradual search into the pathogenesis of NEC identified a bacterial receptor subsequently called â&#x20AC;&#x2DC;Toll-like-receptor 4â&#x20AC;&#x2122;(TLR4). TLRs are glycoproteins and important component of innate immune response. They are called Toll-like receptors as they resemble Toll protein of fly immune system[2]. Out of ten TLRs identified in humans TLR1 and TLR2 are linked with RNA and TLR9 recognizes DNA of bacteria. However, TLR4 is the receptor for bacterial lipopolysaccharide(LPS)[3,4].Usually newborn intestine colonized with gram negative bacteria(having LPS in its membrane) is followed by NEC. So an important role of TLR4, in the pathogenesis of NEC was suspected and later on a number of evidences confirmed the suspicion[3]. Inhibition of TLR4 signaling may prevent or treat NEC in premature babies.

Importance of TLR4 in Pathophysiology of NEC Neonates with NEC are associated with increased TLR4 levels in their gut mucosa[5,6]. As compared to term babies, premature babies have increased TLR4 levels and TLR4 is more active[7,8]. Mice lacking TLR4 or mutations in TLR4 do not develop NEC[9]. Stress effects such as hypoxia, lipopolysaccharide, infection and formula feeding sensitise the epithelium and cause TLR4 up-regulation[3,4,10]. This is followed by intestinal injury due to up-regulation of inflammatory cytokines along with decreased mucosal repair because of the effect of TLR4 on enterocyte migration, proliferation of stem cells and apoptosis[8]. Normally, there occurs the migration of enterocytes from nearby healthy sites after intestinal mucosal injury and cell death. Platelet activating factor(PAF) level is raised in NEC babies and the values returns to normal as the disease subsides. It also increases in fecal matter of neonates with onset of NEC[11,12]. PAF suggested in pathogenesis of NEC is related to TLR4 up-regulation[4]. TLR4 activation inhibits enterocyte migration by complex molecular mechanisms. Activation of Rho-GTPase and Focal Ad-

hesion Kinase(FAK) cause increased adhesiveness of enterocyte to underlying basement membrane[13]. The cell movement is limited after TLR4 activation due to increased cell- matrix adhesiveness[14]. NEC is associated with death of enterocytes, i.e., apoptosis[15,16]. TLR4 activation can cause enterocyte apoptosis[17]. Following epithelial cell death, there occurs transmural migration of microbes from the intestinal lumen. Loss of enterocytes are followed by proliferation from precursor stem cells. This process is impaired after TLR4 activation in neonatal intestine[14,17]. Substances that can promote proliferation of enterocytes may be tried for treatment of NEC. TLR4 signaling also cause increased apoptosis and decreased proliferation of stem cells in intestinal crypts[7]. Therefore, TLR4 activation causes intestinal mucosal injury and decreased epithelial repair in premature neonates, proving its importance in pathophysiology of NEC[3,8].

Molecules likely to provide therapeutic benefit in NEC Probiotics and CpG-DNA: Probiotics are live organisms which competitively inhibit enteric pathogens. They decrease pro-inflammatory response and produce antimicrobial as well as antioxidant substances, thereby having health benefit. Commonly used organisms are bifidobacteria, lactobacillus, streptococcus strains and saccharomyces species [4,18,19]. Bifidobacterium bifidium decreases epithelial cell death by up regulating TLR2 in the intestine of NEC babies. During exposure of neonatal gut epithelium to the commensal bacteria, the inflammatory process towards pathogenic invasion is limited and innate immunity is not activated [19]. Combination of probiotic organisms may be more effective than single strain[4]. Enteral supplementation of probiotics attenuate the risk and severity of NEC in preterm babies and use of inactivated /heat killed probiotics is under evaluation[20,21,22]. Increased TLR4 and decreased TLR9 expression is detected in NEC babies. Probiotics are rich in bacterial DNA andTLR9 recognises the CpG motifs of DNA. CpG-DNA activates TLR9, thereby inhibiting TLR4 signaling and reducing NEC severity[11]. Administration of CpG-DNA may provide therapeutic benefit to NEC neonates. MDP activating NOD2 : Nucleotide Oligomerization Domain 2(NOD2) is the receptor for bacterial component of muramyl-di-peptide (MDP)[3]. By competitive effect NOD2 activation decreases TLR4 signaling [23]. MDP activating NOD2 limits TLR4 induced intestinal injury and may serve as a potential therapeutic agent for NEC.

http://www.pacificejournals.com/aabs

R-3 Heat Shock Protein 70 (Hsp70): Heat shock proteins are intracellular proteins activated by different stressors. Hsp 70 is an important member of this group, which decreases TLR4 signaling and cell death [16,24]. This intracellular component is one of the factors supposed to counter-regulate increased TLR4 expression normally occurring in many preterm babies, thereby preventing the development of NEC[8]. TLR4 induces expression of Hsp 70, which then reduces TLR4 signaling in intestinal epithelium[8,16]. Decreased activity or loss of Hsp 70 causes enterocyte apoptosis and release of pro-inflammatory cytokines from uncontrolled TLR4 activity[16]. This will cause development of NEC in premature neonatal intestine. Severity of NEC can be decreased by up regulating Hsp70, thereby offering potential benefit in this disease[8,16]. Amniotic fluid rich in EGF: Fetus maintains an increased TLR4 level due to developing gut mucosa. This increased TLR4 and its subsequent risk to immature intestine might be balanced by amniotic fluid swallowed by fetus. Amniotic fluid is rich in epidermal growth factor(EGF), which decreases TLR4 signaling[25]. Premature neonates with high basal TLR 4 and lacking protection from amniotic fluid are at risk of NEC. Hypoxia and stressed conditions increase EGF which in turn helps in mucosal repair after damage. Although the clinical trial of EGF in NEC babies is limited in humans, extended work on this growth factor supplementation is required for prevention of NEC[26]. EGF administration up-regulates release of protective and anti-inflammatory cytokine IL-10[11]. Thus EGF, by decreasing NEC severity may be a potential treating agent. Heparin binding EGF(HB-EGF): HB-EGF, a member of EGF family protect intestinal epithelium from cytokine induced apoptosis. HB-EGF helps in proliferation of crypt cells, maintains mucosal integrity following injury and reduces translocation of pathogenic microorganisms[26]. Chen et al, described the role of HB-EGF in limiting injury to stem cells of intestine from hypoxia and ischemia[27]. It also decreases nitric oxide production, thereby having protective role. HB-EGF have beneficial effect by replacement of damaged enterocytes by stem cells and enhancing enterocyte migration. Increased enterocyte proliferation and decreased apoptosis observed following HB-EGF supplementation suggests its beneficial effects in decreasing incidence and severity of NEC[26]. Scientists also found, HB-EGF and mesenchymal stem cells(MSC) act synergistically when administered in animals with NEC, thereby upgrading the protective role[28]. As it reduces NEC severity, HB-EGF administration may be a potential therapeutic benefit.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 Anti-inflammatory cytokine IL-10: Pro-inflammatory cytokines(IL-1β, IL-2, IL-12, TNFα), platelet activating factor(PAF) and anti-inflammatory cytokine(IL-10) play important role in pathophysiology of NEC. IL-1β may serve as marker for the progression of disease. PAF is released after hypoxia, local injury and infection. PAF receptor activation cause release of inflammatory mediators, increased mucosal permeability and apoptosis of epithelial cells. PAF upregulates TLR4 in intestinal epithelium[18]. The pro-inflammatory cytokine, PAF may induce NEC and PAF antagonists decrease severity of NEC in animal models[12]. The anti-inflammatory cytokine, IL-10 decreases pro-inflammatory cytokine release, thereby having beneficial effect in NEC. Nitric oxide produced from l-arginine by high concentration of inducible nitric oxide synthase (iNOS) during inflammation cause enterocyte apoptosis and inhibit mucosal repair [29]. Inducible NOS is expressed in high concentration in NEC and IL-10 decreases iNOS level. IL-10 is found in breast milk and might be preventing NEC. This protective cytokine may be considered as a therapeutic target in NEC. Breast milk and breast feeding schedule: Breast milk having digestive enzymes, secretory IgA, EGF, lactoferrin, oligosaccharide and immunomodulating substances gives protection against development of NEC [11,21]. It is well absorbed than formula and have less allergic effect in intestine. Insulin like growth factor in milk helps in bowel growth. Presence of glutamine in breast milk maintains structural integrity and decreases bacterial translocation of neonatal intestine. TLR4 expression of enterocyte is increased with formula feeding and stress as compared to breast feeding. Breast fed babies are rich in enteric commensals like Bifidobacteria, whereas pathogenic bacteria like coliforms and Enterococci are found in formula fed babies[21]. Breast milk helps in colonization of neonatal intestine with commensal bacteria like Bifidobacterium and Lactobacillus[11] and prevent colonization by pathogenic bacteria[19]. The role of PAF in pathophysiology of NEC is well established. PAF which activates TLR4 expression is present at higher levels in formula fed neonates[18]. Human milk contains considerable amounts of PAF-acetylhydrolase(PAF-AH), which is implicated in PAF degradation. This contributes to the protective role of mother’s milk against development of NEC[4]. Anti-inflammatory cytokines, IL-10, found in breast milk is considered to have protective role in prevention of NEC[19]. Human milk decreases the risk of NEC as compared to formula[21,30]. Debate is still continuing on the development of NEC and enteral feeding. The association seems to have rationale

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article

R-4

with formula feeding. Early breast feeding practice have potential effect on NEC prevention. Prebiotics and postbiotics: Prebiotics have beneficial effect on bacterial flora of babies and they are basically non digestible food supplements. Prebiotic oligosaccharide present in breast milk help in host defense[31]. Oligosaccharide supplementation in formula have beneficial effect in NEC[21]. Prebiotics and probiotics may have similar action and may be tried in preterm neonates for NEC prevention[18]. The combination of probiotics and prebiotic is sometimes called ‘synbiotics’[1]. Postbiotics are metabolites of bacteria having potential benefit for enteric flora of NEC infants[21]. In contrast to probiotics, prebiotics and postbiotics are not live microbes, therefore there is no risk of potential sepsis.

CONCLUSION NEC remains a challenging disease in low birth weight babies due to its multifactorial etiology, rapid systemic involvement and high morbidity /mortality. Recent knowledge about the bacterial receptor, TLR4 in pathophysiology of NEC may lead to better management strategies. Preterm are prone for NEC as their underdeveloped intestine is associated with elevated levels of TLR4. Absence of host influences like Hsp70 along with bacterial colonization of bowel and stress effects causing TLR4 signaling are added to the pathology. Breast milk containing secretory IgA, EGF, anti-inflammatory cytokine (IL-10) etc., may prevent bacterial colonization of intestine and NEC development. Probiotic supplementation have a preventive potential for the disease. Activation of TLR9 by probiotic preparations rich in bacterial DNA might reduce TLR4 signaling and severity of NEC. CpG-DNA and MDP reduce TLR 4 signaling and NEC intensity through their roles on TLR9 and NOD2 respectively. The use of prebiotics and postbiotics cannot be ignored. Another potential approach for the management of NEC may be expected by up-regulation of intracellular protein Hsp70, which will cause down-regulation of TLR4. Administration of EGF and more potentially HB-EGF can attenuate the development and intensity of NEC by reducing TLR4 signaling. NEC is a common cause of ‘short bowel syndrome’. Further research on tissue engineering will expand our vision to solve intestinal failure following NEC. The recent understanding of immunology of NEC and the role of TLR4 offers new look into the pathophysiology of NEC. Modified management approach based on these findings will increase our ability to treat and more successfully to prevent this serious disease in fragile neonates.

ACKNOWLEDGEMENTS Head of Department of Paediatric Surgery, SVPPGIP, Cuttack, Odisha

FUNDING None.

COMPETING INTERESTS None declared.

REFERENCES 1.

Patel BK and Shah JS. Necrotizing Enterocolitis in Very Low Birth Weight Infants: A Systemic Review. ISRN Gastroenterology 2012; 2012:1-7. Article ID 562594.doi:10.5402/2012/562594. 2. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997; 388: 394-397. 3. Afrazi A, Sodhi CP, Richardson W, Neal M, Good M, Siggers R, Hackman DJ. New Insights Into the Pathogenesis and Treatment of Necrotizing Enterocolitis: Toll-Like Receptors and Beyond. Pediatric Research 2011; 69(3): 183-188. 4. Frost BL, Caplan MS. Necrotizing Enterocolitis: Pathophysiology, platelet-activating factor, and probiotics. Seminars in Pediatric Surgery 2013;22(2):88-93. 5. Chan KL, Wong KF, Luk LM. Role of LPS/CD14/TLR-4 mediated inflammation in necrotizing enterocolitis: pathogenesis, and therapeutic implications. World J Gastroenterol 2009; 15:4745-52. 6. Lu J, Jiling T, Li D, Caplan MS. Polyunsaturted fatty acid supplementation alters proinflammatory gene expression and reduces the incidence of necrotizing enterocolitis in a neonatal rat model. Pediatr Res 2007; 61:427-432. 7. David J. Hackam, Amin Afrazi, Misty Good, and Chhinder P. Sodhi. Innate Immune Signaling in the Pathogenesis of Necrotizing Enterocolitis. Clinical and Developmental Immunology Vol. 2013, Article ID 475415, 10 pages, 2013. doi:10.1155/2013/475415 8. Curtis MD, Terrin G. Necrotizing enterocolitis(NEC): what’s going on. J Pediatr Neonat Individual Med 2013;2(2):e020218. 9. Jiling T, Simon D, Lu J, Meng FJ, Li D, Schy R, et al. The roles of bacteria and TLR4 in rat and murine models of necrotizing necrocolitis. J Immunol 2006; 177(5):3272-82. 10. Alcamo AM, Schanbacher BL, Huang H, Nankervis CA, Bauer JA, Glannone PJ. Cellular strain am-

http://www.pacificejournals.com/aabs

R-5

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

AABS; 3(1): 2016 plifies LPS-induced stress signaling in immature enterocytes: potential implications for preterm infants NCPAP. Pediatric Research 2012; 72(3): 256-261. doi: 10.1038/pr.2012.77. Chu A, Hageman JR, Caplan MS. Necrotizing Enterocolitis: Predictive Markers and Preventive Strategies. NeoReviews. 2013;14(3):e113-118. Fox TP, Godavitarne C. What really causes Necrotizing Enterocolitis? ISRN Gastroenterology 2012; 2012:1-9. doi:10.5402/2012/628317. Cetin S, Ford HR, Sysko LR, Agarwal C, Wang J, Neal MD, et al. Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions. J Biol Chem 2004; 279(23):24592-24600. Dai S, Sodhi C, Cetin S, Richardson W, Branca M, Neal MD, et al. Extracellular high mobility group box-1(HMGB1) inhibits enterocyte migration via activation of toll-like receptor-4 and increased cell-matrix adhesiveness. J Bio Chem 2010; 285(7):4995-5002. doi: 10.1074/jbc.M109.067454. Siggers RH and Hackman DJ. The role of innate immune-stimulated epithelial apoptosis during gastrointestinal inflammatory diseases. Cell Mol Life Sci 2011; 68(22):3623-3634. Afrazi A, Sodhi CP, Good M, Jia H, Siggers R, Yazji I, et al. Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium. J Immunol 2012; 188(9):4543-4557. Sodhi CP, Shi XH, Richardson WM, Grant ZS, Shapiro RA, Prindle T Jr, et al. Toll-like receptor-4 inhibits enterocyte proliferation via impaired beta-catenin signaling in necrotizing enterocolitis. Gastroenterology 2010; 138(1): 185-196. Caplan MS. Probiotic and prebiotic supplementation for the prevention of neonatal necrotizing enterocolitis. J Perinatol 2009; 29(Suppl2):S2-6. doi: 10.1038/jp.2009.21. Chen CC, Walker Allan. Probiotics and the mechanism of necrotizing enterocolitis. Seminars in Pediatric Surgery 2013; 22(2):94-100. Al Faleh K, Anabrees J. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2014;4:CD005496. doi:10.1002/14651858. CD005496.pub4. Coran AG, Caldamone A, Adzick NS, Krimmel TM, Laberge JM, Shamberger R. Pediatric Surgery. 7th Edition, Philadelphia, USA, Elsevier Saunders, 2012, Chapter 94;2: 1187-1207. ISBN: 978-0-323-07255-7.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

22. Tarnow Mordi W, Soll RF. Probiotic supplementation in preterm infants: it is time to change practice. J Pediatri 2014; 164(5):959-60. 23. Richardson WM, Sodhi CP, Russo A, Siggers RH, Afrazi A, Gribar SC, et al. Nucleotide-binding oligomerization domain-2 inhibits Toll-like receptor-4 signaling in the intestinal epithelium. Gastroenterology 2010; 139(3):904-917. 24. Joly AL, Wettstein G, Mignot F, Ghiringhelli and Garrido C. Dual role of heat shock proteins as regulators of apoptosis and innate immunity. J Innate Immun 2010; 2(3):238-247. 25. Good M, Siggers RH, Sodhi CP, Afrazi A, Alkhudar F, Egan CE, et al. Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium. Pro Natl Acad Sci U S A 2012;109(28):11330-11335. 26. Rowland KJ, Choi PM, Warner BW. The role of growth factors in intestinal regeneration and repair in necrotizing enterocolitis. Seminars in Pediatric Surgery2013;22(2):101-111. 27. Chen CL, Yu X, James IO, et al. Heparin-binding EGF-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing enterocolitis. Lab Invest 2012;92:331-344. 28. Yang J, Watkins D, Chen CL, Bhushan B, Zhou Yu Besner GE. Heparin-binding epidermal growth factor like growth factor and mesenchymal stem cells act synergistically to prevent experimental necrotizing enterocolitis. J AM Coll Surg 2012; 215:534-545. 29. Heather J.L. Brooks, Michelle A. McConnell and Roland S. Broadbent. Microbes and the Inflammatory Response in Necrotising Enterocolitis, Preterm Birth. Dr. Offer Erez (Ed.), 2013. ISBN: 978-953-51-0952-5, 30. Morgan J, Young L, McGuire W. Delayed introduction of progressive enteral feeds to prevent necrotizing enterocolitis in very low birth weight infants. Cochrane Database Syst Rev 2013;5:CD001970.doi:10.1002/14651858. 31. Dai D, Nanthkumar NN, Newburg DS, Walker WA. Role of oligosaccharides and glycoconjugates in intestinal host defense. J Pediatr Gastroenterol Nutr 2000; 30: S23-S33.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article Urine Cytology: A Review Archana Bhat, Manjunath J, Manjunath Shettty 1 Department of Pathology, Father Muller Medical College, Kankanady, Mangalore, India Department of Nephrology, Father Muller Medical College, Kankanady, Mangalore, India 3 Department of Urology, Father Muller Medical College, Kankanady, Mangalore, India

Keywords: Urine Cytology, the Paris System, Urine Cytology Review

ABSTRACT Urine cytology is being studied since many years. This simple and cost effective test can help in diagnosis of urothelial malignancies. It has undergone a series of modifications in accordance with changes in histopathological classifications. Various systems for reporting urine cytology were being followed. But still there were difficulties and confusion in patient management. As such there were no clear cut criteria which led to lack of interobserver reproducibility. With the upcoming of The Paris System of reporting urine cytology, many of the doubts cleared. This system mentions addresses the problem of sample adequacy and clearly mentions the criteria for the various categories. To top it up, it also gives clear cut guidelines for the management of each of the categories. This review gives a glimpse of the various reporting systems with special emphasis on The Paris System of urine cytology.

*Corresponding author: Dr Archana Bhat, Department of Pathology, Father Muller Medical College, Kankanady, Mangalore 575002, India Phone: +91 9035572134 E-mail: archibhat3@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

R-7

Introduction

The ‘matula’ ,a flask for inspection of urine used by physicians of the Middle ages is even to this day depicted in the official emblems of the American Urological Association, the German Society for Urology and the Professional Association of German Urologists. 1 Bladder cancer can be diagnosed by cystoscopic evaluation and biopsy, urine cytology and urinary biomarkers. Urine cytology is simple, cost effective and non invasive test. The presence of neoplastic cells in urine was described by Sanders in as early as 1864.2 However urinary oncocytology gained popularity with the publication by Papanicolaou and Marshall in 1945.3 Bladder cancer is the most common cancer of the urinary tract and the 9th most common cancer overall.4 There are about 74000 new cases of bladder cancer diagnosed for 2015 as per the American Cancer Society Estimate and also about 16000 deaths from bladder cancer.5

Discussion Types of urine samples Voided urine sample is the most common type of sample sent for cytological analysis. The first morning sample is avoided due to the degenerating effects produced by overnight stagnation of urine. The second morning sample is preferred. Voided urine samples the entire urinary tract from the pelvis to urethra which is referred to as the “funnel effect”. But the disadvantage is the contamination by squamous cells especially in the females.6 Other type of sample is the catheterized urine sample which is more cellular than voided urine and lacks the contamination by squamous cells. Wash and brush samples from the bladder, ureter or pelvis can be done along with cystoscopy. These provide better cellularity , targeted sampling and lack of contamination. Other less common samples include ileal conduit and neobladder samples.

AABS; 3(1): 2016 membrane filtration and Monoprep. Most of the studies suggest that ThinPrep is the preferred method overall due to the better cytomorphologic details, cleaner background and less obscuring inflammation.7 The various Reporting systems for urine cytology: The reporting systems for urine cytology went through a series of modifications in accordance with the changes in the histopathological classification of urothelial neoplasms. Lack of a uniform reporting system caused significant interobserver variability and created a great deal of confusion to the treating surgeons. The earliest system was proposed by G. Papanicolaou8 way back in 1947 which included 5 classes as follows: Class 1- Absence of abnormal or atypical cells Class 2 - Atypical cells present but without abnormal features Class 3- Cells with abnormal features but not sufficiently pathognomonic Class 4- Fair number of pathognomonic cells and cell clusters Class 5- Large number of conclusive cells and cell clusters The positive predictive value was 94.8% for positive cytology (Class 4 or 5), 47.4% for suspicious cytology (Class 3), and 7.4% for negative cytology (Class 1 or 2) in the study published by G.Papanicolaou.8 With the introduction of new WHO classification of urothelial neoplasms in 1973, Professor Koss proposed a cytological classification as follows9,10 in table 1. TABLES: Table 1 Histology

Cytopathology

Benign

Benign urothelial cells, few ATY 1 cells

Inflammatory conditions and instrumented urine

Bland clusters/fragments; ATY 1 cells

Papilloma, grade 1 papillary carcinoma

Clusters, nuclear elongation

Grade 2 and 3 papillary carcinoma, CIS

Malignant cells; numerous ATY 2 cells

Indications for urine cytology 1. Used as a screening test for urothelial malignancies, especially in people with occupational exposure to carcinogens. 2. Used in evaluation of patients presenting with hematuria. 3. Used to monitor patients of urothelial neoplasms post treatment. 4. Used to detect infection especially of polyoma virus in patients who have undergone renal transplantation. Processing of urine samples for cytology: Various methods used for urine cytology include ThinPrep, AutoCytePREP, Shandon Cytospin, nitrocellulose

Murphy and colleagues came up with another classification11 of thiers. They were of the opinion that low grade neoplasms cannot be differentiated from reactive processes and that a modearate rate of false positivity should be tolerated. They suggested that the term ‘dysplasia’ can be better used as an alternative. They reported that large cells with preserved nucleus-to-cytoplasmic ratios, smooth nuclear contours, and vacuolated cytoplasm support a benign process. The

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article

R-8

classificationscheme proposed by Murphy et al is given below. Classification Scheme Proposed by Murphy Negative/reactive

II. General categorization Negative for epithelial cell abnormality Epithelial cell abnormality present (see Descriptive Diagnosis) III. Descriptive diagnosis Negative for epithelial cell abnormality Infectious agents (bacterial organisms, fungal organisms, viral changes) Nonspecific inflammatory changes Cellular changes associated with chemotherapy or radiation Epithelial cell abnormalities

Dysplastic cells Abnormal cells, suspicious for malignancy Malignant tumor cells –

Low-grade neoplasm

–

High-grade neoplasm

–

Squamous cell carcinoma

–

Undifferentiated malignant tumor

Atypical urothelial cells (see Comments below)

–

Nonepithelial neoplasm.

Low-grade urothelial carcinoma

Later Ooms and Veldhuizen , in 1992 came up with another classification stating that there was lack of consensus on cytologic criteria for the term ‘dysplasia’ put forth by Murphy et al. This classification eliminated the term ‘dysplasia’. The classification proposed by them is as follows: 12

High-grade urothelial carcinoma Squamous cell carcinoma Adenocarcinoma IV. Others

Classification Scheme Proposed by E. C. Ooms Negative cytology

Comments section is included at the discretion of the cytopathologist.

Atypical cells, significance uncertain

Findings from ancillary studies can be incorporated in this section.

Atypical cells, suspicious for malignancy

Then came the Diagnostic categories of the Hopkins Template for Urine Cytology Samples proposed by Owens et al14 which is as follows:

Neoplastic cells present – Grade 1 carcinoma –

Grade 2 carcinoma

No urothelial atypia or malignancy identified (NUAM)

–

Grade 3 carcinoma

Urothelial carcinoma (specify)

–

Carcinoma in-situ

High-grade (HGUC)

–

Squamous cell carcinoma

Low-grade (LGUC)

– Adenocarcinoma –

Atypical urothelial cells of uncertain significance (AUCUS)

Small cell carcinoma

– Other In 2003, then came the Papanicolaou Society of Cytopathology Task Force classification.13 This classification included only one equivocal category –‘atypical urothelial cells’. However the criteria for this equivocal category were not conclusive and they felt the need for more studies on this category. They also mentioned about the extension of ancillary studies like FISH on urine cytology specimens. Papanicolaou Society of Cytopathology Practice Guidelines Task Force classification I. Adequacy statement

Atypical urothelial cells, cannot exclude HGUC (AUC-H)] Other But the discrepancy, the controversy and the lack of uniformity in reporting urine cytology continued. This led to a new system of reporting urine cytology which is comparable to the Bethesda system for reporting cervical cytology and thyroid cytology. This new system was put forth by a panel of international cytopathologists and urologists at the 18th International Congress of Cytology held at Paris in May, 2013. This came to be named as “The Paris System for reporting Urine Cytology”.(TPS)15

http://www.pacificejournals.com/aabs

R-9

AABS; 3(1): 2016

The Paris System for reporting urinary tract Cytology: Adequacy : Specimen is considered Adequate if atypical, suspicious or malignant Adequate if there is appropriate benign urothelial cellularity. Adequate if there is adequate volume in absence of appropriate benign urothelial cellularity. Inadequate if non-urothelial factors are obscuring urothelial cells. Inadequate if there is no appropriate benign urothelial cellularity in instrumented specimen. Categories of The Paris System:

5. High Grade Urothelial Carcinoma

Unsatisfactory/Non diagnostic

Conclusion

1. Negative for High grade Urothelial Carcinoma (NHGUC) 2. This does not exclude the possibility of low grade urothelial neoplasms. These patients should be again screened in the next scheduled check-up. 3. Atypical urothelial cells(AUC)- the criteria for AUC are – Non-superficial and non-degenerated urothelial cells with a N:C ratio of >0.5 – Along with one of the three below mentioned features – Hyperchromasia – Irregular coarse, clumped chromatin – Irregular nuclear membrane(contours)

The cytological features are high cellularity, loose clusters, singles, moderate to marked pleomorphism, increased N:C ratio, irregular clumped chromatin, irregular nuclear membrane, eccentrically located large pleomorphic nuclei, prominent nucleoli, squamous/glandular differentiation,.

6. Low Grade Urothelial Neoplasm

The features for this category are subtle and easily missed. The important feature that can be relied upon is the presence of well defined fibrovascular cores with capillaries within.

7. Other malignancies- Primary and secondary. Hopefully with the upcoming of The Paris system of reporting urine cytology, there will be uniformity and good reproducibility resulting in better treatment and patient outcome.

Acknowledgements None

Funding None

Competing Interests None declared

Reference

1. Rathert P. History of Urinary Cytology. Berlin Heidelberg. Springer 1993.

This category of patients should be followed up closely. In the context of previously documented urothelial neoplasm, this category should be subjected to ancillary studies like FISH, microsatellite studies, etc. If the N:C ratio is >0.7 along with two of the three features, then a diagnosis of “suspicious for high grade urothelial carcinoma” should be made. 4. Suspicious for High Grade Urothelial Carcinoma The criteria for this category include – N:C ratio of >0.7 and – Hyperchromasia Along with one of the two below mentioned features – Irregular clumped chromatin – Irregular nuclear contours. This category of patients should be followed up closely with cystoscopy, ureteroscopy and surgical biopsies.

2. Sanders WR. Cancer of the bladder. Fragments forming urethral plugs discharged in the urine: Concentric colloid bodies. Edinb J Med 1864;10:273‑4

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

3. Papanicolaou GN, Marshall VF. Urine sediment smears as a diagnostic procedure in cancers of the urinary tract. Science 1945;101:519‑20. 4. Ploeg M, Aben KK, Kiemeney LA. The present and future burden of urinary bladder cancer in the world. World J Urol 2009; 27:289. 5. www.cancer.org 6. Sullivan PS, Chan JB, Levin MR, Rao J. Urine cytology and adjunct markers for detection and surveillance of bladder cancer. American Journal of Translational Research. 2010;2(4):412-440. 7. Jesse S. Voss, Benjamin R. Kipp, Angela K. Krueger, Amy C. Clayton, Kevin C.Halling, R. Jeffrey Karnes, Michael R. Henry, Thomas J. Sebo.

Review Article

R-10

Changes in Specimen Preparation Method May Impact Urine Cytologic Evaluation. American Journal of Clinical Pathology Sep 2008, 130 (3) 428-433 8. Papanicolaou GN. Cytology of the urine sediment in neoplasms of the urinary tract. J Urol. 1947;57:375-379. 9. Koss LG, Bartels PH, Sychra JJ, Wied GL. Diagnostic cytologic sample profiles in patients with bladder cancer using TICAS system. Acta Cytol. 1978;22:392-397. 10. Koss LG. Diagnostic Cytopathology and Its Histopathologic Basis. 5th ed. Vol 2. Philadelphia, PA: JB Lippincott Company; 2006. 11. Murphy WM, Soloway MS, Jukkola AF, Crabtree WN, Ford KS. Urinary cytology and bladder cancer. The cellular features of transitional cell neoplasms. Cancer. 1984;53:1555-1565.]

12. Ooms EC, Veldhuizen RW. Cytological criteria and diagnostic terminology in urinary cytology. Cytopathology. 1993;4:51-54. 13. Layfield LJ, Elsheikh TM, Fili A, Nayar R, Shidam V; Papanicolaou Society of Cytopathology. Review of the state of the art and recommendations of the Papanicolaou Society of Cytopathology for urinary cytology procedures and reporting: the Papanicolaou Society of Cytopathology Practice Guidelines Task Force. Diagn Cytopathol. 2004;30:24-30. 14. Owens, C. L., VandenBussche, C. J., Burroughs, F. H. and Rosenthal, D. L. (2013), A review of reporting systems and terminology for urine cytology . Cancer Cytopathology, 121: 9–14. doi: 10.1002/cncy.21253 15. Rosenthal DL, Wojcik EM, Kurtycz DFI. The Paris System for reporting Urine Cytology. New York. Springer; 2016.

http://www.pacificejournals.com/aabs

Original Article

Diagnosis of Endophthalmitis with amplification of syndrome specific signature genes by Syndrome Evaluation System: A Retrospective Analysis Anoop Sivaraman1, Pandurang Kulkarni1, Sundaram Natarajan1, Rishi Bhardwaj1, Ashad Sivaraman2 1 2

Department of Vitreo Retina Services, Aditya Jyot Eye Hospital, Wadala, Mumbai, India Department of Vitreo Retina, Precise Eye Care, Trivandrum, Kerala, India Keywords: Endophthalmitis, Syndrome Evaluation System, Vitrectomy

ABSTRACT

Background: To evaluate the microbiological concordance of clinically diagnosed endophthalmitis cases using isolation of genetic material of the causative agent from either aqueous or vitreous fluid and simultaneous amplification of syndrome specific signature genes. Methods: Thirteen cases clinically diagnosed as endophthalmitis at tertiary centre of Vitreo-Retina care, in whom isolation of genetic material of the causative agent from either aqueous or vitreous fluid and simultaneous amplification of syndrome specific signature genes termed as Syndrome evaluation system (SES) was carried out were included in this retrospective analysis. The vitrectomy samples or Aqueous taps were sent to for SES analysis and results were obtained within 24 hours of vitrectomy and results considered for deciding treatment. Results: 7 out of 13 cases were positive for bacteria or fungi or virus when tested on SES. Out of Seven positive two were also culture positive and concordant. Three out of these seven positives did not improve in visual acuity while two cases improved and in two cases there was deterioration in visual acuity noted. Six out of the 13 cases SES did not detect any organisms. It can also be seen that 5 out of six negatives improved in visual acuity with treatment. Conclusion: SES was an accurate, quick and reliable diagnostic method in endophthalmitis whose sensitivity was much higher than culture and Gramâ&#x20AC;&#x2122;s staining. This diagnostic technique can help in administering targeted therapies at a much earlier stage and hence improve the overall outcome of the endophthalmitis cases.

*Corresponding author: Dr. Anoop Sivaraman, Department of Vitreo Retina Services, Plot no 153, Road no. 9, Major R Paramesweran Road, Opposite S.I.W.S. College Gate No.3, Wadala, Mumbai, India. 400031 email:anoop.sivaram@gmail.com; Ph: +91-9400004404

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-2

Original Article

INTRODUCTION Endophthalmitis is the bacterial or fungal infection of the vitreous and/or aqueous humors[1]. This condition is usually categorized based on several aspects including clinical course (acute or chronic), aetiology (infectious or non-infectious), route of entry of the causative agent (exogenous - noted following eye surgery or penetrating eye trauma or endogenous - occurring due to bacteraemic or fungaemic seeding of the eye) and organism(s) involved (bacteria, fungi, parasites and rarely, viruses)[2]. Prompt and accurate diagnosis is vital to guide the initial therapy and minimize damage as disorders such as bacterial endophthalmitis can often progress rapidly, while fungal endophthalmitis can worsen with the empiric use of corticosteroids (without specific antimicrobial therapy)[3]. Diagnosis of endophthalmitis relies on clinical symptoms as well as laboratory tests[1]. Gram stain of the aspirate of aqueous and/or vitreous is commonly followed, which often is not highly sensitive and is considered helpful only when positive for organisms. Vitrectomy cultures although highly reliable, often have poor concordance when comparing anterior chamber culture with vitreous isolates [2, 4]. Polymerase chain reaction (PCR), used to amplify deoxyribonucleic acid (DNA) sequences, has been reported to have a vital role in the diagnosis of endophthalmitis[2]. However, there is a need for multiple PCR tests in order to point to the aetiological agent in a given case of endophthalmitis [5]. The major obstacle for performing multiple PCRs to arrive at a diagnosis in an algorithmic approach is the paucity of clinical specimen, aqueous or vitreous. Moreover a false-positive rate of 5% has been noted with PCR tests reported hitherto, which in turn resulted in cautious use of molecular diagnostics for endophthalmitis[6]. Hence is the requirement of high specificity built in to the methodology. Syndrome evaluation system (SES), a patented technology involving isolation of genetic material of the causative agent from relatively small volume of either aqueous or vitreous fluid and simultaneous amplification of syndrome specific signature genes of all probable organisms, followed by syndrome specific hybridization, allows for simultaneous detection of all probable pathogens in a single test and can prove to be a valuable tool for accurate and rapid identification of the causative microorganisms in endophthalmitis in a short time.[7] . Multiplex amplification of genes in SES confers high sensitivity to this molecular diagnostic tool while syndrome specific hybridization conferred specificity to the test.

This retrospective analysis of 13 continuous cases clinically diagnosed as endophthalmitis was performed to compare the microbiological concordance of results of SES test with those of culture, Gram’s stain and Calcofluor white staining for its sensitivity and concordance.

MATERIALS AND METHODS Thirteen cases clinically diagnosed as endophthalmitis at Aditya Jyoth Hospital, Mumbai between January 2009 and October 2013, in whom SES evaluation was carried out were included in this retrospective analysis. Initial evaluation of these patients were conducted using culture, Gram’s stain and Calcofluor white staining. The SES evaluation was advised on the day of vitrectomy in cases where no clinical improvement was noted. The vitrectomy samples or Aqueous taps were sent to XCyton lab in Bangalore wherein SES analysis was done and results were conveyed to the surgeon within 24 hours after the vitrectomy. The treatment in these 13 cases was then decided based on the SES results. SES endophthalmitis detects Gram +ve organisms S. aureus, Coagulase Negative Staphylococci, Streptococcus spp, Entrococcus spp, Propionibacterium acnes, Gram negative Bacteria E. coli, Klebsiella, Enterobacter aerogenes, H. inluenzae, Pseudomonas aeruginosa and fungi Candida Spp, Aspergillus spp and Fusarium Spp. a) Nucleic acid extraction: Nucleic acid was extracted from the standard strains using commercial columns (Qiagen, USA) as per the procedure specified in the instruction manual provided by the manufacturer. b) Nucleic acid amplification: Nucleic acid amplification was standardized in a 50µl volume containing 4 mM magnesium chloride, 0.2 mM deoxynucleoside triphosphates, 50 to 300 nM concentration of each primer set and 1U of Taq polymerase (ABI, USA). The initial denaturation step was carried out at 950C for 10 minutes followed by 40 cycles of denaturation at 950C for 45 seconds, annealing at 600C for 45 seconds and extension at 720C for 45 seconds in a thermal cycler (Bio-Rad, UK). c) Hybridization: Signature gene sequences chosen as probes for each of the pathogen were commercially synthesized (Metabion Inc., Germany). 20µM of probes for each of the pathogen were transferred on to a pre-determined position on the SES platform according to the templates. The SES platform comprised of a plastic frame mounted on a charged membrane on to which probes were arrayed at predetermined positions For each gene amplified a single probe was used for hybridization. The whole process from Gene Extraction to results takes 7 hours.

http://www.pacificejournals.com/aabs

A-3

AABS; 3(1): 2016

Table 1:Results of diagnostic evaluation of samples obtained. Sl. No.

Vision before treatment Pl Pr

Vision after 3 months of treatment 6 / 60

Pl Pr

Hm Cf

Pl Pr

no Pl

Negative

Hm Pl Pr

6./9 no Pl

negative

Negative

Cf 3mt Missing data

Negative Negative Candida

negative negative negative

Negative

Cf 1 mt Missing data Pl Pr 6/.60 6./24

Negative Negative Negative

negative negative negative

Negative

6/.60 Cf 2 M Cf 1 mt

6./6 CF 2 M Cf 1 mt (6 weeks post op)

15.38%

7.69%

Sample

SES

Culture

Gram’s Staining

Gram Positive Bacteria

Negative

negative

Negative

negative

Negative

negative

4 5

Vitreous humor Vitreous humor Vitreous & Tissue Vitreous tap Vitreous asp

Negative enterococci

negative

6 7

Vitreous asp Vitreous Tap

Gram Positive Bacteria Gram Positive Bacteria Streptococcus/ Enterococcus Negative Gram Positive Bacteria

Negative

8 9 10

Vitreous tap Vitreous tap Capsular bag

11 12 13

Vitreous tap Vitreous tap Vitreous tap

Negative Negative CONS, Candida, E.aerogenes, P.aeruginosa* Negative S. aureus positive Varicella Zoster Virus

Sensitivity

53.85%

1 2 3

Calcofluor Staining

few fungal filaments

Hm Cf 6/.24 6./24

*CONS: coagulase-negative staphylococci; E. aerogenes: Enterobacter aerogenes; P.aeruginosa: Pseudomonas aeruginosa. Abbreviations: Pl newer techniques are considered to be mainly perception of light; Pr – projection of rays; Hm – hand motion in front of face;These Cf – counting of fingers

Patients were reassessed three months after discharge. Institutional review board (IRB) approval was obtained for this analysis.

RESULT The study sample included 61.5% men and 38.5% women, with a mean age of 60.62 years (range: 28 years -78 years). Three of these patients had been diagnosed with diabetes while two of them had hypertension. One patient had both diabetes and hypertension. The results of the diagnostic evaluation have been given in table 1. As can be seen from the table 1, 7 out of 13 cases were positive for bacteria or fungi or virus when tested on SES. Out of Seven positive two were also culture positive and concordant. Three out of these seven positives did not improve in visual acuity while two cases improved and in two cases there was deterioration in visual acuity noted. Six out of the 13 cases SES did not detect any organisms. It can also be seen that 5 out of six negatives improved in visual acuity with treatment.

DISCUSSION Along with conventional culture techniques, molecular biology techniques have now become an important complementary diagnostic method in the diagnosis of endophthalmitis.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

beneficial while evaluating bacteria that are impossible or difficult to grow owing to their intrinsic properties, presence in a small inoculum, sequestration on prosthetic materials, or inactivation by prior antibiotic therapy[8].

While the syndrome specific hybridization made possible with the SES method allows for higher sensitivity, re-naturation of amplified signature gene to its chemically identified complementary gene sequence on the SES ensures higher specificity[7]. However the organisms detected in SES- Endophthalmitis cover most of the pathogens known to cause postoperative endophthalmitis and endogenous endophthalmitis In the current analysis, the SES assisted detection rate was 3.5 times higher than that of culture (sensitivity with SES and culture was 53.85% and 15.38%, respectively) while the concordance with culture was 100% (SES was able to detect enterococci and candida as noted with culture results). Additionally, SES was able to detect the following pathogens in comparison to culture testing: i) Enterococci and Gram positive bacteria Streptococcus ii) E. aerogenes, P. aeruginosa and Coagulase-Negative Staphylococcus

In a similar retrospective review of consecutive cases with infective endogenous endophthalmitis in Hong

e-ISSN: 2349-6991; p-ISSN: 2455-0396

A-4

Original Article

Kong, the overall culture positive rate with intraocular specimen was 24% and was considered to be relatively low by the authors. They also noted that such a low rate would make it difficult to arrive at a microbiological diagnosis in endogenous endophthalmitis[6].

COMPETING INTERESTS

Discordance was noted with 1 case of calcofluor white staining showing few fungal filaments. However, the sample was culture negative, as well as SES negative. Viral detection was possible in one case with SES wherein the presence of varicella zoster virus (as suspected) was identified in the vitreous sample.

The recruitment of cases discussed in this report occurred after an initial trial of intra-ocular antibiotic. Only the cases with no clinical improvement were taken in to this study and thus the improvements seen due to SES guided therapy are not dramatic. Molecular diagnostics like SES, if used early enough during the episode of endophthalmitis, may therefore prove to be crucial for better patient outcomes. The treatment decisions were taken based on the results obtained following SES. Accurate diagnosis followed by early therapy improves prognosis, especially in rare conditions such as endogenous endophthalmitis[9]. Molecular diagnostic techniques promise lesser diagnostic times with more accurate diagnosis than other modalities. Such methods would enable initiation of targeted therapies at a much earlier stage of the disease to ensure better prognosis[2].

None declared.

REFERENCES

CONCLUSION In conclusion, SES is an accurate, quick and reliable diagnostic method in endophthalmitis whose sensitivity is much higher than culture and Gram’s staining. This diagnostic technique can help in administering targeted therapies at a much earlier stage and hence improve the overall outcome of the endophthalmitis cases.Although this study involved a small number of patients, a larger study with more number of patients is required to accurately assess the impact of SES in patient outcomes.

ACKNOWLEDGEMENTS None.

FUNDING

Durand ML. Endophthalmitis. Clin Microbiol Infect. 2013 Mar;19(3):227-34. Safneck JR. Endophthalmitis: A review of recent trends. Saudi J Ophthalmol. 2012 Apr;26(2):181-9. doi: 10.1016/j.sjopt.2012.02.011. Epub 2012 Mar 3. Davis JL. Diagnostic dilemmas in retinitis and endophthalmitis. Eye (Lond). 2012 Feb;26(2): 194-201. Almeida DR, Miller D, Alfonso EC. Anterior chamber and vitreous concordance in endophthalmitis: implications for prophylaxis. Arch Ophthalmol. 2010 Sep; 128(9):1136-9. Ravikumar BV. “Syndrome evaluation system (SES)” A paradigm shift in diagnosis of critical infections. Kerala Journal of Ophthalmology. 2011; 23(1); 77-78. Wu ZH, Chan RP, Luk FO, Liu DT, Chan CK, Lam DS, Lai TY. Review of Clinical Features, Microbiological Spectrum, and Treatment Outcomes of Endogenous Endophthalmitis over an 8-Year Period. J Ophthalmol. 2012;2012:265078. doi: 10.1155/2012/265078. Epub 2012 Feb 23. Mahalingam P, Sambhav K. Diagnosis of post-operative polymicrobial endophthalmitis by xcyton analysis. J Clin Ophthalmol Res 2013;1:21-2. Cornut PL, Boisset S, Romanet JP, Maurin M, Carricajo A, Benito Y, Vandenesch F, Chiquet C. Principles and applications of molecular biology techniques for the microbiological diagnosis of acute post-operative endophthalmitis. Surv Ophthalmol. 2013 Dec 17. pii: S0039-6257(13)00200-2. doi: 10.1016/j.survophthal. 2013.08.002. Asencio-Egea MA, Huertas-Vaquero M, Carranza-González R, Cells-Sánchez J, González-del Valle F, Tenías-Burillo JM, Barberá-Farré JR. Endogenous endophthalmitis: case report and brief review of a serious ocular disease. Rev Chilena Infectol. 2013;30(5):516-21.

None.

http://www.pacificejournals.com/aabs

Original Article

A Case Series of Retinochoroidal Toxoplasmosis from Western India: Guide to Salient Features, Diagnosis and Treatment Deepen Sheth, Pandurang Kulkarni, Sundaram Natarajan Department of Vitreoretinal & Uveitis Services, Aditya Jyot Eye Hospital Pvt. Ltd., Wadala, Mumbai, India. Keywords: Toxoplasmosis, Posterior Uveitis, Retinochoroiditis, Floaters

ABSTRACT

Background: To study a series of cases of retinochoroidal toxoplasmosis with special mention of its clinical features, investigations & treatment. Although rare, it represents a significant number of all causes of posterior uveitis. Settings and Design: Retrospective Observational study. Methods: 5 cases of primary and 2 cases of recurrence of acquired ocular toxoplasmosis in immunocompetent adults, from western India are included in these series. We study the clinical features & optical coherence tomography & fundus fluorescein angiography characteristics and the outcome with the quadruple drug therapy. Results: In our study, we confirm that presence of specific symptoms and classical signs along with typical fundus fluorescein angiography (FFA) & optical coherence tomography (OCT) findings makes the diagnosis precise. A positive serology further aids in the diagnosis. Treatment with the quadruple regimen resulted in good visual prognosis in all but one case complicated with scarring at macula. Conclusions: This study revives the diagnostic findings and helps as a guide to identify this potentially treatable cause of visual morbidity.

*Corresponding author: Dr Deepen Sheth; Aditya Jyot Eye Hospital Pvt. Ltd., Plot No. 153, Road No. 9, Major Parmeshwaran Road, Opp S.I.W.S. College Gate No. 3, Wadala, Mumbai 400 031. India. Phone numbers : Tel : +91-22-2417 7600. Mobile : 09619850581. E-mail:deepensheth@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-6

Original Article

INTRODUCTION Toxoplasma gondii.is an ubiquitous obligate intracellular protozoa, infecting upto one third of world’s population.1-3 Once infected, it remains chronically persistent throughout life. It has recently been concluded that eye is an important site for the acquired disease.4-7 Thus toxoplasmosis is an important cause of visual impairment, it being responsible for a third to a half of all posterior uveitis cases.7-13 Acquired infections in immunocompetent individuals are usually asymptomatic. Diagnosis of such cases helps us to administer a treatment regimen which has proven good results. We present this series in an attempt to provide a guide in managing these patients.

MATERIALS AND METHODS A total of 7 eyes, of 7 patients who were diagnosed to have retinochoroidal toxoplasmosis, are included in this series. 5 of them were diagnosed as acquired primary disease and 2 had recurrent disease. The patients underwent thorough ophthalmic evaluation including visual acuity, intraocular pressure, anterior segment examination at slit lamp and posterior segment examination with indirect ophthalmoscopy at all visits. After provisional clinical diagnosis, they underwent optical coherence tomography & fundus photography (including autofluorescence) on day 1 & on 6 weeks after completion of the treatment regimen. Fundus fluorescein angiography was performed before commencing the treatment. Serological tests for IgG & IgM were also conducted for all the patients. Classic regimen include a combination of oral sulfadiazine + pyrimethamine (or sulfamethoxazole + trimethoprim) and prednisolone.14,15 Oral clindamycin is added as a part of the quadruple therapy.16,17 All our patients recieved a regimen as in the table. (Table 1) Table 1. Quadruple treatment regimen. Oral drug Sulfamethaxazole - Trimethoprim (double strength) Prednisolone

Dosage 800 mg – 160 mg BD x 6 weeks

Remarks Well tolerated.

40/30/20/10/5 mg OD ½ hour after breakfast (8 am) x 3 days each

Clindamycin

300 mg QID x 6 weeks

To be started 2 days after the antibiotics, tapered and stopped before them*. Side effect : Pseudomembranous colitis†.

*Avoided in Case 7 as the lesion was away form macula & optic disc and the patient was not keen for steroid intake. †Clindamycin is added to the classic regimen for the quadruple therapy. 2 patients (Case 2 & Case 6) reported loose stools while on the therapy. The drug was discontinued in the first week for Case 2 due to intolerance.

Case 1: A 52-year-female patient came to us with blurring of vision and floaters in the right eye (RE) since 5 days. Anterior chamber showed minimal inflammation with occasional cells. Posterior segment showed attached retina, retinochoroiditis lower nasal to disc with localised vitritis& adjoining vasculitis (Fig. 1a). FFA RE showed a hypofluorescent lesion with border becoming hyperfluorescent at late phase (Fig. 2). OCT RE showed hyperreflective lesion with localised vitritis (Fig. 3a). Patient’s response to treatment was excellent, as is evident with resolution of vitritis and lesion (Fig. 1b &1c). Case 2: A 17-year-male presented with a 6 day history of left eye (LE) decreased vision and nasal field appearing hazy. Upon examination he was diagnosed as paramacular active toxoplasmosis temporal to fovea (Fig. 3a & 3b) and trace vitreous cells. OCT showed evidence of submacular fluid (Fig. 5b). FFA examination demonstrated the classical progression of hypofluorescence to hyperfluorescence in the periphery of the lesion. Patient was discontinued of clindamycin within a week, due to loose stools. Treatment with the triple therapy resulted in an inactive retinochoroidal scar (Fig. 3e & 3f). Case 3: A 35-year-male presented with recent onset of blurring and floaters in LE since 15 days. He gave a history of ocular toxoplasmosis 5 years back. On examination he was found to have recurrence of retinochoroidal toxoplasmosis, localised vitritis with serous retinal detachment and pigmented scar upper temporal quadrant (UTQ) in LE. Upon treatment his lesion resolved with residual vitreous debris, an UCVA of 6/5 in LE and resolution of the subretinal fluid. Case 4: A 32-year-female came to our hospital with redness and floaters in LE since 10 days. She was diagnosed with active retinochoroiditis with overlying localised vitritis and vasculitis in LE. Case 5: The above patient presented at 6 years with a recurrence at the margin of a previous pigmented scar (Fig. 3c & 3g) with diminution of vision. BCVA increased from 6/60 to 6/12p in LE at 6 weeks following resolution of the lesion. Case 6: A 57-year-male complained of central blurring in RE since few days. He was diagnosed as acute retinochoroiditis with a positive serology. At 5 weeks of the therapy, clindamycin was stopped as he reported with complaints of frequent loose stools. His vision gradually deteriorated as opposed to other cases reported in our series. At 6 weeks, examination showed an inactive scar with a speck of hemorrhage (Fig. 4b &5a). Patient was not keen on angiography & was lost to follow up.

CASES :

http://www.pacificejournals.com/aabs

A-7

AABS; 3(1): 2016

Figure 1 : Active whitish retinochoroiditis lesion (white arrowhead) with associated vasculitis (black arrows) {Figure 1a}. Colour fundus photo {Figure 1b} and autofluorescence photo {Figure 1c} showing resolved vitritis and healing lesion with hypofluorescent ring in the periphery (grey arrowhead). Vasculitis is also resolved (red oval).

Figure 2 : Fundus Fluorescein Angiography showing the classical masking in the initial phase {Figure 2a} with late hyperfluorescence at the periphery {Figure 2b & 2c} (grey arrow).

Figure 3 : Colour and red free photos showing active retinochoroiditis {Figure 3a, 3b, 3c, 3d} and inactive scar {Figure 3e, 3f, 3g, 3h}. ILM striae are evidently lost. Pigmented scars (white arrows) with recurrence & resolution (black arrows) seen near the margins.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

A-8

Original Article

Case 7: A 29-years-male came for a check up for floaters in RE since 15 days. He was diagnosed with retinochoroiditis. FFA showed the classical hypo to hyperfluorescence of the lesion. Steroids were avoided as the patient was not keen & the lesion was away from the fovea and optic disc (Fig. 3d & 3h). OCT showed presence of localised vitritis&epiretinal membrane (Fig. 4c). Institutional review board / Ethics Committee: This series was registered and approved by the institutional review board / ethics committeeas it was a retrospective observational study.

RESULT Symptoms : In our series, sudden onset of floaters and blurring of vision were reported as the most common presenting symptoms, while 2 patients presented with diminution of vision (Graph 1).

Figure 4 : Optical Coherence Tomography {Figure 4a & 4c} shows an elevatedhyperreflective lesion with localised vitreitis and an epiretinal membrane.OCT of the right eye {Figure 4b} shows scar formation .

Symptoms

7 6 5 4 3 2 1 0 Blurring

Floaters

Dimunition of vision

Redness

Graph 1 : Symptoms distribution

Complications 7 6 5 4 3 2 1 0

Complications

Graph 2 : Complications distribution.

Figure 5 : Fundus photo {Figure 5a} showing evidence of a macular scar with a speck of hemorrhage (black arrow). OCT macula {Figure 5b} showing presence of subretinal fluid at the macula. Temporal to it is seen a hyperreflective lesion with localised vitreitis.

Investigations : FFA & OCT provided sufficient confirmatory findings to diagnose the cases. IgG was positive in all the patients, while IgM was positive in Case 3. Treatment : Most patients in the series received the complete course of the quadruple therapy. However, clindamycin was stopped in the first week for Case 2, due to complaints of severe loose stools. Case 6 also complained of loose stool, though he could tolerate the treatment and completed the course (Table 1).

http://www.pacificejournals.com/aabs

A-9

AABS; 3(1): 2016

Outcome : All patients showed visual improvement at the completion of therapy, except Case 6 (Table 2) which progressed to developing macular scar. Noted complications in our series include vasculitis, epiretinal membrane & serous retinal detachment (Table 2 & Graph 2). Table 2. Patient data with complications & visual outcomes. Cas e No. 1.

Age/ Sex

Laterality

Pre Rx

Post Rx

Complications

52/F

6/6

17/M

6/24

6/5

35/M

6/9

6/5

4. 5. 6.

32/F 37/F 57/M

LE LE RE

6/18 6/60 6/6p

29/M

6/6

6/9 6/18 FC @ ½m 6/5

Vasculitis, Epiretinal membrane. Serous retinal detachment Serous retinal detachment Vasculitis CNVM suspect Epiretinal membrane

lead to development of an epiretinal membrane (Fig. 4a & 4c) or vitreoretinal traction adjacent to the area of retinochoroiditis.. Retinochoroiditis :  



DISCUSSION Toxoplasma exists in three possible forms : 1. Oocysts (highly infective form) which results from the sexual cycle occurring in the intestinal epithelium of felines, 2. Tissue cysts (latent form in the chronic phase & contains bradyzoites), persisting in the skeletal muscles and brain, and, 3. Tachyzoites (actively replicating form), results in quick dissemination of the infection through the blood stream to various tissues, & in some reports, from brain to the eye via the optic nerve.1 Humans acquire the infection either through the ingestion of poorly cooked meat containing tissue cysts, or ingestion of water or food contaminated with oocysts or through transplacental transmission of tachyzoites.1 Acute retinochoroiditis results from active replication of tachyzoites in the tissue, predilection for retinal tissue is confirmed by its high affinity for retinal vascular endothelium.18 Active lesions upon resolution result in scar formation, which may harbour tissue cysts and result in a recurrence at a later date. Symptoms :The common signs and symptoms of this cause of infectious uveitis are summarised below19 Anterior segment : Granulomatous uveitis Vitireitis : Inflammation of the vireous may be diffuse or more commonly localised (Fig. 4a & 4c) , resulting in blurring and/or floaters. Localised vitritis over the lesion may Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016



Primary: fluffy whitish lesion suggestive of focal retinal necrosis (Fig. 1a, 3a, 3b, 3c & 3d). Satellite lesion: active lesion at the edge of a well demarcated retinochoroidal pigmented scar (Fig. 3c& Fig. 3g). Headlight in fog (coined by Richard O Connor) : appearance of a white reflex in the presence of severe vitritis when a bright light is shown on the fundus (Fig. 1a). Vasculitis (Fig.1a) : vascular sheathing, exudation and arteriolar plaques (Kyrieleis’ arteriolitis). Periphlebitis is noted more commonly than arteritis. Resolution of the active lesion results in atrophic retinochoroidal scar which heals from the periphery to the centre resulting in pigmentation (Fig. 3e, 3f, 3g & 3h).19

In our study, most patients presented with classical signs and symptoms suggestive of the condition, which was confirmed on investigations and treatment response. Investigations : Serology: A battery of serology tests include detection of antiToxoplasma antibody IgG, IgM, IgA &IgE. IgM antibodies appear in the first week of infection and generally decline over a few months. IgG antibodies appear in the second week of infection, peak in 6 to 8 weeks, and remain detectable for life, hence is not diagnostic. IgA and IgE antibodies can be detected during the acute phase of the infection and may be used for the identification of recently acquired infections.20,21However, as Toxoplasma infects a third of the world’s population, serology faintly plays an active role in deciding upon treatment.22,23 Detection of Tgondii. : PCR technique or isolation from body fluids including aqueous & vitreous increases sensitivity & specificity.24These are expensive and not easily accessible methods, but may be useful in visually threatening non classical suspicious lesions. Fundus fluorescein angiography: Hypofluorescence (due to masking by the lesion) at the early phase of the angiography, is followed by hyperfluorescence progressing towards the center of the lesion (Fig. 2). Optical coherence tomography: Demonstrates localised vitritis, epiretinal membrane, tractional vitreous attachment, retinal edema, serous retinal detachment (Fig. 5b) and neovascular membrane. Treatment : The main goals of the treatment are to reduce the duration and severity of symptoms of acute intraocular inflammation, and the risk of permanent vise-ISSN: 2349-6991; p-ISSN: 2455-0396

A-10

Original Article

ual loss and the risk of recurrent episodes. Various treatment regimen including triple/quadruple drugs are being followed all over the globe. Classic regimen include a combination of oral sulfadiazine + pyrimethamine (or sulfamethoxazole + trimethoprim) and prednisolone. Oral clindamycin is added as a part of the quadruple therapy. Other drugs that have been used include spiramycine, minocycline, azithromycin, atovaquone, and clarithromycin. Among all these drugs, atovaquone is the only drug which is active against cystic forms as well. However it is shown to only prolong reactivation and not prevent recurrences.25 Recent reports have suggested an alternate promising treatment option with multiple intravitreal clindamycin (1.5mg/1mg, weekly or biweekly) +/- dexamethasone.15,26,27 Recently acquired cases have shown a better response profile with classic v/s intravitreal therapy.27 Complications : Although rare,these include vasculitis, vascular occlusions, neovascularisation, epiretinal membrane, serous retinal detachment, macular edema, optic disc involvement, etc...28

10.

11.

12.

ACKNOWLEDGEMENTS None.

13.

FUNDING None.

14.

COMPETING INTERESTS None declared.

15.

REFERENCES 1.

Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217–58. Holland GN. Ocular toxoplasmosis: a global reassessment. Part I: epidemiology and course of disease. Am J Ophthalmol 2003;136:973–88. Jones J, Kruszon-Moran D, Wilson M, et al.Toxoplasmagondii infection in the United States. Am J Epidemiol 2001;154:357–65. Bowie WR, King AS. Outbreak of toxoplasmosis associated with municipal drinking water. Lancet 1997;350:173–8. Burnett AJ, Shortt SG, Isaac-Renton J, et al. Multiple cases of acquired toxoplasmosis retinitis presenting in an outbreak. Ophthalmology 1998;105:1032–7. Holland GN, Muccioli C, Silveira C, et al. Intraocular inflammatory reactions without focal necrotizing retinochoroiditis in patients with acquired

16.

17.

18.

19.

20.

systemic toxoplasmosis. Am J Ophthalmol1999;128:413–20. Glasner PD, Silveira C, Kruszon-Moran D, et al. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am J Ophthalmol 1992;114:136–44. Silveira C, Belfort R Jr, Burnier M, et al. Acquired toxoplasmosis infection as the cause of toxoplasmicretinichoroiditis in families. Am J Ophthalmol 1988;106:362–4. Matos K, Muccioli C, Belfort R Jr, et al. Correlation between clinical diagnosis and PCR analysis of serum, aqueous, and vitreous samples in patients with inflammatory eye disease. Arq Bras Oftalmol 2007;70:109–14. Woods AC, Jacobs L, Wood RM. A study of the role of toxoplasmosis in adult chorioretinitis.Am JOphthalmol 1954;37:163–77. McCannel CA, Holland GN, Helm CJ, et al. Causes of uveitis in the general practice of ophthalmology. UCLA Community-Based Uveitis Study Group. Am J Ophthalmol 1996;121:35–46. Arevalo JF, Belfort R, Muccioli C, et al. Ocular toxoplasmosis in the developing world. Int OphthalmolClin 2010;50(2):57–69. de-la-Torre A, López-Castillo CA, Rueda JC, et al. Clinical patterns of uveitis in two ophthalmology centres in Bogota, Colombia. ClinExpOphthalmol 2009 Jul;37(5):458–66. Acers TE. Toxoplasmicretinochoroiditis: a double-blind therapeutic study. Arch Ophthalmol 1964;71:58–62. Soheilian M, Sadoughi MM, Ghajarnia M, et al. Prospective randomized trial of trimethoprim/sulfamethoxazole versus pyrimethamine and sulfadiazine in the treatment of ocular toxoplasmosis. Ophthalmology 2005;11:1876–82. Holland G, Lewis K. An update on current practices in the management of ocular toxoplasmosis. Am JOphthalmol 2002;134(6):102–14. Stanford MR, Gilbert RE. Treating ocular toxoplasmosis: current evidence.MemInstOswaldo Cruz 2009;104(2):312–15. Smith JR, Franc DT, Carter NS, et al. Susceptibility of retinal vascular endothelium to infection with Toxoplasma gondiitachyzoites. Invest Ophthalmol Vis Sci 2004;45:1157–61. Emmanuelle Delair, Paul Latkany, A. Gwendolyn Noble, et al. Clinical Manifestations of ocular toxoplasmosis. Ocular Immunology & Inflammation 2011;19(2):91–102. Adan A, Giralt J, Alvarez G, et al. Pars planavitrectomy for vitreoretinal complications of ocular toxoplasmosis. Eur J Ophthalmol 2009;19:1039–43.

http://www.pacificejournals.com/aabs

A-11 21. Francis JM, Joynson DH. Duration of specific immunoglobulin A antibody following acute toxoplasmosis as determined by enzyme immunoassay and immunosorbent agglutination assay. Eur J ClinMicrobiol Infect Dis 1993;12:556–9. 22. Gaete MIL, Malaguenio E, Cordeiro F, et al. Relacao entre IgA excretoranalagrima X IgA sericanatoxoplasmose ocular com lesaoativa. Dissertacao de Mestradodefendidaemjunho de 2003;2007:03-08. 23. Holliman RE, Stevens PJ, Duffy KT, Johnson JD. Serological investigation of ocular toxoplasmosis. Br J Ophthalmol 1991;75:353–5. 24. Chapman DJ, Ashburn D, Ogston SA, et al. The relationship between ocular toxoplasmosis and levels of specific toxoplasma antibodies.Epidemiol Infect 1999;122:299–303 25. Bou G, Figueroa MS, Marti-Belda P, et al. Value of PCR for detection of Toxoplasma gondiiin aqueous humor and blood samples from immunocompetent

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 patients with ocular toxoplasmosis. J ClinMicrobiol 1999;37:3465–8. 26. Winterhalter S, Severing K, Stammen J, et al. Does atovaquone prolong the disease free interval of toxoplasmicretinochoroiditis? Graefes Arch ClinExpOphthalmol 2010;248:1187–92. 27. Soheilian M, Ramezani A, Azimzadeh A, et al. Randomized trial of intravitreal clindamycin and dexamethasone versus pyrimethamine, sulfadiazine, and prednisolone in treatment of ocular toxoplasmosis. Ophthalmology 2011;118(1):134-41. 28. Kishore K, Conway MD, Peyman GA. Intravitrealclindamycin and dexamethasone for toxoplasmicretinochoroiditis. Ophthalmic Surg Lasers 2001;32(3):183–92

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

Positive feedback between climate change, forest pests and the carbon cycle Anne l.M. Arnold1, Annett Reinhardt1*, Ignacy Korczynski2, Maren Grüning1, Carsten Thies3 1

Institute of Soil Science of Temperate and Boreal Ecosystems, Büsgen-Institute, University Göttingen, Germany Poznan University of Life Sciences, Department of Forest Entomology, Ul. Wojska Polskiego 71c, PL-60-637 Poznan, Poland 3 Natural Resources Research Laboratory, Bremer Str. 15, 29308 Winsen, Germany 2

Keywords: Forest, Carbon cycle, Tree

ABSTRACT

Forest trees under climate stress increasingly become more vulnerable to insect pests resulting in vastly defoliated swaths of forest land. Here, we simulated a forest pest mass outbreak using a microcosm incubation experiment, and show a positive feedback between climate change, forest pests and the carbon cycle. Treatments with insect faeces showed 16-fold higher fluxes of carbon dioxide (CO2) and 8-fold higher fluxes of dissolved organic carbon (DOC) compared to treatments without insect faeces (control) across a four weeks period, presumably due to the input of limited nitrogen (N) and fastly decomposable carbon (C) compounds that accelerate soil decomposition processes.

*Corresponding author: Annett Reinhardt, Institute of Soil Science of Temperate and Boreal Ecosystems, Büsgen-Institute, University Göttingen, Germany. email: areinha@gwdg.de

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-13

INTRODUCTION

Feedback effects between climate change and plant-insect interactions are rarely considered, but may linked to forest pest mass outbreak in the course of destruction of the forest vegetation across wide swaths of land. The chemical composition of matter inputs and the climate are major determinants of decomposition rates and nutrients mineralisation [1]. Moreover, herbivore-by-products such as frass pellets and the subsequent occurrence of altered throughfall chemistry (leachates) can have distinct chemical properties that change rates of organic matter decomposition and nutrient release within ecosystems [2,3,4], with changing quantity and temporal distribution of organic matter inputs from the canopy to the soil [5,6]. In contrast, the effects of insect derived organic matter inputs on soil decomposition processes are largely unknown, such as priming effects, the function as co-substrate, or filling budgeting gaps as reported for nitrogen [7]. Insect derived input are expected to lead to accelerate or decelerate carbon and nitrogen cycles, and studies assessing soil microbial feedbacks to insect residues report ambiguous results (6,7, 8,9, 10,11,12,13,14]. In this study, we analysed C fluxes in soils under controlled conditions in a microcosm experiment by adding amounts of insect faeces that have been recorded under naturally occurring heavy pest outbreak conditions. We expected that (1) soil organic matter decomposition would be accelerated due to the input of nitrogen and, thereby (2) carbon fluxes would be increased in treatments with insect faeces.

MATERIALS AND METHODS

In our experiment, we simulated an insect infestation with vastly defoliated 120-years old oak stand (Quercus petrea L.) of the Bramwald-Brackenberg forest (301-350m a.s.l) in southern Lower Saxony, Germany. We used forest floors with an Ol-, Of-, and Oh- horizon of a soil developed from a loess layer from solifluction over Triassic lime stone, classified as Luvisol according to the FAO. The forest floor is 5-6 cm thick and classified as raw humus with a content of 39.0% C and 1.3% N, resulting in a C/N ratio of 30. For the experiment, forest floors were cutted out and transferred in soil core boxes to the laboratory. Subsequently, the soil was dried at 25°C for one week, sieved and homogenised. We used soil columns with a diameter of 15,5cm and a height of 7cm. Each column was filled with 200g dried soil to simulate the forest floor. Microcosm treatments with and without insect faeces supply were replicated 5-fold. Within a pre-incubation phase (two weeks) all columns were irrigated synchronously with 60ml at the beginning of the experiment and irrigated weekly with volumes that were calculated based on the water-holding-capacity-test of Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 soil according of [15] and adjusted to the weight loss of the sample due to evaporation. Incubation was realised in an air-conditioned chamber with 21°C for four weeks. Microcosm treatments were supplemented with 103g dry mass of insect faeces derived from larvae of the gypsy moth (Lymantria dispar L.) feeding on sessile Oak leaves. This amount of insect faeces corresponded to amounts that been measured under field conditions during a mass outbreak in a vastly defoliated 120-years old oak forest, with inputs of 500-800 kg/ha faeces in a period of five month (le Mellec et al. 2011). The faeces in our experiment contained 49.5% C and 3,0% N, resulting in a C/N ratio of 17. Soil gas samples (CO2) were automatically taken by the continuous flow system. CO2 analyses were obtained with a gas-phase chromatograph (GC Shimadzu 14 B). Concentrations of dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC) in soil percolates (0.45-µm membrane-filtered with Cellulose-acetate filters, Sartorius) were determinated by thermal oxidation (Dimatoc 100, Dimatec, Essen, Germany). Element fluxes were calculated by multiplying volumes with concentrations across a four weeks study [16]. Statistical analyses were performed using chi-square tests. In the text, arithmetic mean and standard deviations are given.

RESULT

The accumulated fluxes of carbon compounds are summarized in figure 1. In treatments without insect faeces (control) the output was 213.7±19.5 mg CO2-C, 152.2±36.8 mg DOC, and 96.8±17.1mg DIC across the four weeks study period. In contrast, in treatments with insect faeces the output of was considerably higher, with 3462.2±320.9 mg CO2-C, 1254.7±324.8 mg DOC, and 110.6±33.8 mg DIC. Transferred to the spatial scale of one hectare, in treatments without insect faeces (control) the output was 113,0±10,3 kg CO2-C; 80,5±19,5 kg DOC and 51,2±9,4 kg DIC across the four weeks study period, and 1831,5±169,8 kg CO2-C, 663,7±171,8 kg DOC, and 58,5 ±17,9 kg DIC in treatments with insect faeces. Budgeting these inputs and outputs, 500 mg C and 30 mg N from insect faeces accelerated a 7-fold output of CO2-C and a 2.5-fold output of DOC.

DISCUSSION

The experimental analysis of organic matter input via insect faeces shows significantly altered gaseous (CO2) and dissolved (DOC) carbon fluxes compared to the control. The increased C outputs clearly suggest an accelerated decomposition process in treatments with insect faeces, probably due to the input of nitrogen (N) that is a limiting factor for decomposition as well as due to the large amounts dissolved organic carbon (DOC) that is fastly decomposable. Although our experiments do not simulate the complexity of carbon processes in real forest floors, such as seasonal fluctuations of soil temperature and humidity, interactions of decomposers e-ISSN: 2349-6991; p-ISSN: 2455-0396

A-14

Original Article

Fig. 1: Cumulative fluxes of C of infested and uninfested microcosm. DOC = dissolved organic matter, DIC, dissolved inorganic matter, CO2-C= carbon dioxide carbon. Level of significance (* = p < 0.05 / ** = p < 0.01 / *** = p < 0.001), indicating differences between infested vs. uninfested fluxes.

with root and root microflora, and tree growth and respiration in the course of defoliations, our results in microcosms without insect faeces (control) correspond with C effluxes in temperate forest under field conditions [5,17]. Therefore, we suggest that insect mass outbreak may also play an important role for translocation of DOC by soil vertical fluxes into deeper and microbiologic less active soil layers, possibly contributing to long-term C sequestration and/or DOC leaching into the ground water [18].

The earthâ&#x20AC;&#x2122;s climate is in a period of rapid change. These changes in temperature and precipitation not only could result in enhanced forest productivity, but also may increase the frequency and intensity of forest disturbances such as mass outbreaks of forest pests (Kirilenko & Sejo 2007) that vastly defoliate swaths of forest land, and thereby apparently trigger large-scale decomposition processes and CO2 effluxes into the atmosphere. A basic understanding of disturbance-induced forest changes may contribute to a forest management that reduces pest outbreaks and improves carbon sequestration.

In summary, forest pest epidemics appear to significantly change nutrient cycling in an accelerating way resulting in reduced carbon storage in the organic horizon and significant release of carbon dioxide (CO2), representing a feedback loop to climate change. This is a new experimental finding and in contrast to the general expectation that forests of the northern hemisphere function as large C sinks [19].

None.

ACKNOWLEDGEMENTS FUNDING None.

COMPETING INTERESTS None declared.

http://www.pacificejournals.com/aabs

A-15

REFERENCES 1.

Meentmeyer V. Macroclimate and lignin control of litter decomposition rates. Ecology 1978 59, 465-472. 2. Hollinger, D.Y. Herbivory and the cycling of nitrogen and phosphorus in isolated California oak trees (1986). Oecologia 1986 :70, 291-297. 3. Lovett, G.M. & A.E. Ruesink. Carbon and nitrogen mineralization from decomposing Gypsy Moth frass . Oecologica 1995 ;104, 133-138. 4. Stadler, B., Solinger, S. & B. Michalzik. Insect herbivores and the nutrient flow from the canopy to the soil in coniferous and deciduous forests. Oecologia 2001;126, 104-113. 5. Michalzik, M., Kalbitz, K., Park, J.H., Solinger, S. & E. Matzner . Fluxes and concentrations of dissolved organic carbon and nitrogen - a synthesis for temperate forests . Biogeochemistry 2001;52, 173-205. 6. le Mellec A., Gerold G. & Michalzik M. Insect herbivory, organic matter deposition and effects on belowground organic matter fluxes in a central European oak forest. Plant and Soil 2011;342, 393-403. 7. Chapman, S.K., Hart, S.C., Cobb, N.S., Whitham, T.G. & G.W. Koch. Insect herbivory increases litter quality and decomposition: an extension of the acceleration hypothesis. Ecology 2003;84, 2867-76. 8. Guggenberger, G. & W. Zech. Composition and dynamics of dissolved organic carbohydrates and lignin-degradation products in two coniferous forests, N.E. Bavaria, Germany. Soil Biol. Biochem 1994;26, 19-27. 9. Christenson L.M., Lovett G.M., Mitchell M.J. & Groffmann P.M. 2002 The fate of nitrogen of gypsy moth frass deposited to an oak forest floor. Oecologia 131, 444-454. 10. Russel C.A., Kosola K.R., Paul E.A. & Robertson G.P. 2004 Nitrogen cycling in poplar stands defoliated by insects. Biogeochemistry 68, 365-81.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 11. Hunter, M.D. 2001 Insect population dynamics meets ecosystem ecology: effects of Herbivory on soil nutrient dynamics . Agricultural and Forest Entomology 3, 77-84. 12. Morehouse K., Johns T., Kaye J. & Kaye M. 2008 Carbon and nitrogen cycling immediately following bark beetle outbreaks in southwestern ponderosa pine forests. Forest Ecology and Management 255, 2698-2708. 13. Cobb R.C. 2010 Species shift drives decomposition rates following invasion by hemlock woolly adelgid. Oikos 119, 1291–1298. 14. Schowalter, T.D, Sabin, T.E., Stafford, S.G. & J.M. Sexton 1991: Phytophage effects on primary production, nutrient turnover, and litter decomposition of young Douglas fir in western Oregon. Forest Ecology and Management 42, 229-243. 15. Carter, M.R. & Gregorich, E.G.(eds.) 2006 Soil Sampling and Methods of Analysis. Canadian Society of Soil Science. CRC Press. Boca Raton. 16. le Mellec, A., Habermann, M. & Michalzik, B. 2009 Canopy herbivory altering C to N ratios and soil input patterns of different organic matter fractions in a Scots pine forest. Plant and Soil 325, 255–262. 17. Mahli Y., Baldocchi D. & Jarvis P.G. 1999 The carbon balance of tropical, temperate and boreal forests. Plant, Cell and Environment 22, 715-740. 18. Qualls RG & Haines BL 1992 Biodegradability of dissolved organic matter in forest throughfall, soil solution, and stream water. Soil Sci Soc Am J 56,578–586. 19. Luyssaert S, Schulze E-D, Bőrner A, Knohl A, Hessenmőller D, Law BE, Ciais P & Grace J. 2008 Old-growth forests as global carbon sinks. Nature 455, 213-5.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Assessment of the health related quality of life in patients suffering from hypertension and diabetes mellitus: A cross sectional study. Ishtpreet Mann1, Navtej Singh2, Manisha1, Ashwani K Gupta3, Prithpal S Matreja3* 1

Under-graduate, Gian Sagar Medical College and Hospital, Village Ram Nagar, District Patiala, Punjab, India Department of Internal Medicine, BPS Government Medical College and Hospital, Khanpur Kalan, District Sonepat, India 3 Department of Pharmacology, Gian Sagar Medical College and Hospital, Village Ram Nagar, District Patiala, Punjab, India 2

Keywords: Hypertension, Diabetes Mellitus, Quality Of Life, Emotional

ABSTRACT Background: Diabetes mellitus and hypertension are chronic disorders, inadequate management of these two disorders leads to several complications and end organ damage that can impair health related quality of life (HRQoL) in these individuals. Several studies in hypertensive patients concluded that hypertension reduced HRQoL and participants with diabetes also reported comparably decreased HRQoL. The data on HRQoL in patients suffering from both hypertension and diabetes is limited hence we designed this study to assess health related quality of life in patients suffering from hypertension and diabetes mellitus. Methodology: This single centre, cross-sectional study was conducted for 2 months between April and August 2013 in patients with hypertension and diabetes mellitus. Patients suffering from hypertension were recruited in study and were divided into two groups, Group 1 consisted of patient suffering from hypertension and diabetes mellitus whereas Group 2 consisted of patients suffering from hypertension. Patients were assessed on Short form health Survey (SF-36) and the WHOQOL â&#x20AC;&#x201C; Bref scores. Results: A total of 85 patients were screened out of which 41 patients were enrolled in the study, 21 patients in Group 1 and 20 patients in group 2. The SF-36 Scores showed significantly (p<0.05) worse pain scores in patients in Group 2. Patients in Group 1 had a better quality of life as compared to other group as evident by higher scores in most of the parameters of SF-36 and WHO-QOL Bref Score, though it was not statistically significant. Conclusion: Both groups had compromised quality of life; patients with hypertension and diabetes had a better quality of life.

*Corresponding author: Dr Prithpal S Matreja, Professor, Department of Pharmacology, Gian Sagar Medical College and Hospital, Village Ram Nagar, Tehsil Rajpura, District Patiala, Punjab 140601 India Phone: +91-9855001847, Fax No.: +91-1762-520024 E-mail: drpsmatreja@yahoo.co.in

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-17

Introduction Hypertension is a major public health problem in India and its prevalence is rapidly increasing among both urban and rural populations (1, 2). The prevalence of hypertension ranges from 20-40% in urban adults and 12-17% among rural adults. The number of people with hypertension is projected to increase from 118 million in 2000 to 214 million in 2025, with nearly equal numbers of men and women (3, 4). According to WHO Health statistics 2012, the prevalence of hypertension in India was 23.1% in men and 22.6% in women in population in the age group of 25 or more than 25 years of age. It caused approximately 51% of death from strokes and 45% from coronary artery disease in 2004; and was considered responsible for about 12.8 percent of the total of all global deaths (5). Recent studies show that for every known person with hypertension there are two persons with either undiagnosed hypertension or pre hypertension (6). A reduction in blood pressure can decrease cardiovascular risk and this can be achieved by lifestyle measures in mild cases and this should be the initial approach to hypertension management in all cases. This includes dietary interventions weight reduction, tobacco cessation, and physical activity (1). But unlike in Western countries, stress management is often not given greater emphasis in India (4). India leads the world with largest number of diabetic subjects earning the dubious distinction of being termed the â&#x20AC;&#x153;diabetes capital of the worldâ&#x20AC;?. According to the Diabetes Atlas 2006 published by the International Diabetes Federation, the number of people with diabetes in India currently around 40.9 million is expected to rise to 69.9 million by 2025 unless urgent preventive steps are taken, World Health Organization (WHO) reports show that 32 million people had diabetes in the year 2000 (7). In chronic diseases such as hypertension and diabetes, healthrelated quality of life (HRQoL) is an especially important outcome, given their lifelong nature and the need for daily self-management (8). Inadequate management of these two disorders leads to several complications and end organ damage that can impair the HRQoL in the individuals (9). A systematic review and meta-analysis of observational studies of HRQoL in hypertensive patients concluded that hypertension reduced HRQoL; this was secondary to the awareness of hypertension, adverse drug effects, newly diagnosed type 2 diabetes mellitus or obesity (10). Participants with diabetes and those with hypertension reported comparably limited HRQoL (8) similarly; another study done to assess the quality of life in American Indians showed that respondents with both diabetes mellitus and

hypertension have lowest HRQoL (11). Hypertensives exhibit higher depression scores, more semantic memory problems and less satisfactory sex lives; they feel less fit physically, less in control of their lives, more tense and score lower on a hardiness scale in comparison with their normotensive counterparts (12). Diabetes, also seriously affect the HRQoL of the patient and it is seen self confidence is most commonly affected by diabetes and all aspects of family life was more negatively impacted (13). The data on Health related quality of life in patients suffering from both hypertension and diabetes is limited in this region and most of the studies have been done on western population hence we designed this study to assess the health related quality of life in patients suffering from hypertension and diabetes mellitus.

Material and Methods This cross-sectional study was conducted in the Department of Internal Medicine, Gian Sagar Medical College and Hospital, Patiala for 2 months between April and August 2013. Patients with hypertension and diabetes mellitus were recruited in the study. The study was approved by the Institutional Ethics Committee and patients were recruited after they gave written informed consent. Patients between the ages of 18 to 60 years, with a known history of hypertension (Blood Pressure > 140/100 mmHg), and registered with diabetes mellitus at any particular centre for 12 months were included in the study. Patients with chronic renal disease or end stage renal disease, history of heart or respiratory failure, recent myocardial infarction (MI), shock, liver disease, chronic alcohol use, pregnant or lactating females were excluded from study. Procedure: The participants were divided into two groups, Group 1 consisted of patient suffering from hypertension and diabetes mellitus whereas Group 2 consisted of patients suffering from hypertension. A detailed history was also taken and the participants underwent a thorough medical examination, they were also given counselling for life style modifications. The patients were given questionnaire of SF-36 and WHO-QOL Bref; they were given time to fill up the questionnaire in a separate room without any interference from the treating physician Parameters Short form health Survey (SF-36): This questionnaire contains 36 items integrated in multi-item scales measuring eight generic health concepts: physical functioning (PF), social functioning (SF), role physical (RP), bodily pain (BP), mental health (MH), role emotional (RE), vitality (VT), and general health (GH).

http://www.pacificejournals.com/aabs

A-18 Scoring included transformation of raw scores for each subscale to a 0-100 scale and a higher scores representing better quality of life (14). The WHOQOL – Bref: was monitored at visit. This is a 26-item self-administered generic questionnaire, a short version of WHOQOL -100 scale. It can be analyzed from perspective of either six domains (physical health, psychological health, level of independence, social relationships, environment, & spiritual) or four domains (physical health, psychological health, social relations, and environment) (15). The QOL index of each domain and their associations with demographic factors were assessed, a higher score indicated a better quality of life (16, 17, 18). Statistical Analysis: The data was tabulated as mean ± standard deviation (SD). Results were analyzed using non parametric tests (Chi-Square Test), parametric tests (two tailed student t-test) and correlation (Pearson correlation coefficients) analysis. A p<0.05 was considered statistically significant.

Results A total of 85 patients suffering from hypertension visiting the OPD in a period of 2 months were screened for enrollment in the study, 32 patients were not enrolled in the study as they did not fulfill the eligibility criteria for enrollment in the study. Around 12 patients were not included because they did not give the informed consent. A total of 41 patients participated in the study, 21 patients suffering from hypertension and diabetes were included in Group 1 and 20 patients suffering for hypertension only were included in Group 2. All the patients gave informed consent and were included in the analysis of result. The baseline characteristics of the patients are shown in Table 1. Both the groups were comparable at baseline except for random blood sugar which was significantly higher (p<0.05)

AABS; 3(1): 2016 in patients in Group1 (180.35±65.64 vs. 121.25±13.96) as compared to group 2. The patients in Group 1 were of lower age group (55.65±9.79 vs. 58.3±12.82) and had slightly higher systolic and diastolic blood pressure though it was not statistically significant. SF-36 Scores: The SF-36 Scores in both groups are shown in Figure 1. There were significantly (p<0.05) worse pain scores in patients in Group 1 (52.03±33.6 vs. 75±22.24) as compared to Group 2. The Group1 had better aspect of physical functioning (50±20.46 vs. 47.5±29.0), role limitation due to physical health (31.25±40.54 vs. 20±35.91), role limitation due to emotional problem (46.67±48.85 vs. 35±46.49), energy/fatigue (37.5±22.09 vs. 34±19.97) as compared to Group2. Whereas, Group2 had better aspect of social functioning (73.75±25.62 vs. 60±28.56) and general health (46.75±21.54 vs. 43.25±15.58) as compared to Group 1. The emotional well being score was comparable in both groups (51.2±22.72 and 51.4±15.86). WHO-QOL Bref S cores: WHO-QOL bref scores are shown in Figure 2. Group 1 had higher scores in all the 4 domains that is, physical health (49.85±14.47 vs. 48.95±18.81), psychological (51.65±16.26 vs. 47.85±14.28), social relationship (69.6±13.2 vs. 64.7±16.62) and environment (68.2±14.03 vs. 62.95±16.39) but it was not statistically significant. As the questionnaires were to be filled up by patients only, hence there was a possibility of interpretation bias based on understanding of the patients. Correlation: Estimates of correlation for SF-36 Scores with WHO-QOL Bref Scores along with their significant levels among patients in Group 1 and 2 are presented in Table 2. It has been observed that SF-36 Score has significant (p<0.05) correlation with physical health, psychological and social relationship in both groups; with environment in Group 1.

Table1. Baseline characteristic of both groups Characteristic

Group 1 (n=21)

Group 2 (n=20)

p value

Age (years) (Mean±SD)

55.65±9.79

58.30±12.82

0.46#

Sex(M:F)

11:9

12:8

1.00ɵ

Random Blood Sugar (mg/dl) (Mean±SD)

180.35±65.64

121.25±13.96

<0.05*#

Systolic Blood Pressure (mmHg) (Mean±SD)

149.3± 13.48

148.3± 18.19

0.84#

Diastolic Blood Pressure (mmHg) (Mean±SD)

96.9±10.31

96.1±12.72

0.82#

*p<0.05 and statistically significant; #using student ‘t’ test; ɵusing Chi Square Test

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-19

Table 2. Correlation coefficients for SF-36 scores with WHO-QOL Bref Scores among patients in both groups. SF-36 Scores Variables

Group 1 (n=21) r

Group 2 (n=20) r

Domain I/ Physical Health

0.41

<0.05*

0.42

<0.05*

Domain II/ Psychological

0.42

<0.05*

0.29

<0.05*

Domain III/ Social Relationship

0.35

<0.05*

0.26

<0.05*

Domain IV/ Envoirment

0.36

<0.05*

0.12

0.13

*p<0.05 and statistically significant

Fig. 1: SF-36 scores in both groups

Fig. 2: WHO-QOL Bref scores in both groups

Discussion

Bref Scores. The patients suffering from hypertension alone had more compromised HRQoL.

Diabetes mellitus and hypertension are chronic disorders which are emerging as major health problems with increasing morbidity and mortality. The prevalence of hypertension is double among diabetics as compared to non-diabetics in the western world (9). Recent guidelines agree on the need for early, aggressive reduction of blood pressure and fasting blood sugar in patients with diabetes (18). In chronic diseases such as hypertension and diabetes, health-related quality of life (HRQoL) is an especially important outcome, given their lifelong nature and the need for daily self-management (10). Inappropriate management of these two disorders leads to several complications and end organ damage that can impairs the health related quality of life (HRQoL) in the individuals (9). The present study was undertaken to assess the HRQoL in patients suffering from hypertension and diabetes mellitus. The results showed that patients with hypertension and diabetes were of lower age group and had higher systolic and diastolic blood pressure and had significantly higher random blood sugar. The QOL was impaired in both groups as evident by low scores in both SF-36 and WHO-QOL

A cross-sectional population-based study demonstrated that patients who were aware of their hypertension had lower scores in physical functioning and general health than patients without hypertension then patients who were unaware of hypertension. The results of our study are in similarity with this study as our results showed that patients had a compromised QOL, the only difference being that in our study patients with both diabetes and hypertension had slightly more compromised QOL (10). A study done to assess health-related quality of life (HRQoL) among people with diabetes or hypertension in the Croatian Adult Health Survey demonstrated that participants with diabetes and those with hypertension reported comparably limited HRQoL in all dimensions of SF-36, compared with healthy individuals. The results of our study agrees with this study as our results showed that patients with both diabetes and hypertension had

http://www.pacificejournals.com/aabs

A-20

AABS; 3(1): 2016

comparable compromised QOL with most significant effect on pain component in SF-36 scores in hypertensive patients (8).

4. Hypertension in India. (Accessed on 10th September, 2013). Available on url. http://www.cadiresearch.org/ topic/hypertension/hypertension-india

Another article focusing on the literature published since 2000, on HRQoL in elderly hypertensive individuals as well as hypertensive’s with co-existent diseases, including chronic kidney disease (CKD), cardiovascular disease and diabetes mellitus showed that most studies found that hypertensive individuals with co-existent co-morbidities tend to have lower HRQOL than those with hypertension alone with the most pronounced effect was noted in the physical function domains of HRQOL. The results of our study are in contrary with this study as our results showed that patients with both diabetes and hypertension had slightly less compromised QOL with most significant effect on pain component in SF-36 scores in hypertensive patients (19).

5. Kanwar S. Prevalence of hypertension high among lower, middle class population in India. Apr 5, 2013 (Accessed on 10th September, 2013). Available on url. http://articles.timesofindia.indiatimes.com/201304-05/chandigarh/38305800_1_anti-hypertensivemedications-uncontrolled-hypertension-high-bp

There are certain limitations in our study, firstly the sample size could have been larger but, the duration of study was only two months hence we tried to include patients who fulfilled the eligibility criteria. Secondly, a comparison with the intervention arm could be done, but any intervention could have prolonged the duration of study and we would not have been able to complete the study in the allotted 2 months. To conclude it was observed in our study that both groups had compromised quality of life, patients suffering from hypertension alone had a worse quality of life as pain was significantly more in patients with hypertension only as per SF-36 Score, physical functioning, role limitation, fatigue was more compromised in patients with hypertension only and social functioning, general health less compromised in patients with hypertension only. Patients with both hypertension and diabetes had less compromised QOL as per WHO-QOL Bref scores. Source of Funding: This projects is a part of ICMRSTS (Indian Council of Medical Research – Short term Studentship Program) 2013. The project has been supported by ICMR-STS 2013 program.

Reference

6. Joshi SR, Saboo B, Vadivale M, et al. Prevalence of Diagnosed and Undiagnosed Diabetes and Hypertension in India-Results from the Screening India’s Twin Epidemic (SITE) Study. Diabetes Technol Ther 2012; 14: 8-15. 7. Mohan V, Sandeep S, Deepa R, Shah B, Varghese C. Epidemiology of type 2 diabetes: Indian scenario. Indian J Med Res 2007; 125: 217-30. url. http://icmr. nic.in/ijmr/2012/october/Most_cited2.pdf 8. Poljičanin T, Ajduković D, Šekerija M, PibernikOkanović M, Metelko Z, Mavrinac GV. Diabetes mellitus and hypertension have comparable adverse effects on health-related quality of life. BMC Public Health 2010; 10: 12. http://www.biomedcentral. com/1471-2458/10/12 9. Adepu R, Madhu S. Influence of post discharge counseling on health outcomes in diabetic and hypertensive patients. Asian J Pharm Clin Res 2011; 4 (3): 28-33. 10. Korhonen PE, Kivelä SL, Kautiainen H, Järvenpää S, Kantola I. Health-related quality of life and awareness of hypertension. J Hypertens 2011; 29: 2070–4. 11. Jiang L, Beals J, Whitesell NR, Roubideaux Y, Manson SM, AI-SUPERP Team. Health-related quality of life and help seeking among American Indians with diabetes and hypertension. Qual Life Res 2009; 18: 709–18. 12. Amir M, Bar-on D. Hypertension and quality of life: The disease, the treatment or a combination of both. Psychol Health 1996; 11: 685-95.

1. Gupta R, Guptha S. Strategies for initial management of hypertension. Indian J Med Res 2010; 132: 531-42. 2. Gupta R, al-Odat NA, Gupta VP. Hypertension epidemiology in India: meta-analysis of 50 year prevalence rates and blood pressure trends. J Hum Hypertens 1996; 10: 465-72. 3. Reddy KS. Regional case studies–India. Nestle Nutr Workshop Ser Pediatr Program. 2009; 63:15-24; discussion 41-16, 259-268.

13. Vijayakumar K, Varghese RT. Quality of Life Among Diabetic Subjects: Indian Perspectives. In Handbook of Disease Burden and Quality of Life Measures. Ed Preedy VR, Watson RR. Springer, New York 2010; p 2071-93.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

14. 36-Item Short Form Survey from the RAND Medical Outcomes Study. Accessed on 10th April, 2013. url. http://www.rand.org/health/surveys_tools/mos/mos_ core_36item.html

Original Article

A-21

15. Skevington SM, Lotfy M, O’ Connell KA, the WHOQOL Group. The World Health Organization’s WHOQOLBref quality of life assessment: psychometric properties and results of the international field trial. A report from the WHOQOL group. Qual Life Res 2004; 13: 299-310. 16. Sainfort F, Becker M, Diamond R. Judgments of quality of life of individuals with severe mental disorders: patient self-report versus provider perspectives. Am J Psychiatry 1996; 153: 497-502. 17. Alshubaili AF, Ohaeri JU, Awadalla AW, Mabrouk AA. Family caregiver quality of life in multiple sclerosis

among Kuwaitis: a controlled study. BMC Health Services Research 2008, 8: 206 doi:10.1186/14726963-8-206. This article is available from: http:// www.biomedcentral.com/ 1472-6963/8/206. 18. Anees M, Hameed F, Mumtaz A, Ibrahim M, Khan MNS. Dialysis-related factors affecting quality of life in patients on haemodialysis. Iranian J Kidney Dis 2011; 5: 9-14. 19. Soni RK, Porter AC, Lash JP, Unruh ML. Healthrelated quality of life in hypertension, chronic kidney disease and coexistent chronic health conditions. Adv Chronic Kidney Dis 2010; 17 (4): e17–26. doi:10.1053/j.ackd.2010.04.002.

http://www.pacificejournals.com/aabs

Original Article Radiological and Biochemical aspect of Alcoholic Liver Disease: A Prospective study Siddharth Bhargava1, Navtej Singh2, Prithpal S Matreja3*, Ashwani K Gupta3, Arshdeep Singh4 1

Keywords: Fatty Liver, Cirrhosis, Alcohol, Ascites, Alcoholic Liver Disease

ABSTRACT Background: Alcoholic cirrhosis is second leading indication for liver transplantation in United States and Europe. The spectrum of alcohol related liver injury varies from simple steatosis to cirrhosis; patients with alcoholic cirrhosis have high prevalence of complications at the time of cirrhosis diagnosis. So there is a need to study the radiology and underlying biochemical changes for early diagnosis to reduce morbidity and mortality in case of alcoholic liver disease. Methodology: the patients visiting the OPD of Medicine and suffering from ALD underwent through medical examination and then the severity of ALD was determined by radiological and biochemical findings. Patients who fulfilled the inclusion and exclusion criteria were enrolled in the study if they are willing to give written informed consent. Results: A total number of 30 male patients were studied, 17 patients had cirrhosis and 9 had fatty liver. Ascites was most common manifestation in both cirrhosis and fatty liver; followed by splenomegaly and portal hypertension in cirrhosis, whereas common bile duct dilatation was seen more in fatty liver. There were derangements in liver function tests associated with different stages of alcoholic liver disease but this was not statistically significant. There seem to be no statistically significant (p>0.05) correlation with the various biochemical parameters. Conclusions: Though a large number of radiological and biochemical changes are seen in cirrhosis and fatty liver, there seems to be no relation between these two. However the most common manifestation in both these conditions is ascites.

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-23

Introduction

ALD (alcoholic liver disease) is damage to liver due to years of heavy drinking. Alcohol causes inflammation in liver, over time, scarring and cirrhosis being the final stage of ALD [1]. Currently, alcoholic cirrhosis is second leading indication for liver transplantation in United States and Europe [2]. Recent epidemiological data suggest consumption of 40g of ethanol per day in men and 10-20 g per day in women for between 10 and 12 years being associated with an increased relative risk of developing liver disease [3]. As per the surveillance report published by National Institute on Alcohol Abuse and Alcoholism showed that liver cirrhosis was 12th leading cause of deaths in United States, with total of 29,925 deaths in 2007, 48% of which were alcohol related with drinking outside mealtimes reported to increase the risk of ALD by 2.7 fold [4]. The spectrum of alcohol related liver injury varies from simple steatosis to cirrhosis, they are not necessarily distinct stages of evolution of the disease, but rather, multiple stages that may be present simultaneously in a given individual [5]. An aspartate transaminase/ alanine transaminase (AST/ ALT) ratio>3 is highly suggestive of ALD [6], imaging studies have been used to diagnose presence of liver disease but do not have role in establishing alcohol as specific etiology of liver disease. However, diagnosis of fatty acid change, established cirrhosis, and hepatocellular carcinoma may be suggested by ultrasound, computerized tomography (CT) scan, or Magnetic Resonance Imaging (MRI) and confirmed by other laboratory investigations [7]. Patients with alcoholic cirrhosis have high prevalence of complications at the time of cirrhosis diagnosis. The presence and type of complications at diagnosis are predictors of mortality, but not of the risk of subsequent complications [8]. These predictors of mortality are often unnoticed and the patient usually presents in hospital with end stage disease. So there is a need to study the radiology and underlying biochemical changes for early diagnosis to reduce morbidity and mortality in case of alcoholic liver disease. There exist geographical variations regarding intake of alcohol. The burden of alcohol-related disease is the highest in the developed world, where it may account for as much as 9.2 % of all disability-adjusted life years However, even in the developing regions of the world, alcohol accounts for a major portion of the global disease burden, and is projected to take on increasing importance in those regions over time. Punjab is among the top three regions of alcohol consumption in India. [9, 10]. Abstinence is the most important therapeutic intervention for patients with ALD [11] and has shown to improve the outcome and histological features of hepatic injury, reduce

portal pressure and decrease progression to cirrhosis, and improve survival at all stages in patients with ALD [11– 14]. The studies done in India are limited, hence this study was designed, to study the radiological and biochemical aspects of ALD.

Methodology This prospective, cross sectional study was conducted in patients suffering from Alcoholic Liver Disease from the Out Patient Department of our hospital. An assessment was done on the basis of AST/ALT from biochemical aspect and features of liver from radiological aspect. Patients of both sexes, more than 18years with history of alcoholism, diagnosed with alcoholic liver disease and willing to give written informed consent were included in the study. All patients with Non Alcoholic Fatty Liver, chronic medical, surgical conditions, organic brain syndrome, and chronic mental illness were excluded from the study. Prior to the enrolment of the patients in the study, approval was obtained from the Institutional Ethics Committee, the patients visiting the OPD of Medicine and suffering from ALD underwent through medical examination and then the severity of ALD was determined by radiological and biochemical findings. Patients who fulfilled the inclusion and exclusion criteria were enrolled in the study if they are willing to give written informed consent. Statistics: The data was presented as mean ± standard deviation (mean ± SD). The results obtained from the scales were compared using appropriate parametric (Student ‘t’ test, ANOVA) and non parametric tests (Chi-Square, Mann Whitney U, Wilcoxon Sign Rank test) wherever applicable. A p <0.05 was considered statistically significant.

Results On the basis of procedure described in this study, radiological aspects, biochemical aspects were studied as well as an effort to establish a relation in between them was also made. A total number of 30 patients were studied and the following observations are made. Out of 30 patients, • • •

No. of patients of cirrhosis seen = 17 No. of patients of fatty liver seen = 9 No. of patients with deranged LFTs but normal radiographic picture = 4 So, incidence of cirrhosis in Alcoholic liver disease =56.66% Incidence of fatty liver disease in Alcoholic liver disease =30%

http://www.pacificejournals.com/aabs

A-24

AABS; 3(1): 2016

Fatty liver, alcohol hepatitis, and cirrhosis are the three stages of alcoholic liver disease. An effort to establish the percentages of underlying manifestations with these stages was made and the following results are noted. Table 1 show the manifestation of patients with cirrhosis of liver. Most of patients with cirrhosis had ascites followed by portal hypertension and splenomegaly. 1. Ascites is seen in 94.10% of cirrhotic patients. 2. Ascites is followed by portal hypertension and splenomegaly.(seen in 70.5% patients) 3. However pleural effusion is seen in very less percentage of patients( 5.80%) Table 2 show the manifestation of patients with fatty liver. Most of patients with fatty liver had ascites followed by common bile duct dilatation. 1. Ascites is seen in 88.88% of fatty liver patients 2. It is followed by manifestations of common bile duct dilation (seen in 44.44% patients) Table 1: Manifestations of patients with cirrhosis of liver Manifestation of patient with Cirrhosis of Liver

N=17 (%)

Ascites

16(94.1)

Portal hypertension

12(70.5)

Splenomegaly

12(70.5)

Hepatoencephalothy Varices Pleural effusion

3. Manifestations of portal hypertension and cholelithiasis were found to be equal(seen in 11.10% patients) From the data collected and observed, it is seen that Ascites is most common manifestation in both cirrhosis and fatty liver, however percentages are different in both of them. Biochemical findings: Table 3 shows comparison of the biochemical parameters and demographic parameters in patients with cirrhosis and fatty liver. All the patients enrolled in the study were males. There were seen derangements in liver function tests associated with different stages of alcoholic liver disease. The patients with cirrhosis were of lower age group had higher serum bilurubin, direct bilurubin, higher total proteins, higher aspartate transaminase and alkaline phosphatase levels but this was not statistically significant (p>0.05). Correlation Estimates of correlation for age with various biochemical parameters was calculated and it was seen that age had no statistically significant (p>0.05) correlation with the various biochemical parameters (Table 4). Table 2: Manifestations of patients with fatty liver Manifestation of patient with Fatty Liver

N=9 (%)

Ascites

8(88.9)

Portal hypertension

1(11.1)

Common bile duct dilatation

4(44.4)

3(17.64)

Cholelithiasis

1(11.1)

1(5.8)

Splenomegaly

3(33.3)

2(11.7)

Table 3: Demographic and biochemical parameters of patients with cirrhosis and fatty liver Cirrhosis (n=17)

Fatty Liver (n=9)

p value

48.29±11.64

53.78±12.31

>0.05*

Serum Bilurubin (Mean±SD)

7.42±9.61

7.39±10.4

>0.05*

Serum Bilurubin Direct (Mean±SD)

4.17±5.07

3.31±5.11

>0.05*

Total protein (Mean±SD)

9.89±16.18

6.68±0.90

>0.05*

Serum Albumin (Mean±SD)

3.21±1.07

3.43±0.85

>0.05*

Serum Globulin (Mean±SD)

3.74±0.57

3.41±0.44

>0.05*

Albumin:Globulin Ratio (Mean±SD)

0.91±0.32

0.96±0.35

>0.05*

Aspartate transaminase (Mean±SD)

133.94±101.63

117±88.14

>0.05*

52.11±22.85

58.44±24.74

>0.05*

184.71±123.11

156.89±81.94

>0.05*

Characteristics Age (Years) (Mean±SD)

Alanins transaminase (Mean±SD) Alkaline phosphatase (ALP) (Mean±SD)

*no statistically significant difference between groups using student ‘t’ test.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-25

Table 4: Correlation coefficients for age with biochemical parameters among patients in both groups Age

Variables Cirrhosis (n=17)

Fatty Liver (n=9)

Serum Bilurubin

0.07

0.87

0.02

0.94

Serum Bilurubin Direct

0.08

0.83

0.11

0.71

Total protein

0.01

0.99

0.20

0.50

Serum Albumin

0.08

0.84

0.22

0.40

Serum Globulin

0.12

0.75

0.18

0.49

Albumin:Globulin Ratio

0.09

0.82

0.15

0.47

SGOT

0.09

0.82

0.35

0.17

SGPT

0.05

0.90

0.31

0.22

ALP

0.15

0.71

0.17

0.52

p>0.05 and not statistically significant

Discussion

Ascites is the main complication of cirrhosis [15] and the mean time period to its development is approximately 10 years [16, 17]. Ascites correlated best with early onset cirrhosis as compared to alcoholic hepatitis, even though both were nearly equally (n=5 vs. n=3) distributed in these subset of patients [18]. Similar finding is seen in our study where ascites is seen frequently in cirrhosis patient. Study from Mumbai noted that among 327 patients that were followed up, 41% had cirrhosis while the remaining 31% had noncirrhotic liver disease [17]. The results of our study also reflect figures similar to this. This study was an attempt to study the radiological and biochemical findings in Alcoholic liver disease. Ascites correlated best with cirrhosis in early stage [18]. Similar finding is observed in our study. Portal hypertension is the underlying cause of cirrhosis in most of the clinical cases and hence increased percentages of ascites in cirrhosis also shows increased percentages of portal hypertension. However this is not as much appreciable in fatty liver. Varices are also most commonly associated with cirrhotic disease. Alcohol acts as a toxic agent (hepatotoxic) and leads to pathological changes in liver. Though 80% of alcohol passes through liver to be detoxified, on chronic consumption of alcohol their occurs secretion of pro inflammatory cytokines (TNF-alpha, Interleukin 8), oxidative stress, lipid peroxidation, acetaldehyde toxicity. These factors cause inflammation, apoptosis, and eventually fibrosis of liver cells. The final stage is cirrhosis in which fibrosis is seen extensively and it shrinks the liver further damaging the liver. Ultrasonography helps to reveal the morphological changes occurring in liver.

Conclusion

The various radiological and biochemical findings reveals the underlying manifestations associated with them. Though a large number of radiological and biochemical changes are seen it is difficult to establish a relation between these two. However this relation is established by some studies that have used samples of liver biopsy. But we have used limited number of resources and no samples are taken. The findings in this study help us to understand various underlying pathophysiological manifestations related to study and also tell about derangements in LFTs seen in alcoholic liver disease. As Punjab is one of the top three states in name of alcohol abuse there is a need of conducting further studies in relation to this and awaring general population about the various clinical effects and fatality chronic alcohol consumption may cause.

References

1. Longstreth GF, Zieve David. “Alcoholic Liver Disease”. MedLinePlus: Trusted Health Information for You. Bethesda, MD: US National Library of Medicine & National Institutes of Health. 2009. Available at url. http://www.nlm.nih.gov/medlineplus/ ency/article/000281.htm (Last Assessed on 13th October, 2014) 2. Iruzubieta P, Crespo J, Fabrega E. Long-term survival after liver transplantation for alcoholic liver disease. World J Gastrenterol 2013; 19: 9198-208. 3. Continuing Education in Anaesthesia, Critical and Pain, Volume 10; Number 3: 2010. Available at url: http://e-safe-anaesthesia.org/e_library/11/Alcoholic_ liver_disease_CEACCP_2010.pdf (Last Assessed on 13th October, 2014)

http://www.pacificejournals.com/aabs

A-26 4. Gao B, Bataller R. Alcoholic liver disease: Pathogenesis and new therapeutic targets. Gastroenterology 2011; 141: 1572-85.

AABS; 3(1): 2016

11. Pessione F, Ramond MJ, Peters L, Pham BN, Batel P, Rueff B, et.al. Five-year survival predictive factors in patients with excessive alcohol intake and cirrhosis. Effect of alcoholic hepatitis, smoking and abstinence. Liver Int 2003; 23: 45–53

12. Borowsky SA, Strome S, Lott E. Continued heavy drinking and survival in alcoholic cirrhotics. Gastroenterology 1981; 80: 1405 – 9. 13. Brunt PW, Kew MC, Scheuer PJ, Sherlock S. Studies in alcoholic liver disease in Britain. I. Clinical and pathological patterns related to natural history. Gut 1974; 15: 52 – 8. 14. Luca A, Garcia-Pagan JC, Bosch J, Feu F, Caballeria J, Groszmann RJ, et.al. Effects of ethanol consumption on hepatic hemodynamics in patients with alcoholic cirrhosis. Gastroenterology 1997; 112: 1284-9. 15. European Association for the Study of the Liver. EASL clinical practice guidelines on the management of ascites, spontaneous bacterial peritonitis, and hepatorenal syndrome in cirrhosis. J Hepatol 2010; 53: 397–417. 16. D’Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 118 studies. J Hepatol 2006; 44: 217–31. 17. Mukhopadhyay P, Saha S, Philips CA, Sinha U. Clinical, biochemical and pathological correlation in alcoholic liver disease among Indian patients. Available at url. http://www.tropicalgastro.com/ articles/33/3/clinical-biochemical-and-pathological. html. (Last Assessed on 13th October, 2014) 18. Narawane NM, Bhatia S, Abraham P, Sanghani S, Sawant SS. Consumption of ‘country liquor’ and its relation to alcoholic liver disease in Mumbai. J Assoc Physicians India 1998; 46: 510–3.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

5. Mendez-Sanchez N, Almeda-Valdes P, Uribe M. Alcoholic liver disease. An update. Ann Hepatol 2005; 4: 32-42. 6. Nyblom H, Berggren U, Balldin J et al. High AST/ ALT ratio may indicate advanced alcoholic liver disease rather than heavy drinking. Alcohol Alcohol 2004;39:336–9. 7. Bird GL. Investigation of alcoholic liver disease. Baillieres Clin Gastroenterol 1993; 7; 663-82. 8. Keane MG, Lai C, Pereira SP. Detecting patients with cirrhosis in primary care. Practitioner 2014; 258 (1773): 15-20, 2-3. 9. Global Status Report on Alcohol 2004. Available at url. http://www.who.int/substance_abuse/publications/ global_status_report_2004_overview.pdf. (Last Assessed on 13th October, 2014). 10. Ezzati M, Lopez A, Rodgers A, Vander Hoorn S, Murray CJ, Comparative Risk Assessment Collaborating Group. Selected major risk factors and global and regional burden of disease. Lancet 2002; 360: 1347–60.

Original Article Etiological and clinical spectrum of pancytopenia based on bone marrow examination and case records: A retrospective study Prashanth B Gandhi1, Tharanath Shankar1, Mohammed Afraz Pasha2*, Mangala Gouri3 1

Dept of Internal Medicine, M.S. Ramaiah Medical College, Bangalore, India Dept of Emergency Medicine, St. Johns Medical College, Koramangala, Bangalore, India 3 Dept. of Pathology, M.S. Ramaiah Medical College, Bangalore, India

Keywords: Pancytopenia, Bone marrow examination, Etiology, Diagnosis, Pathology

ABSTRACT Background: Pancytopenia is a common entity seen in clinical practice. This study aims to determine the spectrum of etiology and clinical presentation of pancytopenia by conducting a retrospective study of case records, peripheral blood smears and bone marrow aspiration/biopsy findings of patients admitted to M S Ramaiah Medical Teaching Hospital, Bangalore, India. Methods: Case records, blood smear reports and bone marrow aspiration/biopsy findings of patients presenting with pancytopenia who fit the inclusion criteria during the period of January 2010 to May 2015 were analyzed. Relevant history, physical and systemic examination and hematological parameters at presentation were recorded using a standard proforma. Results: Among 134 cases of pancytopenia at presentation mean age was 28 years. Male to female ratio was 1.16. The etiological break up was according to the following order â&#x20AC;&#x201C; megaloblastic anemia (46.27%), leukemia (20.15%), aplastic anemia (9.7%), hypersplenism (7.46%), infections (6.72%), myelodysplastic syndrome (5.97%) and HIV (3.37%). The clinical spectrum in patients with each of these etiologies has been elucidated. Conclusion: The commonest cause of pancytopenia in this part of the India is megaloblastic anemia which is a curable deficiency. This is followed by leukemia, where an early diagnosis and intervention will improve their quality of life dramatically.

*Corresponding author: Dr. Mohammed Afraz Pasha, Dept of Emergency Medicine, St. Johns Medical College, Koramangala, Bangalore-560034, Karnataka, India Phone: +91 7406648141 E-mail: drafrazmohammed@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-28

Introduction Pancytopenia is a common entity seen in clinical practice. It is a condition where all the three formed blood elements (red blood cells, white blood cells and platelets) are reduced below the normal reference range. It has numerous causes. The pattern of diseases leading to pancytopenia may vary with differences in age and geographic distribution, nutritional status and prevalence of infection. The etiology ranges from simple treatable ones like infections and megaloblastic anaemia to serious conditions like leukemia. Studies conducted during various timeline have given variable results about its proportional distribution .This study is aimed at understanding the etiological spectrum of pancytopenia in the southern part of India. Although all the cell-lines of the blood are depleted, the clinical manifestations are varied and mutually different from one another. This calls for a need to identify a pattern in the symptoms so as to pick up the clues to the etiology. A correlation between the clinical features of individual patients and the blood picture, bone marrow examination is being reviewed in this study in order to achieve the same. Such a correlation will help the clinicians to gauge the diagnosis early and easily. Importance of the study lies in the timely intervention for the causes of pancytopenia which can either bring about a complete cure or at least a remission from the disease. In a nutshell, this study is aimed at obtaining a key symptoms and signs, which, when coupled with Peripheral blood smear of pancytopenia; can help the clinician to arrive at a diagnosis. This can be confirmed with a bone marrow examination. An early and an affordable method of diagnosis is the key to better prognosis. The objective of this study is to determine the spectrum of etiology and clinical presentation of pancytopenia by conducting a retrospective study of case records, peripheral blood smear and bone marrow aspiration/biopsy findings.

Materials and Methods Study design: retrospective record analysis Study site: M. S. Ramaiah medical teaching hospital and Memorial Hospital, Bangalore.

AABS; 3(1): 2016 Statistical analysis: From the data gathered, statistical analysis was done and results regarding the distribution of etiology and clinical presentation of pancytopenia were summarized using descriptive statistics like proportions. Quantitative data such as age and blood investigations will be summarized using descriptive statistics like mean, median and standard deviation. Inclusion criteria: Pancytopenia as per the criteria (1): 1. Anemia 2. Leucopenia and Neutropenia 3. Thrombocytopenia: platelet count Exclusion criteria: 1. 2. 3. 4.

Patients on chemotherapeutic agents Patients on radiation therapy Patients who did not undergo bone marrow analysis Obvious causes of pancytopenia like Dengue fever, other viral fevers where bone marrow aspiration was not indicated and the changes were transient.

Result There were 134 cases of pancytopenia at presentation for which bone marrow aspiration/biopsy was done to ascertain the diagnosis. Samples where the diagnosis could not be arrived at were excluded from study. Most common causes of pancytopenia where there was no clinical decision made to do a bone marrow analysis were Swine flu (n=76) and dengue fever (n=103). Here the improvement in count was expected in due course of the illness. The mean age of presentation of pancytopenia was 28 years. Male to female ratio was 1.16. The etiological break up was in the following order of megaloblastic anemia (46.27%), leukemia (20.15%), aplastic anemia (9.7%), hypersplenism (7.46%), infections (6.72%), myelodysplastic syndrome (5.97%) and HIV (3.37%). The descending order of symptom presentation was as follows: fatigue (80.6%), shortness of breath (52.24%), bleeding manifestation (48.51%), fever (45.52%), frequent infections (44.71%), palpitation (42.54%), headache (23.13%), dizziness (22.4%) and chronic diarrhea (11.19%).

Method: After obtaining institutional review boardâ&#x20AC;&#x2122;s approval, case records, blood smear reports and bone marrow aspiration/biopsy findings of patients presenting with pancytopenia who fit the inclusion criteria during the period of January 2011 to May 2015 were analyzed. Relevant history, physical and systemic examination and hematological parameters at presentation were recorded using a standard proforma.

Around 88.71% of megaloblastic anemia patients presenting as pancytopenia were lacto vegetarians. The most common presenting sign was pallor (99.25%), followed by tachycardia (61.94%), fever (52.99%), splenomegaly (44.03%), hepatomegaly (39.55%), petechiae (36.52%), bone tenderness (18.66%), lymphadenopathy (11.94%) and jaundice (6.72%).

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-29

The clinical features of individual etiologies are shown in table 1 and table 2. Based on analysis of peripheral smear report, the hematological parameters during presentation were as follows: hemoglobin (mean 5.5g/dL), WBC (mean 3.04x109/L), neutrophils (mean 0.9x109/L) and platelets (48.85x109/L).

4. Destruction of cells due to action of antibodies. 5. Trapping of normal cells due to overactive and hypertrophied reticulo-endothelial system. The etiological break up of pancytopenia in this study is comparable with similar studies done in India. The following table compares the two most common causes according to few similar studies (Table 3).

Based on bone marrow cellularity, 67.91% of cases were associated with hypercellular causes and 14.92% each were associated with normocellular and hypocellular causes.

Based on our analysis of peripheral smear reports, the hematological parameters during presentation were as follows: hemoglobin (mean 5.5g/dL), WBC (mean 3.04x109/L), neutrophils (mean 0.9x109/L) and platelets (48.85x109/L). These are comparable with the results produced by a study in Nepal (13).

Discussion Pancytopenia is a common clinical presentation. According to Bashawri (2), 11.9% is the frequency of bone marrow examination done as pancytopenia being the indication. From pathogenesis point of view, the causes of pancytopenia can be broadly categorized as follows:

The most common cause of pancytopenia in the present study was megaloblastic anemia (46.27%). The increased incidence of megaloblastic anemia can be correlated to the increased prevalence of nutritional deficiency in our country. The mean age of presentation was 38 years. The male to female ratio was 0.82. This was the only etiology

1. Reduced formation in the marrow. 2. Cell death in marrow due to ineffective erythropoiesis. 3. Defective cell formation which is avidly removed by circulation. Table 1: Symptom presentation of individual etiology: Megaloblastic anemia [n, (%)]

Leukemia [n, (%)]

Aplastic anemia [n, (%)]

Hypersplenism [n, (%)]

Infection [n, (%)]

MDS [n, (%)]

HIV [n, (%)]

Fatigue

51(82.26)

22(81.48)

9(69.23)

8(80)

7(77.78)

6(75)

5(100)

Shortness of breath

37(59.68)

12(44.44)

5(38.46)

5(50)

4(44.44)

4(50)

3(60)

Dizziness

15(24.19)

3(11.11)

2(15.32)

4(40)

2(22.22)

1(12.5)

3(60)

Palpitation

29(46.77)

9(33.33)

6(46.15)

5(50)

1(11.11)

5(62.5)

2(40)

Headache

14(22.58)

7(25.93)

3(23.08)

1(10)

3(33.33)

0(0)

3(60)

Fever

19(30.65)

20(74.07)

5(38.46)

4(40)

8(88.89)

2(25)

3(60)

Frequent infections

26(41.93)

17(62.96)

4(30.77)

3(30)

2(22.22)

3(37.5)

5(100)

Bleeding manifestations

27(43.55)

14(51.85)

6(46.15)

7(70)

5(55.56)

2(25)

4(80)

chronic diarrhea

10(16.13)

0(0)

1(11.11)

1(12.5)

3(60)

Table 2: Signs in individual etiologies Megaloblastic anemia [n, (%)]

Leukemia [n, (%)]

Aplastic anemia [n, (%)]

Hypersplenism [n, (%)]

Infection [n, (%)]

MDS [n, (%)]

HIV [n, (%)]

Tachycardia

42(67.74)

16(59.26)

6(46.15)

6(60)

6(66.67)

5(62.5)

2(40)

Fever

23(37.1)

20(74.07)

5(53.85)

5(50)

8(88.89)

4(50)

4(80)

Pallor

62(100)

27(100)

13(100)

9(90)

9(100)

8(100)

5(100)

Jaundice

2(3.23)

0(0)

4(40)

3(33.33)

0(0)

Petechiae

21(33.87)

11(40.74)

4(30.77)

5(50)

4(44.44)

1(12.5)

3(60)

1(1.61)

9(33.33)

0(0)

1(12.5)

4(80)

Splenomegaly

34(54.84)

7(25.92)

0(0)

10(100)

3(33.33)

1(12.5)

3(60)

Hepatomegaly

34(54.84)

6(22.22)

0(0)

6(60)

1(11.11)

2(25)

2(40)

Bone tenderness

12(19.35)

8(29.63)

1(7.69)

0(0)

2(25)

2(40)

Lymphadenopathy

http://www.pacificejournals.com/aabs

A-30

AABS; 3(1): 2016

Table 3: Other studies Country

Year

Number Commonest cause of cases

2nd Most common cause

International agranulocytosis Israel & and aplastic anemia group(3) Europe Keisu and Ost(4) Israel & Europe

1987

319

Hypoplastic anemia (52.7%)

MDS (4.5%)

1990

100

Neoplastic disease, radiation (32%)

Hypoplastic anemia(19%)

Verma and Dash(5) Tilak and Jain(6) Kumar et al(7) khunger et al(8) Khodke et al(9) Memom et al(10)

India India India India India Pakistan

1992 1999 1999 2002 2000 2008

202 77 166 200 50 230

Hypoplastic anemia(40.6%) Megaloblastic Hypoplastic anemia Megaloblastic anaemia (72%) Megaloblastic Aplastic anemia (23.9%)

Megaloblastic Hypoplastic anemia Megaloblastic Aplastic anaemia (14%) Hypoplastic anemia Megaloblastic anemia (13.04%), leukemia (13.05%)

Istiaq et al (11) Savage et al(12) Jha et al(13) Gamal and Safa Ramesh et al(14)

Pakistan Zimbawe Nepal Yemen Nepal

2004 1999 2008 2008 2009

100 134 148 75 28

Megaloblastic anemia (39%) Megaloblastic anemia Hypoplastic anemia (29.05%) Hypersplenism (28%) Megaloblastic anemia (42.855)

Hypersplenism (19%) Aplastic anaemia Megaloblastic anemia (23.64%) Malaria (17.3%) Aplastic anemia (35.71%)

present study

India

2011

134

Megaloblastic anemia (46.27%) Leukemia (20.15%)

Study

Fig. 1: Representation in bar graph:

Fig. 2: Age distribution of individual etiologies

which affected females more than males. The overall sex ratio of pancytopenia was 1.16. Around 88.17% of these patients were lacto vegetarians, showing that dietary factor is an important contributing entity. 16.3% patients had chronic diarrhea which can be a cause for dietary losses. The only etiology where an even higher percentage of chronic diarrheas of 80% were seen was in case of HIV infection. Tropical sprue is endemic in southern India. It is readily corrected by folate and antibiotics. It primarily affects distal small intestine (15), therefore incriminated as the cause of cobalamine deficiency (16).

diarrhea. Due to gradual development of anemia in this condition, the symptoms of anemia are seen only when hematocrit is significantly low.

The most common symptom in patients with megaloblastic anemia was fatigue followed in descending order by shortness of breath, palpitation, bleeding manifestation, frequent infections, fever, dizziness, headache and chronic

The most common presenting sign was pallor followed in descending order by tachycardia, splenomegaly, hepatomegaly, fever, petechiae, bone tenderness, jaundice and lymphadenopathy. On blood smear examination, significant parameters were hyper segmented neutrophils (82.26%), macrocytic hypochromic blood picture (80.64%) and anisopoikilocytosis (77.42%). Circulating normoblasts were seen in 22.58% patients. On bone marrow examination, 67.91 % of the cases demonstrated hypercellularity of which the predominant ones were of erythroid series. Findings of megaloblastosis clinch the diagnosis.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-31

The second most common cause of pancytopenia was leukemia which constituted 20.15% of patients. Among these AML constituted 44.44% and ALL constituted 55.56%. The mean age of diagnosis of AML was 72 years in contrast to that of ALL which was 8 years. AML did not show any gender preponderance but the sex ratio of ALL was 1.5 in favor of male: female. The descending order of symptoms at presentation was fatigue, fever, recurrent infections, bleeding manifestations, shortness of breath, palpitation, headache and dizziness. The frequencies of clinical signs elicited were (in descending order): pallor, fever, tachycardia, petechiae, lymphadenopathy, bone tenderness, splenomegaly and hepatomegaly. On peripheral blood smear, 92.59% showed circulating immature cells, 74.07% showed normochromic normocytic anemia and 55.56% showed circulating normoblasts. On marrow examination, 85.18% showed hypercellular marrow. These findings correlate well with observations of other studies (17, 18, 19 and 20). The third most common cause of pancytopenia was aplastic/ hypoplastic anemia of unknown cause which was seen in 9.7% patients. The mean age of presentation was 24 years. The male to female ratio was 2.25. The descending order of symptom presentation was as follows: fatigue, bleeding manifestations, palpitation, fever, shortness of breath, frequent infections, headache and dizziness. The descending orders of clinical signs were: pallor, fever, tachycardia, petechiae. No significant lymphadenopathy, splenomegaly and hepatomegaly were noted. Hence if any of the above mentioned finding is seen with pancytopenia, then it rules out aplastic anemia. On peripheral smear, 92.55% showed normocytic normochromic anemia with pancytopenia. On bone marrow examination, 100% showed hypocellular marrow. The fourth most common cause of pancytopenia in this study was hypersplenism (7.46%). The mean age of presentation was 45 years. The male to female ratio was 2.33. Most of these patients were alcoholics and had decompensated liver disease, whose independent association with hypersplenism is shown by a study (21). In alcoholic liver cirrhosis, megaloblastic anemia is usually caused by folate deficiency (22 and 23). Alcohol acutely depresses serum folate levels and this accelerates megaloblastic anemia. Alcohol also causes marrow suppression of reticulocytes, granulocytes and platelets (24 and 25). The next most common cause of pancytopenia in our study was infections (6.72%). One third of these patients had malaria, enteric fever and viral fever with sepsis each.

These findings correlate well with studies done by Menon et al (10). Maximum infections occurred in the age group of 0 to 9 years. The male to female ratio was 2. Myelodysplastic syndrome (MDS) was the next most common cause of pancytopenia seen in 5.97% of patients. In a study done in Israel and Europe, MDS was the second most common cause of pancytopenia (3). In this study, the mean age of presentation was 62 years and the male to female ratio was 1. Patients with multi-lineal cytopenia are a subset of MDS with increased morbidity and reduced life expectancy. Immune and inflammatory syndrome is seen in around 10% patients. A symptom complex which simulates systemic lupus erythematosus (fever, arthritis, pleurisy and pancytopenia with hypercellular marrow) may precede AML (26). HIV accounted for 3.32% of cases presenting as pancytopenia. Of the 5 patients, two were in terminal stage of the disease. Pancytopenia in HIV has been attributed to many causes which include inflammatory cytokines or HIV virus itself, infiltration by lymphoma or infections, polypharmacy, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, auto-antibodies and malnutrition (27 and 28).

Conclusion The commonest cause in the southern part of India is megaloblastic anemia. It is a curable deficiency which if intervened at an early stage will prevent severe neurological and hematological sequelae. Adequate replenishment by vitamin B12 is enough to bring back the normal counts in these patients by which significant morbidity and mortality can be prevented. The second most common cause was leukemia, where it is needless to say the miracle that can be achieved on timely treatment. Early diagnosis and intervention will lead to remission of disease and better quality of life. Time and again, the investigation of choice to diagnose unexplained pancytopenia or to rule out grave conditions like neoplastic infiltration of bone marrow has been bone marrow aspiration/biopsy. Infective etiologies are easily diagnosable since it only needs a fairly good knowledge about the endemicity of an area like malaria and typhoid fever.

Acknowledgements Special Thanks to Dr A C Ashok, Principal and Dean of the Hospital whose constant enthusiasm and permission to carry out the study was very pivotal for successful completion of the study.

Funding None

http://www.pacificejournals.com/aabs

A-32

Competing Interests None declared

Reference

AABS; 3(1): 2016 14. Ramesh K Makaju1, Mohamad Ashraf, Sushama Bhatta, Ram Gurung, Babu R Pokharel.Pancytopenia: A Memorable Manifestation of Megaloblastic Anemia. JNAMLS 2009 Dec;10(1).

1. Donald LY, Catherine MB, Sharon MG. Normal blood values. In: Hoffman R, editor. Hematology: Basic principles and practice, 5th ed. Churchill livingstone Elsevier; 2008. 2. Bashwri L A. Bone marrow examination. Indications and diagnostic value. Saudi Medi J. 2002;23:191-6. 3. International agranulocytosis and aplastic anemia study. Incidence of aplastic anemia, the relevance of diagnostic criteria. Blood 1987;70:1718-21. 4. Keisu M, Ost A. Diagnosis in patients with severe pancytopenia suspected of having aplastic anemia. Eur J Haematol 1990;45:11-4. 5. Varma N, Dash S. A reappraisal of underlying pathology in adult patients presenting with pancytopenia. Trop Geogr Med 1992;44:322-7. 6. Tilak V, Jain R .Pancytopenia-a clinico-hematologic analysis of 77 cases Indian J Pathol Microbiol. 1999 Oct;42(4):399-404. 7. Kumar R, Kalra SP, Kumar H, Anand AC, Madan H. Pancytopenia . A six year Study. JAPI 2001;49:10781081. 8. Khunger JM, Arulselvi S, Sharma U, Ranga S, Talib VH. Pancytopenia-a clinic haematological study of 200 cases. Indian J Pathol Microbiol. 2002 Jul;45(3):375-9. 9. Kishor Khodke, S Marwah, G Buxi, RB Yadav, NK Chaturvedi. Bone Marrow Examination in Cases of Pancytopenia. Journal, Indian Academy of Clinical 10. Medicine. 2001Jan-June;2;1-2. Memon S, Shaikh S, Nizamani MA. Etiological spectrum of pancytopenia based on bone marrow examination in children. J Coll Physicians Surg Pak. 2008 Mar; 18(3):163-7. 11. Ishtiaq O, Baqai HZ, Anwer F, Hussain N. Patterns of pancytopenia patients in a general medical ward and a proposed diagnostic approach.J Ayub Med Coll Abbottabad. 2004 Jan-Mar;16(1):8-13. 12. Savage DG, Allen RH, Gangaidzo IT, Levy LM, Gwanzura C, Mayo et al. Pancytopenia in Zimbabwe. Am J Med Sci. 1999 Jan;317(1):22-32. 13. Jha A, Sayami G, Adhikari RC, Panta AD, Jha R-Bone Marrow Examination in Cases of Pancytopenia. J Nepal Med Assoc 2008;47(169):12-7.

15. Klipstein FA. Progress in gastroenterology. Tropical sprue. Gastroenterology 1968; 54:275.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

16. Sheehy TW, Perez-Santiago E, Rubini ME. Tropical sprue and vitamin B12. N Engl J Med 1961;265:1232. 17. 17. Pui C-H, Crist WM. Acute lymphoblastic leukemia, in Childhood Leukemia. New york:Cambridge University Press;1999.p288. 18. McKenna SM, Baehner RL. Diagnosis and treatment of childhood acute lymphoblastic leukemia, in Neoplastic Diseases of the Blood. 3rd ed.edited by PH Wiernik, GP Canellos, JP Dutcher, RA Kyle. New York Churchill Livingstone; 1996.p271. 19. Reiter A, Schrappe M, Ludwig WD, et al. Chemotherapy in 998 unselected childhood acute lymphoblastic leukemia patients. Results and conclusions of the multicenter trial ALL-BFM 86. Blood 1994;84:3122. 20. Chessells JM, Hall E, Prentice HG, et al.The impact of age on outcome in lymphoblastic leukemia; MRC UKALL X and XA compared: A report from the MRC Paediatric and Adult Working Parties. Leukemia 1998;12:463. 21. Peck-Radosavljevic M: Hypersplenism. Gastroenterol Hepatol 2001;13:317.

Eur

22. Savage D, Lindenbaum J: Anemia in alcoholics. Medicine (Baltimore) 1986;65:322. 23. Eichner ER, Hillman RS: Effect of alcohol on serum folate level. J Clin Invest 1973;52:584. 24. Liu YK. Effects of alcohol on granulocyte and lymphocytes. Semin Hematol 1980;17:130. 25. Post RM, Desforges JF. Thrombocytopenia and alcoholism. Ann Intern Med 1963;68:1230. 26. Hebbar M, Hebbar-Savean K, Fenaux P. Systemic diseases in myelodysplastic syndromes. Rev Med Intern 1995;16:897. 27. Zon LI, Arkin C, Groopman JE. Hematologic manifestations of the human immunodeficiency virus. Semin Hematol 1988;25:208. 28. Harriman GR, Smith PD, Horne MK, et al. Vitamin B12 malabsorption in patients with acquired immunodeficiency syndrome. Arch Intern Med 1989 149:2039.

Original Article Prevalence of molar pregnancy (a three year retrospective study) in a tertiary care hospital Jangbhadur Singh1*, Shaveta Sharma1, Kirpal Kour2 Shazia Bashir 1

Dept. of Pathology. Shere Kashmir Institute of Medical Sciences MCH Bemina, Srinagar, India 2 Dept. of Gynae & Obs , G. B. Pant Hospital, Srinagar, India Keywords: Hydatidiform Mole, Trophoblastic Hyperplasia, Cisterns.

ABSTRACT Hydatidiform mole is an abnormal gestation characterized by trophoblastic hyperplasia and overgrowth of placental villi. H. mole is classified as complete (CHM) and partial (PHM). The diagnosis is based on histopathology and genetic origin. In our set up, we used only histopathological diagnostic criteria. The incidence of molar pregnancy varies in different parts of the world. The malignant potential of the disease is higher in South East Asia as compared to western countries. Objective of the present study was to determine the frequency, clinical presentation and morphological features of H. mole and compare them with those of other studies. This is a 3 year retrospective descriptive case series conducted in the Department of Pathology, SKIMS Medical College, Bemina from 31st December 2011 to 31st December 2014. The case records of all the molar pregnancies during the study period were analysed regarding patientâ&#x20AC;&#x2122;s history, clinical presentation and morphological features. A total of 50 cases were examined during the study period which included 38 cases of complete mole and 11 cases of partial mole. One case was labeled as complete mole with atypical trophoblastic proliferation. Frequency of CHM was higher as compared to partial mole. The disease was common in extremes of ages. A number of histopathological diagnostic criterias are used to distinguish CHM from PHM. We concluded that there is no single criterion to differentiate CHM from PHM.

*Corresponding author: Dr Jangbhadur Singh Assistant professor, Dept. of Pathology. Shere Kashmir Institute of Medical Sciences MCH Bemina, Srinagar, India Phone: +91 9419032757 E-mail: jangbhadursinghsarna@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-34 Introduction Molar pregnancies represent a significant burden of disease on the spectrum of gestational trophoblastic disease. The incidence appears to be higher in women from South Asia including a trend towards recurrent molar pregnancies1,2. H. mole is an abnormal gestation characterized by trophoblastic hyperplasia and overgrowth of placental villi. H. mole is classified as complete and partial hydatidiform mole. The diagnosis is based on histopathology and genetic origin 3 .Accurate diagnosis and classification of H. mole is important as the risk of persistent gestational trophoblastic disease including choriocarcinoma is significantly high. The risk of choriocarcinoma in CHM is 10-30% and in PHM is 0.5%-5% 4.Histological examination is the main tool in the diagnosis of molar pregnancy including the degree of trophoblastic hyperplasia, villous contours and scalloping, presence of distinct cisterns, trophoblastic inclusions and presence or absence of nucleated RBCâ&#x20AC;&#x2122;s in fetal vessels5,6.However,there is considerable overlap in the histological features between CHM and PHM resulting in significant interobserver variability in the diagnosis. Moreover, molar pregnancies are being evacuated early in gestation before the development of well established classical morphological features thus adding to the difficulty in diagnosis7.

Materials and Methods

AABS; 3(1): 2016 TABLE 1: This table depicts the types of molar pregnancy seen in 50 cases. Type of hydatidiform moles

No. of patients

1) Complete H.moles

38 (76%)

2) Partial H.moles

11(22%)

3) Complete mole with atypical trophoblastic proliferation

1(2%)

TABLE 2: This table gives the information regarding the reproductive age of patients. No. of patients

Age in years 1) <20

17(34%)

2) 21-35

11(22%)

3) >35

22(44%)

TABLE 3: Gestational age noted in 50 cases is depicted in the following table. Gestational age in months

No. of patients

1-2months

16(32%)

2-5months

26(52%)

>5months

8(16%)

A hydatidiform mole is a pregnancy in which the placenta contains grape like vesicles that are visible to the naked eye. The vesicles arise by the hydropic change within the chorionic villi which are seen as trophoblastic hyperplasia. The diagnosis of hydatidiform mole is clinically important because of its potential to give rise to persistent

gestational trophoblastic disease including invasive mole, choriocarcinoma and placental site trophoblastic tumour. Furthermore, the distinction between complete and partial mole is also significant10. In the present study, out of 50 cases noted, 38 cases were labeled as complete mole, 11 cases were labeled as partial mole and one case was labeled as complete mole with atypical trophoblastic proliferation. Therefore the frequency of complete moles was 76%. Jaffer11 in his study of 60 cases of molar pregnancy also reported that frequency of CHM was higher as compared to PHM. Maternal reproductive age is the most important risk factor for H. mole in every region and ethnic group. In this study, disease was more common at extremes of reproductive ages with highest frequency seen in women of more than 35 years of age group (44%) and less than 20 years of age group (34%). It is consistent with the findings of studies of Nizam12 and Jaffer11. The available evidence suggests that H. mole arises as a consequence of defective ova. It is premature in young and postmature in old ages12. Gestational age was also noted in the present study. Maximum number of patients approximately 52% presented during 2-5months of gestational age i.e during first and mid second trimester. Koirala13 in his study of 64 cases reported 2nd trimester as the most common period of presentation. This is probably due to increase in gynaecological, sonographicand

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

This retrospective, descriptive case study was conducted at the Department of Pathology, SKIMS Medical College, Bemina from 31stDecember; 2014 to 31stDecember;2011. The case records of all these patients with molar pregnancy were analysed regarding age of patients, gestational age, symptoms and histopathology. All patients having molar pregnancy with elevated Î˛- HCG levels, histopathological evidence of the disease were included in the study. The criteria for the diagnosis of CHM and PHM were those of Szulman and Surti8-9. The histological diagnosis was attempted in all cases, even when material was scanty. The following features were graded-(1) Hyperplasia of trophoblasts; diffuse (perivillous,circumferential); focal(perivillous,multifocal); absent. (2) Cistern:present ;absent.(3) Pseudoinclusions of

Result

A total of 50 cases were identified during the study period.

Discussion

Original Article

A-35

TABLE 4: Histopathological criterias in complete and partial moles. Types of mole Complete mole Partial

Trophoblastic hyperplasia Focal Diffuse 7 32 9 2

Pseudoinclusions Present Absent 36 3 1 10

Cisterns Present 37 2

Absent 2 9

Vessels in villous stroma Present Absent 0 39 10 1

Fig. 1: Grape like vesicles in H . Mole

Fig. 2: Choionic villi (H&E, 20X)

histopathological services over the years which have led to early recognition of the disease. Histological examination forms the main diagnostic tool in the diagnosis of molar pregnancy. Mainly four diagnostic tools were used in the present study which included trophoblastic hyperplasia, pseudoinclusions,cistern formation and vessels in the villous stroma. Abnormal trophoblastic hyperplasia is a requirement for the diagnosis of molar pregnancy. In the present study, the degree of trophoblastic hyperplasia was more marked in CHM as compared to PHM and it exhibited a circumferential pattern. Cistern formation was mainly seen in CHM. Mayun14 in his study of 73 cases of molar gestation reported trophoblastic hyperplasia in 80% cases of both CHM and PHM. Cistern formation occurred in 80% cases of CHM and 40% cases of PHM. However there is considerable overlap in the histological features between complete and partial mole resulting in significant interobserver variability in the diagnosis. Moreover molar pregnancies are being evacuated early in gestation before the development of well established classical morphological features thus adding to the difficulty in diagnosis4.

criterias for their recognition. The disease was common in extremes of ages. Frequency of CHM was higher as compared to PHM. Morphological features of CHM differ from PHM but with some overlap. Biological variability and scarcity of available tissue however will sometimes impose difficulties in the differential diagnosis between CHM and PHM.

Conclusion

We concluded that CHM and PHM have widely different prognosis and therefore require very accurate diagnostic

Acknowledgements NONE

Funding None

Competing Interests NONE

Reference

1. Lorigan P. C., Sharma S., Bright N., Coleman R. E. and Hancock B.W., “Characteristics of women with recurrent molar pregnancies”, Gynaecologic oncology, 78(3); 288-292, 2000. 2. Khaskheli M, Khushk I.A, Baloch S and Shah H.,”Gestational trophoblastic disease:experience at a tertiary care hospital of Sindh”,Journal of the college of physicians and surgeons Pakistan,17(2);81-83,2007.

http://www.pacificejournals.com/aabs

A-36 3. Fisher RA , Hodges MD , Rees HC , Sebire NJ , Seckl MJ , Newlands ES , Genest DR , Castrillon DH. The maternally described gene p57kip2 (CDN1KC) is abnormally expressed in both androgenetic and biparental complete hydatidiform moles. Human molecular genetic ,11; 3267-72, 2002. 4. Merchant SH , Amin MB , Vishwanatha DS, Malhotra RK , MoehLenkamp , Joste NE . p57kip2 Immunohistochemistry in early molar pregnancies . Emphasis on its complementary role in differential diagnosis of hydropicabortuses. Human Pathol , 36 ;180-86,2005. 5. Hoffner L , Dunn J , Esposito N , Macpherson T , Surti U. p57kip2 Immunostaining and molecular cytogenetics. Combined approach aids in diagnosis of morphologically challenging cases with molar phenotype and in detecting androgenetic cell lines in mosaic/chimeric conceptions. Human Pathol ,39; 6372, 2008. 6. SebireJN ,Petson DR , Seckl JM , Newlands SE , Fisher AR . p57kip2. Immunochemical staining of gestational trophoblastic tumors does not identify the type of causative pregnancy. Histopathology, 45; 13541, 2004. 7. LeGallu RD , Stelow EB , Ramixz NC , Atkins KA , Diagnosis of H.moles using p57 IHC and HER2

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016

10.

11.

12.

13.

14.

Fluorescent insitu hybridization. Am J clinPathol , 129;749-55,2008. Szulman AE, Surti U. The syndrome of H.mole cytogenetic and morphological correlations I .Am J obstetGynaecol , 131 ; 665-71 ,1978. SzulmanAE ,Surti U . The syndromes of hydatidiform mole II.Morphological evolution of the complete and partial mole. Am J obstetGynaecol ,132 ; 20-7 ,1978. Di CintioE ,Parazzini F, Rosa C , Chatenoud L , Benzi G .â&#x20AC;? The epidemiology of gestational trophoblastic disease â&#x20AC;&#x153;. Gen DiagnPathol ,143 (2-3); 103-8, 1997. JaffarRozina, KalsoomRahat and QuershiAsmaa. Histopathological review of partial and complete hydatiform moles in a tertiary care hospital , Lahore. 27 ,76-80; 2011. Nizam K, Haider G, Memon N, Haider A. Gestational trophoblastic disease. Experience at NawabshahHospital . J Ayub Med Coll .21; 2009. Koirala A, Khatiwada P, Giri A , Kandel P, Rigmi M, Upreti D. The demographiesof molar pregnancy in BPKIHS, Kathmandu Univ Med J ,36(4); 298300;2011. Mayun AA, Rafindadi AH , Shehu MS. Pathomorphology of molar gestation in Zaria. 51(1) ; 1-4, 2010.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Effect of Tetracycline therapy on Microfilaraemia and Antigenemia in Raipur city, Chhattisgarh state, India Santosh Kumar Agrawal Dept. of Zoology, Vivekanand Govt. P.G. College, Manendragarh, Korea, (C.G.) India

Keywords: Lymphatic filariasis, Wuchereria bancrofti, Microfilaraemia, Antigenemia

ABSTRACT Background: Lymphatic filariasis caused by Wuchereria bancrofti is one of the major health problems and is transmitted in India by Cx. quinquefasciatus. Diethylcarbamazine remains the drug of choice in selective as well as mass chemotherapeutic programs against lymphatic filariasis. Treatment of filarae infected animals with tetracycline resulted in the elimination of Wolbachia endosymbiont from the filarial tissues, prevented parasite establishment, filarial growth and rendered adult worm sterile. Method: Tetracycline capsules (200 mg/day) were given to microfilaraemic patients for 30 days and mf count and antigen level in their blood and infection & infectivity rates in their house hold Culex mosquitoes were assessed post tetracycline therapy. Results: It was found that mf counts and antigenemia in the blood & infection & infectivity rates in the mosquitoes were reduced gradually up to 24th months post tetracycline therapy. Conclusion: Targetting endosymbiont Wolbachia using antibiotics might be effective controlling measure in lymphatic filariasis.

*Corresponding author: Dr. Santosh Kumar Agrawal, Dept. of Zoology, Vivekanand Govt. P.G. College, Manendragarh, Korea, (C.G.) 497442, India Phone: +91 9827122495 E-mail: skazoology@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-38

Introduction

Bancroftian filariasis is a mosquito-transmitted parasitic disease of humans characterized by lymphangitis, hydrocele, lymphoedema, and elephantiasis, and is one of the most common causes of global disability. The disease has been considered to be potentially eradicable due to the fact that anti filarial drugs currently could break the cycle of transmission in endemic areas. Diethylcarbamazine plus albendazole are proved more effective than DEC alone [1, 2] for treating lymphatic filariasis. It is an effective microfilaricidal compound in vivo and its adulticidal function is yet to be proved. However, the drug is nonfunctional in vitro either against microfilariae or adult filarial parasites. Ivermectin is another potential filaricidal drug tested for efficacy and side reactions [3]. At a single dose of 200-400 µg/kg body weight, ivermectin showed promising results [4]. Tetracycline has been demonstrated to have anti-filarial function against microfilariae as well as adult filarial parasites in vitro as well as in vivo [5]. The tetracycline is primarily targeted to the intracellular bacteria viz., Wolbachia in filarial worms. Wolbachia are intracellular microorganisms that form maternally inherited infections with numerous arthropod species [6]. These bacteria have drawn much attention due to the reproductive alterations they induce in their hosts including cytoplasmic incompatibility, feminization and parthenogenesis. The bacteriostatic activity of tetracycline on Wolbachia symbionts appears responsible for altered fertility and embryogenesis [7]. Targeting Wolbachia is becoming an active area of research for control of lymphatic filariasis. Oxytetracycline exhibited macrofilaricidal activity against O. ochengi [8].

Materials And Methods

Study population: Individuals enrolled from a colony of Raipur city (C.G.) in the study. A total of 10 (6 males and 4 females) microfilaraemic patients were included in this study. Written informed consent was obtained from all participants. Individuals eligible for participation were adults of both sexes aged 18-50 years, with a minimum body weight of more than 40 kg, in good health and without any clinical condition requiring chronic medication. Hepatic and renal functions were assessed by urinal test. Treatment regimen: Participants received 2 x 100 mg capsule of tetracycline for a total of 4 weeks. Treatment was done and monitored by a trial clinician in the form of daily observed treatment (DOT). The drug regimen was assessed for microfilaricidal effect by assessing the following parameters

1. The rate of successful treatment: the proportion of mf positive persons treated in whom there was a reduction in the microfilarial count. 2. The percentage of cure rate- the proportion of mf positive persons treated who became negative for microfilariae after treatment. Determination of microfilarial load: For a quick screening in the night, the microfilarial load was determined by microscopic examination of finger-prick blood samples. Subsequently, eligible patients donated 10 ml of venous blood for accurate quantification using Whatman Nucleopore filter method. The same volume of blood was taken from each patient 1, 6, 12, 24 months after the commencement of tetracycline treatment. At each time point, plasma was taken from the remaining sample and frozen at -80 °C for later analysis of antigenemia (filarial adult worm antigens).The mean microfilaraemia load was found 830 (175-1600) / ml of blood. Determination of circulating filarial antigenemia: For determination of circulating filarial antigenemia (CFA), W. bancrofti antigen was measured with the TropBio ELISA test kit (TropBio, Townsville, Australia). The manufacturer’s protocol was followed except that the samples were diluted (1:20 ratio) with the diluent before pipetting into the TropBio ELISA test plates. Samples were tested in duplicate before treatment and at 6, 12 and 24-months follow-ups. The optical density at 414 nm was recorded from plasma samples. Antigen units were calculated with a standard curve from standards provided by the manufacturer, and the final units multiplied by the dilution factor of 20. USG examination: Ultrasonography (by professional ultrasonography centre) of the scrotal area was undertaken to detect adult W. bancrofti. Each patient’s scrotum was scanned in transverse and longitudinal section of the right and left sides. Worm nests were detected by the typical movement of the adult worms (known as Filarial Dance Signs-FDS). Determination of pre and post therapy vector infection & infectivity rates of Cx. quinquefasciatus of the microfilaraemic patient’s houses: Indoor resting Cx. quinquefasciatus will be collected before and after tetracycline therapy from all 10 microfilaraemic patient’s houses, dissected and assessed for mf (infection rates) and L3 larva (infectivity rates) load in the mosquitoes post tetracycline therapy.

http://www.pacificejournals.com/aabs

A-39

AABS; 3(1): 2016

Result

Four weeks course of tetracycline (200 mg/day) was given to 10 microfilaraemic patients, and they were monitored for mf count and antigenemia up to 24 months following tetracycline therapy. The treatment was well tolerated. There were no major adverse reactions of tetracycline therapy to any of the subjects. Some of them faced loose motion for 1 or 2 days which was controlled automatically without any treatment. Table1.1 shows the significant change in microfilaraemia and antigenemia from baseline and at follow-up time points. The mean mf count and antigenemia (CFA) units per ml of blood before treatment were 830 ± 145 and 9447 ± 2397 respectively. Both of these units decrease substantially one month after the start of treatment. The infection reduces gradually up to 24 months. When assessed, the rate of successful treatment was 100% throughout the study period up to 24 months follow up. The cure rate was found 0% when assessed one month after the treatment. 6 months after treatment it was 10% and was 20% after 12 and 24 months post therapy. Measurement of Adult Worm Vitality in Microfilaraemic patients by USG: Presence or absence of worm nest in ultrasound examination was used to asses’ adult worm vitality in microfilaraemic patients before and after tetracycline therapy. Ultrasonography to detect the filarial dance signs (FDS) was performed only in male patients since FDS is detected less frequently and probably less reliably in women. FDS in the scrotal region before treatment showed that three of the five male patients examined had between 1-4 worm nests. All three male patients became FDSnegative at 24 months after treatment. Determination of pre and post therapy vector infection & infectivity rates of Cx. quinquefasciatus

of the microfilaraemic patient’s houses: The impact of tetracycline therapy on the vector infection & infectivity rates of Cx. quinquefasciatus of the microfilaraemic patient’s houses was examined. Indoor resting Cx. quinquefasciatus were collected before and after tetracycline therapy from all 10 microfilaraemic patient’s houses. The mean vector infection & infectivity rates before therapy were 40.3 & 5.23 respectively. Gradual reduction the rates were found after tetracycline therapy. One month post therapy the vector infection & infectivity rates were found to be 33.07 (82%) & 4.48 (86%), 6 month after 14.35 (36%) & 1.83 (35%), 12 months after 7.31 (18%) & 0.84 (16%) and 24 months after treatment 4.08 (10%) & 0.45 (9%) respectively. (Table: 1.2).

Discussion

The past decades has seen major advances that have changed lymphatic filariasis from a neglected disease in to a disease now accepted as potentially eradicable. The main reason is the identification of ivermectin, DEC and albedazole, as effective antifilarial agents. a 4-week course of tetracycline against W. bancrofti induced both sustained reductions in microfilaraemia and, most notably, macrofilaricidal activity according to reductions in antigenemia and absence of adult worms by ultrasonography. This is especially important since the adult worms cause the disease pathology in lymphatic filariasis and no safe, effective macrofilaricidal treatment exists. In this study, tetracycline treatment resulted in almost complete elimination of microfilaraemia, which was sustained from at least 6 to 24 months after treatment. This gradual reduction over months is different to the rapid efficacy seen within a few days with diethylcarbamazine and ivermectin. This slow rate of microfilarial loss is most probably due to the effect of Wolbachia depletion on embryogenesis and the loss of microfilariae from the circulation through natural attrition, as recorded in onchocerciasis and lymphatic filariasis [9].

Table 1.1: Effect of Tetracycline Treatment on Microfilaraemia & Antigenemia Terms Mean Mf count ± SE % Mean Mf count p-value* No. of Mf +ve patients treatment rate No. of cured patients Cure rate Antigenemia Mean ± SE % Antigenemia Mean p-value*

Before Treatment 830 ± 145 100% 10 100% 0 0% 9447 ± 2397 100%

1 month post treatment 495 ± 82 60% 0.00043 10 100% 0 0% 4887 ±1277 52% 0.0014

6 month post treatment 300 ± 63 36% 0.00009 10 100% 1 10% 1956 ± 489 21% 0.0019

12 month post treatment 140 ± 38 17% 0.00010 10 100% 2 20% 717 ± 206 8% 0.0016

24 month post treatment 85 ± 22 10% 0.00014 10 100% 2 20% 187 ± 53 2% 0.0017

*p-value = t-Test

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-40

Table 1.2: Pre & post therapy vector infection & infectivity rates Megaloblastic anemia [n, (%)]

Leukemia [n, (%)]

Aplastic anemia [n, (%)]

Hypersplenism [n, (%)]

Infection [n, (%)]

MDS [n, (%)]

HIV [n, (%)]

Fatigue

51(82.26)

22(81.48)

9(69.23)

8(80)

7(77.78)

6(75)

5(100)

Shortness of breath

37(59.68)

12(44.44)

5(38.46)

5(50)

4(44.44)

4(50)

3(60)

Dizziness

15(24.19)

3(11.11)

2(15.32)

4(40)

2(22.22)

1(12.5)

3(60)

Palpitation

29(46.77)

9(33.33)

6(46.15)

5(50)

1(11.11)

5(62.5)

2(40)

Headache

14(22.58)

7(25.93)

3(23.08)

1(10)

3(33.33)

0(0)

3(60)

Fever

19(30.65)

20(74.07)

5(38.46)

4(40)

8(88.89)

2(25)

3(60)

Frequent infections

26(41.93)

17(62.96)

4(30.77)

3(30)

2(22.22)

3(37.5)

5(100)

Bleeding manifestations

27(43.55)

14(51.85)

6(46.15)

7(70)

5(55.56)

2(25)

4(80)

chronic diarrhea

10(16.13)

0(0)

1(11.11)

1(12.5)

3(60)

One benefit of a gradual decline in microfilaraemia and the depletion of endosymbionts would be the avoidance of inflammatory adverse events after the rapid destruction of parasites and release of bacterial symbionts into the blood and tissues [10, 11, 12].

Conclusion

In this study, tetracycline treatment resulted in almost complete elimination of microfilaraemia, which was sustained from at least 6 to 24 months after treatment. Advantages of antibiotic treatment are that tetracycline and other antirickettsial antibiotics are already licensed for human use and are available in endemic areas. Additionally, the drugs are cheap and have known safety and pharmacological activities.

Acknowledgements

[It should include persons who provided technical help, writing assistance and departmental head that only provided general support. Financial and material support and conflict of interests must be written in this section.]

Funding

If you have no declaration to make please insert the following statements into your manuscript: None

Competing Interests

If you have no declaration to make please insert the following statements into your manuscript: None

Reference

1. VCRC : Report of a National workshop on “Community Oriented Chemotherapy Strategies for Filariasis Control” jointly organized by Vector Control Research Centre and National Filariasis Control Programme, 17-19, February, 1999.

2. Ottesen, E.A., Ismail, M.M. and Horton: Parasitology Today, 15, 382- 386, 1999. 3. Addiss, D.G., Eberhard, M.L., Lammie, P.J. Hitch, W.L. and Harrison, C.S.: Tolerance of single high dose ivermectin for treatment of lymphatic filariasis. Transaction of the Royal Society of Tropical Medicine and Hygiene, 85, 265-266, 1991. 4. World Health Organization: Fifth Report of the Expert Committee on filariasis. Technical Report Series, 821, 1992. 5. Bandi, C., Anderson, T.J.G., Genchi, C. & Blaxter, M.L.: Phylogeny of Wolbachia in filarial nematodes. Proc R Soc Lond, B 265, 2407-2413, 1998. 6. Dobson, S.L., Bourtzis K., Braig, H.R., Jones, B.F., Zou, W., Rousett, F. and O’Neill, S.L. : Wolbachia infections are distributed through insect somatic and germ line tissues. Insect Biochemistry and Molecular Biology, 29, 153-160, 1999. 7. Hoerauf, A., Volkman, L., Hamelmann, C., Adjei, O., Autennrieth, I.B., Fleischer, B., Buttner, D.W.: Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet, 355, 12421243, 2000. 8. Langworthy, N.G., Renz, A., Mackenstedt, U., Henkle-Duhrsen, K., De Bronnsvoort, M.B., Tanya, V.N., Donnelly, M.J. and Trees, A.J.: Macrofilaricidal activity of tetracycline against the filarial nematodes Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship. Proceeding of the Royal Society of London Biological Sciences, 7, 267 (1448), 10631069, 2000. 9. Hoerauf, A., Mand, S., Fischer, K. et al: Doxycycline as a novel strategy against bancroftian filariasis-depletion

http://www.pacificejournals.com/aabs

A-41

AABS; 3(1): 2016

of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilaria production. Med Microbiol Immunol (Berl), 192, 211-16, 2003. 10. Cross, H.F., Haarbrink, M., Egerton, G., Yazdanbakhsh, M., Taylor, M.J.: Severe reactions to filarial chemotherapy and release of Wolbachia endosymbionts into blood. Lancet, 358, 1873-1875, 2001.

11. Keiser, P.B., Reynolds, S.M., Awadzi, K., Ottesen, E.A., Taylor, M.J., Nutman, T.B. : Bacterial endosymbionts of Onchocerca volvulus in the pathogenesis of posrtreatment reactions. J Infect Dis, 185, 805-811, 2002. 12. Chopra, I., Roberts, M.: Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev, 65, 232-260, 2001.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Quantification of oligo-elements and heavy metals in the fruits (pods and seeds) of the introduced tree Gleditsia triacanthos L. in Algeria Benhamiche Samia1*, Benhassaini Hachemi1, Chabane Kheira1, Romane Abderrahmane2, Arjouni Moulay Youssef2 1

Department of environmental sciences, Laboratory of vegetal biodiversity: conservation & valorization, University Djillali Liabes, Sidi Bel Abbes, Algeria 2 Department of chemistry, Laboratory of Applied Organic Chemistry, University Cadi Ayyad, Marrakech, Morocco Keywords: Gleditsia triacanthos L., oligo-elements, heavy metals, valorization.

ABSTRACT Gleditsia triacanthos L. is a moderately fast growing tree. It was used like feedstock since it can provide a source of proteins and metabolic energy. The tree was introduced in Algeria since1949 by the French colonists. In order to valorize the natural substances of this introduced species, the coupled plasma mass spectrometry (ICP-MS) was employed for the determination of essential and nonessential elements in both seeds and pods. The results showed that the quantification of heavy metals using the ICP-MS method showed that the fruits of G. triacanthos L. are very rich on potassium (178,68± 7,31mg.kg-1 for seeds and 164,27± 7,78mg.kg-1 for pods), phosphorus (75,027± 4,17 mg.kg-1 for seeds and 13,06± 0,16 mg.kg-1 for pods) and calcium (58,36± 15,66mg.kg-1 for seeds and 60,64± 4,52 mg.kg-1 for pods). The concentrations of oligo-elements and heavy metals in both seeds and pods decreased in the following order: K>P>Ca>Mg>Na>Fe>Si>Zn>Mn>Al>Cu>Cd>Pb>As>Cr. It arises that the fruits are rich in oligo-elements and the concentrations of heavy metals is lower than the normal range which confer a great interest for many industries.

*Corresponding author: Benhamiche Samia, Department of environmental sciences, Laboratory of vegetal biodiversity: conservation & valorization, University Djillali Liabes, Sidi Bel Abbes, Algeria Phone: +91 (+213)553451425 E-mail: benhamiche1981@hotmail.fr

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-43

Introduction Honey locust (G. triacanthos L.) is a woody species (genus Gleditsia) in Leguminosae family, native to North America. This species can tolerate a wide range of climatic and soil conditions [1] and is spread in America, Middle Europe and Mediterranean countries [2]. It is drought resistant, light lover, thorny and show moderate to fast growth speed [3]. It is considered by numerous authors [4] as common invader. Although, G. triacanthos L. can be competitive for the native species and can form an ecologic danger. Its seeds are composed of the testa (27%), the embryo (29%) and the endosperm (34%) [5].The pods are edible and can be used as vegetable or fermented to produce alcoholic or non-alcoholic beverage. Pods and foliage are valuable fodder for all classes of livestock [1]. The honey locust tree flowers are attractive sources of nectar for bees and, thus, good honey source. The trunk, pods, and bark are also used in many ethno-medicines [1]. There is a growing interest for the mineral content and nutrition of higher plants and their importance in agriculture, foods and Human health. Experiments in cell culture and in intact organisms reveal the importance of trace elements in many metabolic processes and functions throughout the life cycle. Human as well as animal studies originally showed that optimal intakes of elements such as sodium, potassium, magnesium, calcium, manganese, copper, zinc, and iodine could reduce individual risk factors, including those related to cardiovascular disease [6]. These heavy metal ions are also essential micronutrients for plant metabolism but when present in excess, these, and nonessential metals such as Cd, Hg, Ag and Pb, can become extremely toxic [7]. Although the efficacy of plants for curative purposes is often accounted for in terms of its organic constituents, many studies showed that intakes of mineral elements could reduce the risk factor of individuals. It is cited that the main function of iron (Fe) is to promote oxygen transport (hemoglobin), electron transport (cytochromes), muscle metabolism and immunity, manganese (Mn) is an enzyme activator: hydrolase, kinases, carboxylases and transferases and zinc (Zn) is used in immunity, endocrinology (insulin, thyroid, etc.), and antioxidant and night vision. Copper (Cu) is used in electron transfer, antioxidant, superoxide, dismutase, etc. and cobalt (Co) is a component of vitamin B12. Chromium (Cr) is a component of glucose tolerance factor, metabolism of fat. Molybdenum (Mo) is used in the metabolism of sulphur/sulphide oxidase, uric acid metabolism (xanthine oxidase). Selenium (Se) as an antioxidant: glutathione peroxidase, thyroid metabolism (deiodinase) and liver. Fluorine in the structure of enamel

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 and bone. Iodine a component of thyroid hormones, as they can play other important roles in alimentary systems, sources of macro and trace elements [8]. As in many other countries over the world, the investigation of heavy metals in herbal products is widely interested. The organic compositions and accompanied inorganic elements in several herbal products have been well determined using different advanced analytical techniques. Among them, Inductively Coupled Plasma Mass Spectrometry (ICPMS) proved to be the technique of choice to analyze and determine a large number of heavy metals in a wide range of samples. The aim of this work was carried out in order to quantify the oligo-elements and heavy metals in Gleditsia triacanthos L. fruits (seeds and pods separately) growing under semiarid conditions, using the ICP-MS technic.

Materials and Methods The pods of G. triacanthos L. were collected from the university of Djillali Liabes (ITMA) Sidi Bel Abbes during November 2014. The pods were dried in the shade during 3 months. The seeds were separated manually then crushed separately. The resulting flour was preserved in glass bottles, safe from the light for further use. The analysis of the samples was realized by metallic dosage in the obtained solution by using inductively coupled plasma mass spectrometry (Jobin-Yvon 7O ICP) ULTIMA AND JY70. A small quantity (0.5 g) of dry matter was mineralized with 2mL of sulphuric acid (H2SO4), 6mL of nitric acid (HNO3) and 6mL of oxygenated water (H2O2). This mixture was heated for 30 min. The mineral deposit was cooled and filtered by Whatmanashless filter and then supplemented to 25mL of 0.1M HNO3. All procedures of handling were carried out without contact with metals, to prevent cross-contaminations. All experiments were carried out in triplicate.

Data Analysis The results were expressed as mean Âą standard deviation (s.d.; n=3). SPSS software (version19.0) was used for the statistical analysis. One-Way Analysis of variance (ANOVA) was performed to test significant differences between seeds and pods composition. The correlations were investigated using a bivariate Pearson correlation method at P<0.05 and P<0.01. Finally the Principal Components Analysis (PCA) was carried out on the data set of oligoelements and heavy metal.

Result and Discussion Inductively coupled plasma mass spectrophotometry (ICPMS) has become a popular technic in the multi element

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-44

analysis since the first commercial instrument became available in 1980s. Semi quantitative analysis by ICPMS has proven to be a powerful tool for fast screening, in addition, it does not require the element of interest to be present in the calibration standard [9], making it especially useful for the analysis of unknown samples. In this study, the analysis of samples was carried out by using ICP-MS technic. The concentration of oligoelements and heavy metals was determined with different mode equipment to compare results in seeds and pods of G. triacanthos L. The concentrations of oligo-elements and heavy metals in both seeds and pods decreased in the following order (Tab.1): K>P>Ca>Mg>Na>Fe>Si> Zn>Mn>Al>Cu>Cd>Pb>As>Cr. The potassium is the major compound with 178.68mg.kg-1 and 164.27mg.kg-1 respectively for seeds and pods. In plants, this element is an essential macronutrient composing up to 10% of the plant dry weight [10]. It plays a vital role in a wide range of biophysical and biochemical processes. It ful-fills a number of important functions related to enzyme activation, as well as the neutralization of negative charges, the maintenance of cell turgor, plant growth and organ movement [11].

Phosphorus is the second most abundant elements in seeds (75.027mg.kg-1); succeed by Calcium, Magnesium and Sodium. Phosphorus (P) is an essential part of the process of photosynthesis and involved in the formation of all oils, sugars, starches, etc [22]. Another essential element for plants is calcium whose role has been well documented [23]. This earth alkali metal has an important role in plant physiology, including involvement in the responses to stress, and controls numerous processes and its availability is essential in the biochemistry of plants. The result (Tab.1) shows that G. triacanthos seeds contain 58.36 mg.kg-1 of calcium. Meanwhile, its pods contain 60.64mg.kg-1.

Table 1: Total concentration of oligo-elements and heavy metals in honey locust (G. triacanthos L.) compared to the normal range in plants cited by different authors.

Gleditsia seeds and pods are also very rich in magnesium, they present about 25.57mg.kg-1, 6.36mg.kg-1 respectively. Exceeding the normal range in plants cited by [19] (Tab.1). It is a part of the chlorophyll in all green plants and essential for photosynthesis. Magnesium is absorbed as the Mg2+ ion and is mobile in plants. It leaches from the soil like calcium and potassium. It serves as an activator for many enzymes required in plant growth processes and stabilizes the nucleic acids. The magnesium concentration of tissues considered as deficient, sufficient, or toxic depends on what growth parameter is being measured in the crops. In many food crops, classification of nutrient sufficiency is based on harvestable yields and quality of the edible plant parts [24].

Potassium and sodium are structurally and chemically very similar elements and exhibit homologous behavior. Both are alkali metals and belong to the s-block in the periodic table [11].

In other hand, reactions between iron and manganese are commonly observed and the ratio of these two metals in both growth medium and plant tissue seems to be more

Table 1: Total concentration of oligo-elements and heavy metals in honey locust (G. triacanthos L.) compared to the normal range in plants cited by different authors. Heavy metals

Seeds (mg.kg-1)

Pods (mg.kg-1)

Al As Ca Cd Cr Cu Fe K Mg Mn Na P Pb Si Zn

0,39± 0,27 0,047± 0,006 58,36± 15,66 0,071± 0,000 0,037± 0,011 0,09± 0,007 1,294±0,436 178,68± 7,31 25,578± 3,78 0,615± 0,036 12,06± 10,80 75,027± 4,17 0,06± 0,02 1,15± 0,67 0,63± 0,10

0,54± 0,04 0,036± 0,005 60,64± 4,52 0,072± 0,002 0,12± 0,018 0,0036± 0,000 0,79± 0,11 164,27± 7,78 6,36± 0,049 0,136± 0,004 2,25± 0,46 13,06± 0,16 0,09± 0,01 1,37± 0,31 0,12± 0,04

http://www.pacificejournals.com/aabs

Normal range in plants (mg.kg-1) 200‒≥1000 5 1830.2 – 2042.5 2 0.006 – 18 0.4 – 45.8 640 ‒ 2486 / 0.73 – 1.41 15 – 100 / / 3 / 1 – 160

Reference [12] [13] [14] [15] [16] [17] [18] / [19] [20] / / [21] / [17]

A-45 important to plant metabolism than their concentrations [17]. The result shows in Tab. 1 that iron in G. triacanthos seeds was 1.294mg.kg-1; however in pods it was 0.79mg. kg-1. Iron (Fe) is an essential micronutrient for plant growth. It is considered as the key metal in energy transformations needed for syntheses and other life processes of the cells [25]. Although iron itself is not considered toxic, it is environmentally significant because of its interaction with metals that are toxic. Iron oxides adsorb many elements and participate in the attenuation of most trace and heavy metals. Other elements like silicium, zinc, manganese, aluminum and copper are also important to plant metabolism. According to Lefaucheur [26], Silicium is one of the most abundant alkali metals in nature, it represent about 28% of the Earth’s crust and plays multiple functions in plant metabolism, structure, solidity and flexibility. It is present in seeds with 1.15mg.kg-1 and in pods with 1.37mg.kg-1. Zinc is a vital element for plant nutrition, structuration and/or enzymes catalysis (superoxide dismutase, alcohol dehydrogenase, and RNA polymerase). According to Kabata-Pendias & Pendias [17], the normal range of Zn in plants is about 1–160mg.kg-1. Our results are lower than the literature data (0.63mg.kg-1for seeds and 0.12mg. kg-1 for pods). Manganese (Mn) is an important plant micronutrient and is required by plants in the second greatest quantity compared to iron. Like any other element, it can have a limiting factor on plant growth if it is deficient or toxic in plant tissue. It is used in plants as a major contributor to various biological systems including photosynthesis, respiration, and nitrogen assimilation. This element is also involved in pollen germination, pollen tube growth, root cell elongation and resistance to root pathogens [27]. As indicated in Tab.1, seeds of G. triacanthos contain 0.615mg.kg-1 and pods contain 0.136mg.kg-1. Aluminum (Al) is the most abundant metal on the planet and the third most common element in the earth’s crust and indicated that the normal range in plants is about 200≥1000 [12]. The result shows in Tab.1 that aluminum in G. triacanthos seeds was 0.39mg.kg-1; however in pods it was 0.54mg.kg-1. In both seeds and pods it was very lower than norms. Copper is a structural and catalytic component of several proteins and enzymes involved in electron transfer and redox reactions [28]. Its presence is necessary for plant growth in low concentration. Gleditsia seeds and pods as shown in Tab. 1 contain 0.09mg.kg-1, 0.0036mg.kg-1 respectively. Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 Cadmium, lead and Chromium are considered as nonessential and toxic metals to plant growth [7]. As indicated in Tab. 1, the maximum values of Cd, Pb, and Cr were proposed by [15,16,21]. The average concentrations of these elements in honey locust seeds and pods are lower than the normal range. In general, the results have shown that accumulation of oligo-elements and heavy metals in seeds and pods of G. triacanthos L. carried out by using ICP-MS instruments did not reach phytotoxic concentrations or toxic levels (see Tab. 1 for reference values). Also, the results have shown that the accumulation of oligo-elements and heavy metals in seeds was more than in pods.

Fig. 1: comparison between oligo-elements and heavy metals concentrations in G. triacanthos L. seeds and pods.

Due to the application of one way analysis of variance (ANOVA), it was possible to record statistically significant difference between oligo-elements and heavy metals composition of G. triacanthos L. seeds and pods. On the content of these two parts of the fruit; there was statistically significant influence on the concentration of P, Mg, Ca, K, Na, Zn, Cr and Mn (α < 0.001) and Cu (α < 0.05). Also, statistically significant correlations (α < 0.05 and α < 0.01) between concentrations of the metals studied in G. triacanthos fruits (seeds and pods separately) revealed significant positive correlations (Tab.2). Significant positive relationships (α < 0.01) were observed inter alia for the following assemblages: Al-Ca-Si, Cu-Mg-Mn-PZn, Fe-Na, Mg-Mn-P-Zn, and P-Zn. Table 2: Single tailed Pearson Correlation of oligoelements and heavy metals in G. triacanthos L. fruits (seeds and pods) In order to confirm the obtained results by ANOVA and Spearman correlations, we have used the principal components analysis (PCA) to compare between Gleditsia seeds and pods mineralogical profiles. In PCA represented in rotated space (Fig.2), the first component F1 explained e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-46

Table 2: Single tailed Pearson Correlation of oligo-elements and heavy metals in G. triacanthos L. fruits (seeds and pods) Ca

-0,570

0,930**

-0,428

0,267

-0,621

0,300

0,375

-0,776

0,113

0,552

-0,450

0,832*

-0,153

-0,333

-0,935**

0,356

0,623

-0,064

-0,658

0,671

-0,456

0,874*

-0,235

-0,586

-0,751

0,801

0,430

-0,217

0,734

0,090

-0,324

-0,943**

0,966**

0,829*

0,739

-0,362

0,790

-0,055

-0,324

-0,947**

0,994**

0,739

0,776

0,988**

0,445

0,294

0,680

-0,172

-0,641

0,587

0,980**

0,387

0,774

0,666

-0,396

0,809

-0,093

-0,333

-0,946**

0,998**

0,714

0,788

0,981**

0,999**

0,637

0,802

-0,715

0,536

0,160

0,671

-0,816*

-0,204

-0,752

-0,661

-0,764

-0,078

-0,783

0,952**

-0,498

0,951**

0,437

0,276

-0,277

0,511

-0,428

-0,052

-0,190

0,548

-0,224

0,653

-0,416

0,838*

-0,111

-0,302

-0,891*

0,988**

0,685

0,853*

0,953**

0,980**

0,592

0,984**

-0,825*

-0,245

**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).

about 61.11% of the total variance, the second component F2 about 28.26% and the third component F3 about 7.25%. Total contribution from these components is 96.63% of the total variation. As can be observed on Fig.1, we have obtained 4 groups: G1 (Mg-Mn-As-K-Cu), G2 (Na-Fe), G3 (Al-Ca-Si), G4 (Pb-Cr-Cd).

Fig. 2: PCA of G. triacanthos L. seeds and pods according to the mineral composition.

The delivery of metals to seeds depends upon uptake by the mother plant, and subsequent transport to developing seeds. Due to the potential toxicity of excess levels of metals, their uptake and distribution and the intracellular concentration must be carefully regulated. Metals are normally found at the highest concentrations in the roots, and at the lowest concentrations in the reproductive

tissues [29]. This is because metals are sequestered into the vacuoles of root and shoot tissue and the subsequent availability of free metals in the symplast can be low. For example, concentrations of Cd and Ni in soybean were 30 and 20 times, respectively, higher in roots than in leaves. Cadmium concentration was lowest in the seeds, whereas the concentration of Ni was the same in both leaves and seeds [29]. The environmental factors and the type of plant influence the bioaccumulation of heavy metals. Thus, the concentration of essential and non-essential heavy metals in medicinal plants beyond permissible limit is a matter of great concern to public safety all over the world. An assessment of heavy metals tolerance should be based upon a comprehensive analysis of the interaction between the accumulation of heavy metals in plants and the metalâ&#x20AC;&#x2122;s status in soils [25]. The maximum values for heavy metals in herbal drugs and extracts have been discussed by several authors. In 1998, Kabelitz [30] published a detailed evaluation of a database on heavy metals, which included more than 12Â 000 samples originating from quality control analyses by several pharmaceutical companies.

Conclusion The seeds and pods of G. triacanthos L. are very rich in basic elements like potassium, calcium and magnesium and at the same time, the concentrations of heavy metals like chromium, lead and cadmium which are considered as toxic elements are very lower than the normal range in plants cited by different authors. However, we note that seeds are rich than pods in potassium, phosphorus, magnesium and

http://www.pacificejournals.com/aabs

A-47

AABS; 3(1): 2016

sodium. The obtained results remain so equilibrated which militate in favor of the use of honey locust fruits (seeds and the pods) in many industries like the production of animal feeds and in the field of the cosmetic.

5. Sciarini LS, Maldonado F, Ribotta PD, Pérez GT, León AE. (2009) Chemical composition and functional properties of Gleditsia triacanthos gum. Journal of Food Hydrocolloids,23, 306-313.

In other hand, G. triacanthos L. well known as an invading and competing the autochthones species, it is very important for us to valorise its co- products especially in developing countries.

6. Sanchez-Castillo CP, Dewey PJS, Aguirre A, Lara JJ, Vaca R, De La Barra PL, Ortiz M, Escamilla I, James WPT. (1998) The Mineral Content Of Mexican Fruits And Vegetables. Journal of food composition and analysis,11, 340–356.

Acknowledgements [It should include persons who provided technical help, writing assistance and departmental head that only provided general support. Financial and material support and conflict of interests must be written in this section.] This work was realized in behalf with the Laboratory of Applied Organic Chemistry, Faculty of Sciences Semlalia, University Cadi Ayyad Marrakech (Morocco). The author gratefully acknowledges Pr.Romane A. A. and Dr.Arjouni M. Y. for their collaboration and their help to realize the oligo-elements and heavy metals analysis and statistical data.

Funding If you have no declaration to make please insert the following statements into your manuscript:

7. Williams LE, Pittman JK, Hall JL. (2000) Emerging mechanisms for heavy metal transport in plants. Journal of Biochimica et Biophysica Acta, 1465, 104126. 8. Bennouna MA, Belaqziz R, Arjouni MY, Romane A. (2012) Quantitative analysis of some oligo-elements and heavy metals in some species of Thymus from Morocco. Journal of Natural Product Research, DOI:1 0.1080/14786419.2012.751597. 9. Woods G, McCurdy E, Wilbur S. (2004) Interferencefree semiquantitative analysis using the 3 Agilent 7500ce ICP-MS. Agilent Application Note, 59891492.

Competing Interests

10. Leigh RA, Wyn Jones RG. (1984) A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell. Journal of New Phytol., 97, 1–13.

If you have no declaration to make please insert the following statements into your manuscript: None

11. Benito B, Haro R, Amtmann A, Cuin TA, Dreyer I. (2014) The twins K+ and Na+ in plants. Journal of Plant Physiol.,171, 723–31.

Reference

12. Jansen S, Broadley MR, Robbrecht E, Smets E. (2002) Aluminum hyperaccumulation in agiosperms: A review of its phylogenetic significance. Journal of Bot. Rev.,68, 235–269.

None

1. Blair RM.1990.Gleditsia triacanthos L. Honey locust. In: Hardwoods Agriculture Handbook. Vol2, Tech. Coords. Silvics.of North America, Washington DC, USDA Forest Service, 358-364. 2. Üner M, Altınkurt T. (2004) Evaluation of honey locust (Gleditsia triacanthos Linn.) gum as sustaining material in tablet dosage forms. Journal of IL FARMACO, 59, 567-573. 3. Danso SK, Zapata F, wonaike AK. (1995) Measurement of biological N2 fixation in field-grown Robinia pseudo acacia L. Journal of Soil Biology and Biochemistry,27. 4. Speroni FC, De Viana ML. (2001) Community characteristics in a mountain forest invaded by Gleditsia triacanthos L. In: Plant invasions: Species ecology and ecosystem management, ed. Backhuys Publishers. Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

13. British Herbal Medicine Association, British Herbal Pharmacopoeia. (1996). 14. Xu QS, Hu JZ, Xie, KB., Yang HY, Du KH, Shi GX.(2010) Accumulation and acute toxicity of silver in Potamogeton crispus L. Journal of Hazardous Materials,173,86–193. 15. Codex Alimentarius. (2001a) Codex maximum levels for Cadmium in Cereals, Pulses and Legumes, Joint FAO/WHO Standards, CAC/GL39-2001. http// www.codexalimentarius. net/standards_search.asp. (Accessed March 2004). 16. Zayed A, Terry N. (2003) Chromium in the environment: factors affecting biological remediation. Journal of Plant Soil, 249, 139–56.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-48

17. Kabata-Pendias A, Pendias H. (1984) Trace Elements in Soils and Plants.CRC Press, Boca Raton, Florida. 18. Lavilla I, Filgueiras AV, Bendicho C. (1999) Comparison of Digestion Methods for Determination of Trace and Minor Metals in Plant Samples. Journal of Agric. Food Chem.,47(12), 5072-5077. 19. Witkowski ETF, Lamont BB. (1996) Disproportionate allocation of mineral nutrients and carbon between vegetative and reproductive structures in Banksia hookeriana. Journal of Oecologia,105, 38–42. 20. Misra SG, Mani D. (1991) Soil pollution. Ashish Publishing House, Punjabi Bagh. 21. Codex Alimentarius. (2001b) Maximum levels for Lead, Joint FAO/WHO Standards, Codex STAN 2302001, http//www.codexalimentarius.net/standards_ search.asp (Accessed March 2004). 22. O’Relly SE, Sims JT. (1995) Phosphorus adsorption and desorption in a sandy soil amended with high rates of coal fly ash. Journal of Commun Soil Sci. and plant anal., 26(17-18), 2983-2993. 23. White PJ, Broadley MR. (2003) Calcium in plants. Journal of Ann. Botanical, 92, 487–511.

24. Walworth JL, Ceccotti S. (1990) A re-examination of optimum foliar magnesium levels in corn. Jonal of Commun.Soil Sci. Plant Ana., 21(13–16), 1457–1473. 25. Erna WatiIbnuHajar, Ahmad Ziad Bin Sulaiman, Mimi Sakinah. (2014) Assessment of Heavy Metals Tolerance in Leaves, Stems and Flowers of Stevia rebaudianaPlant. Journal of Procedia Environmental Sciences,20, 386 – 393. 26. Lefaucheur L. (1988) Forms chimiques et mécanismes d’absorption du silicium par les plantes. Thèse Doc. Phys. Biol. Org. Pop.: Montpellier. 27. Schäfer U. 2004. Manganèse. In: Elements and their Compounds in the Environment. 2nd edition. Wiley-VCH Weinheim (D). Vol2, Metals and their Compounds, 901-930. 28. Marschner H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London. 29. Malan HL, Farrant JM. (1998) Effects of the metal pollutants cadmium and nickel on soybean seed development. Journal of Seed Sci. Res.,8, 445–453. 30. Kabelitz L. (1998) Heavy metals in herbal drugs (Zur Schwer metall belastung von Arznei- und Kräuter drogen). Journal of Pharm. Ind.,60, 444-5.

http://www.pacificejournals.com/aabs

Original Article Pros And Cons Of Different Methods Of Leucoreduction And Its Scope Of Implementation In The Cost Constrained Settings Ankita A Katara1*, Abhishek Pandey2, Jagdish D Dalsania1, Rohit R Bhalara1, Milan Purohit1, Sanjay Talwelkar1 1

Dept. of Pathology, P.D.U. Govt. Medical College Rajkot, Gujarat (India) Dept. of Orthopedics, Era’s Lucknow Medical College, Lucknow (India)

Keywords: Leucoreduction, Leucofilters, Alloimmunisation, Apheresis

ABSTRACT Half a century ago, most of the blood transfused was whole blood. However, since the 1960s, whole blood has been separated into its various components such as RBCs, platelets and plasma. Rapidly growing size of patients in our country requiring multiple transfusion of blood and components pose a great challenge to transfusion services to provide them red cell and platelet antigen matched products in alloimmunized subjects. However, it has been shown that the removal of leucocytes from various blood products can minimize the risks[1–5] associated with these , which are: Nonhemolytic febrile transfusion reactions, Human leukocyte antigen (HLA) alloimmunization , Platelet refractoriness observed in multi-transfused patients and Transmission of leucotropic viruses such as EBV and CMV. Thus removal of leucocytes below a certain threshold, ≤ 5 × 106 in a blood component certainly helps in prevention of associated risks in these patients. Currently, the best Leucoreduction can be achieved with the help of 3rd generation leucofilters, both in laboratory and patient bed side and apheresis devices.We did a 6 month comparision study of transfusing leucodepleted RCC (Red Cell Concentrate) in Thalassaemia patients prepared by both the methods of leucodepletion ( manual buffy coat removal and leucofiltration) in P.D.U. Medical College and Hospital, Rajkot ,Gujarat ( India) and assesed the effectiveness of both in preventing NHFTRs in multiply transfused patients ,especially in cost contrained settings. Although the terms, leucoreduction and leucodepletion are used interchangeably in literature, Leucoreduction technically implies removal of leukocytes by gross removal method, whereas, Leucodepletion connotes removal of leukocytes with the help of certain specific filters or devices.

*Corresponding author: Ankita A Katara , Shekhar Hospital, B-Block, Church Road, Indira Nagar , Lucknow, U.P.-226016 Phone: +91 9198616408 E-mail: dr.ankitakatara@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-50

Introduction Half a century ago, most of the blood transfused was whole blood. However, since the 1960s, whole blood has been separated into its various components such as RBCs, platelets and plasma. Rapidly growing size of patients in our country requiring multiple transfusion of blood and components pose a great challenge to transfusion services to provide them red cell and platelet antigen matched products in alloimmunized subjects. However, it has been shown that the removal of leucocytes from various blood products can minimize the risks[1–5] associated with these , which are: Nonhemolytic febrile transfusion reactions, Human leukocyte antigen (HLA) alloimmunization , Platelet refractoriness observed in multi-transfused patients and Transmission of leucotropic viruses such as EBV and CMV. Thus removal of leucocytes below a certain threshold, ≤ 5 × 106 in a blood component certainly helps in prevention of associated risks in these patients. Currently, the best Leucoreduction can be achieved with the help of 3rd generation leucofilters, both in laboratory and patient bed side and apheresis devices.We did a 6 month comparision study of transfusing leucodepleted RCC (Red Cell Concentrate) in Thalassaemia patients prepared by both the methods of leucodepletion ( manual buffy coat removal and leucofiltration) in P.D.U. Medical College and Hospital, Rajkot ,Gujarat ( India) and assesed the effectiveness of both in preventing NHFTRs in multiply transfused patients ,especially in cost contrained settings. Although the terms, leucoreduction and leucodepletion are used interchangeably in literature, Leucoreduction technically implies removal of leukocytes by gross removal method, whereas, Leucodepletion connotes removal of leukocytes with the help of certain specific filters or devices.

Materials and Methods

The original leucocyte depletion filter contained sterile cotton wool as a filtering agent and was designed by Diepenhorst who published his work in 1972.[6] Subsequently cellulose acetate filters were discovered and found to be more suitable. Other methods included red cell washing, centrifugation and buffy coat removal, freezing and deglycerolization of red cells and blood component collection through apheresis technology.[7] Of all these methods, leucoreduction by leucofilters (third generation) and components collected through apheresis devices meet the current standards of leucocyte depletion, that is < 5 × 106 WBC/unit of blood component,[8] whereas, other methods achieve leucocyte depletion to a variable extent, as follows: i. Centrifugation and buffy coat removal — 1-2 log leucodepletion

ii. Filtration- 3-4 log leucodepletion iii. Washed red cell concentrate — 1-2 log leucodepletion iv. Frozen deglycerolized red cells — 2-3 log leucodepletion Centrifugation and Buffy Coat Removal This is one of the easiest and least expensive methods and it can be done in closed system. This causes 1-2 log reduction, but sacrifices RBCs upto 20%. Leucofiltration Current generation of leucofilters (third and fourth) have excellent leucocyte removal efficiency (99.99%) as compared to the first and second generation filters (9096%). Presently we have depth and screen-type filters. Depth filter (non woven) has filter material in the form of compressed wool fibers arranged in an irregular fashion, whereas, screen filters (woven type) have fibers arranged in multiple layers in a regular fashion. The primary mechanism [9] of leukocyte removal is the charge-based adhesion of negatively charged leukocytes to the filter material by Vander Waals and electrostatic forces. Timing of leucofiltration Leucofiltration of blood components can be done either at the time of collection and processing, post processing (within the blood bank), or by the side of the patient (post storage). Each of them has their own advantages and disadvantages. However, pre-storage leukoreduction is currently the most widely accepted mode. The advantages of pre-storage over post-storage leucoreduction are as follows: It eliminates the scope of inflammatory cytokine (interleukin-1, interleukin-6, tumor necrosis factor) accumulation due to leucocytes, during storage, and hence, is quite efficient in the prevention of febrile non-hemolytic transfusion reactions.[10-12] It also minimizes the risk of HLA-alloimmunization in multitransfused patients, as it removes the intact leucocytes.[13,14] Pre-storage leucofiltration can also minimize the risk of leucotropic virus transmission as leucocytes disintegrate and release the intracellular organisms after 72 hours of storage in blood components.[15,16] It is always easier to perform leucocyte quality control in the laboratory rather than by the patient’s bedside. Hence during pre-storage leucoreduction, blood components can be thoroughly studied and evaluated for quality control, and various factors affecting the process of leucofiltration modified accordingly.[17,18] At present these factors favour pre-storage leucoreduction, either in the form of universal leucoreduction for all the

http://www.pacificejournals.com/aabs

A-51

AABS; 3(1): 2016

patients or as a selective protocol for a special group of patients. The major drawback with universal leucoreduction is the cost involved; however, selective leucoreduction has its inherent issues of inventory management, as it is quite difficult to predict the requirement of leucoreduced blood components at the time of component preparation. Washing red cells This will achieve a 2 log removal, provided the Buffy coat is removed before or during the washing procedure. This product is generally not recommended as it is more expensive and less effective than LDF(Leucodepletion by Filters) and the lifespan of the product is very short. Freezing and deglycerolizing red cells The level of leucocyte depletion approximates that of LDF(Leucodepletion by Filters). This process is very expensive and is not recommended for the routine preparation of a leucocyte depleted product. Use of this product should be reserved for patients for whom long term storage is required either because they have a rare blood type or have multiple antibodies Leucacytapheresis Blood components collected with the help of the current generation low leucacytapheresis devices are generally 3 logs reduced and hence require no further filtration to meet the standards of leucoreduced blood components. Currently Accepted Clinical Indications Leucoreduced Blood Components[19] Proven benefits Reduced frequency and severity of FNHTRs Reduced risk of CMV transmission

Probably clinically relevant Reduced infectious risk associated with immunomodulation (TRIM) Reduced organ dysfunction and mortality

for

Unproven clinically Avoidance of vCJD transmission Avoidance of HTLV I/II, EBV etc.

Reduced risk of HLAalloimmunization and platelet refractoriness

Reduced direct risk of transfusiontransmissible bacteria

Reduced risk of GVHD

Reduced risk of TRALI

Current accepted standards for leucodepleted blood components American Association of Blood Banks (USA) European Council criteria Director General of Health Services (India) criteria

RED CELL CONCENTRATE <5 × 106 WBC/Unit (red cell loss not more than 15%) <1 × 106 WBC/Unit <5 × 106 WBC/unit (red cell loss not more than 10%)

Result We collected monthwise data of transfusion of RCC( Red Cell Concentrate) to Thalassaemia patients [ TABLE-1] and the number of blood transfusion reactions noted among them [TABLE-2] at P.D.U. Medical College and Hospital, Rajkot ,Gujarat ( India). 92.5% of the RCCs issued were prepared by Manual and Buffy Coat Removal following standard protocols and 7.5%RCC were prepared by using 3rd generation leucofilters[ TABLE-1]. Total 3 Blood Transfusion Reactions were noted in the period of 6 months ,out of that 2 (66.7%)reactions ocurred in thalassaemia patients who were given RCC prepared by Centrifugation and Buffy Coat Removal method of leucodepletion and 1 (33.3%)reaction was noted in patient who was transfused with RCC prepared by leucofiltration method of leucodepletion[TABLE-2].

Discussion

Though, today many new more efficient techniques have arrived , but if this method of leucoreduction ( i.e. centrifugation and manual buffy coat removal ) if performed under standard protocols and with expertise , reduction of leucocytes by approximately 2 log can be

TABLE 1: Month-wise Data of RCC(Red Cell Concentrate) Issued to Thalassaemia Patients Leucodepleted RCC prepared by centrifugation and manual buffy coat removal

Leucodepleted RCC prepared by using 3rd generation filters

SEPTEMBER 2013

232

OCTOBER 2013

252

NOVEMBER 2013

223

DECEMBER 2013

220

JANUARY 2014

264

FEBRUARY 2014

377

TOTAL

1568 (92.5%)

126 (7.5%)

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-52

TABLE 2: No. of Blood Transfusion Reactions Occured by Different Methods of Leucodepletion No. of Blood Transfusion Reactions occured by Leucodepleted RCC prepared by centrifugation and manual buffy coat removal

No. of Blood Transfusion Reactions occured by leucodepleted RCC prepared by using 3rd generation filters

SEPTEMBER 2013

OCTOBER 2013

NOVEMBER 2013

DECEMBER 2013

JANUARY 2014

FEBRUARY 2014

TOTAL

02(66.7%)

01(33.3%)

achieved easily and may be sufficient to prevent most febrile nonhaemolytic transfusion reactions( NHFTRs) in patients who have previously experienced these reactions(20). Rather, it will be cost effective also especially for the government set ups, where it is practically difficult to do leucodepletion by using filters for masses.

Funding

My institute (P.D.U. Medical College and Hospital , Rajkot , Gujarat), follow standard protocols regarding component separation ,through which we have been able to achieve upto 2 log reduction of leucocytes. These protocols are as follows,

1. CA, Dutcher JP, Aisner J, Hogge D, Wiernik PH, Reilly JP. A randomized trial of leukocyte-depleted platelet transfusion to modify alloimmunization in with leukemia. Blood. 1983;62:815–20. 2. Sniecinski I, O’ Donnel MR, Nowicki B, Hill LR. Prevention of refractoriness and HLAalloimmunization using filtered blood products. Blood. 1988;71:1402–7. 3. Andreu G, Dewailly J, Leberre C, Quarre MC, Bidet ML, Tardivel R, et al. Prevention of HLA immunization with leukocyte-poor packed red cells and platelet concentrates obtained by filtration. Blood. 1988;72:964–69. 4. Yazer MH, Podlosky L, Clarke G, Nahirniak SM. The effect of Prestorage WBC reduction on the rates of febrile nonhemolytic transfusion reaction to platelet concentrates and RBC. Transfusion. 2004;44:10–5. 5. Reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusion. The Trial to Reduce Alloimmunization to platelet Study Group. N Engl J Med. 1997;337:1861–9. 6. orst P, Engelfriet CP. Removal of leucocytes from whole blood and erythrocytes suspension by filterations through cotton wool. V, Results after transfusion of, units of filtered erythrocytes. Vox Sang. 1975;29:15–8 7. VM, van Doorn R, Witvliet MD, Claas FH, Brand A, et al. Occurrence of allogenic HLA and non HLA antibodies after transfusions of Prestorage filtered platelets and red blood cells: A Prospective study.

–

All components are prepared with in 24 hrs. of blood collection, even if it is plasma too. – FFP (Fresh Frozen Plasma) and PC (Platelet Concentrate) is prepared within 6hrs of blood collection. – Plasma is prepared within 24hrs of blood collection. – All the procedures are done under strict aseptic conditions and optimal temperature control. So, RCC prepared is free from WBC fragments and cytokines release which have been mentioned as major culprits for NHFTRs (Non Hemolytic Febrile Transfusion Reactions) in multiple transfused patients.

Conclusion Removing the buffy coat from red cells at source results in approximately a 2 log removal of leucocytes and may be sufficient to prevent most febrile nonhaemolytic transfusion reactions in multiply transfused patients and who have previously experienced these reactions and specially in cost constrained settings.

Acknowledgements I am highly indebted to all my wonderful teachers, colleagues and last but not the least my Head of the Department for their guidance, constant supervision and immense support in completing the project.

None

Competing Interests Not declared

REFERENCE

Blood. 1995;85:1736–41

http://www.pacificejournals.com/aabs

A-53

AABS; 3(1): 2016

8. Laforet M, Isola H, Cazenave JP. Leucodepletion of platelet concentrates and plasma collected with haemonetics MCS + apheresis system. Experience of EFS- Alsace. Transfus Apheresis Sci. 2002;25:55–9. 9. Dzik S. Leukodepletion blood filters: Filter design and mechanisms of leukocyte removal. Transfusion Med Rev. 1993;7:65–77. 10. N M, Blajchman MA, Meyer RM, Lipton JH, Walker IR, Sher GD, et al. A randomized controlled trial comparing the frequency of acute reactions to plasma -removed platelets and Prestorage WBC- reduced platelets. Transfusion. 2002;42:556–66. 11. Muylle L, Joos M, Wouters E, De Bock R, Peetermans ME. Increased tumor necrosis factor alpha (TNF alpha), interleukin 1, and interleukin 6 (IL-6) levels in the plasma of stored platelet concentrates: Relationship between TNF alpha and IL-6 levels and febrile transfusion reactions. Transfusion. 1993;33:195–9. 12. Rebulla P, Dzik WH. Multicenter evaluation of methods for counting residual white cells in leukocyte-depleted red blood cells. Vox Sanguinis. 1994;66:25–32. 13. on LM, Wimperis JZ, Williamson P, Copplestone JA, Gooi HC, Morgenstern GR, et al. Besides filtration of blood products in the prevention of HLA alloimmunization-a prospective randomized study. Blood. 1994;83:3028–35. 14. Aas K, van Marwijk Kooij M, van Prooijen HC, van Dijk BA, van Putten WL, Claas FH, et al. Leukocyte depletion of random single- donor platelet transfusions

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

15.

16.

17.

18.

19.

20.

does not prevent secondary human leukocyte antigenalloimmunization and refractoriness: A randomized prospective study. Blood. 1995;85:824–8. Dzik WH, Anderson JK, O’Neill EM, Assmann SF, Kalish LA, Stowell CP. A prospective, randomized clinical trial of universal WBC reduction. Transfusion. 2002;42:1114–22. JC, Pomper GJ, Fisch GS, Champion MH, Snyder EL. Reduction of febrile but not allergic reactions to red cells and platelets following conversion to universal Prestorage leukoreduction. Transfusion. 2004;44:16– 24. E, Shirey RS, Thoman SK, Bensen-Kennedy D, Tanz WS, Ness PM. Universal leukoreduction decreases the incidence of febrile nonhemolytic reactions to red cells. Transfusion. 2004;44:25–9. En L, Taaning E, Mynster T, Hvolris J, Drachman O, Nielsen HJ. Bioactive substancse in buffy-coat-derived platelet pools stored in platelet-additive solutions. Br J Haematol. 1998;103:445–8. Blajchman MA. The Clinical Benefit of the Leukoreduction of Blood products. J Trauma. 2006;60:S83–90. Rupankar Sanyal.Advantages of Leukodepleted Concentrated Red Blood Corpuscles (Packed Cell). 2013(Mollison’s Blood Transfusion in Clinical Medicine, Rossi’s Principles of Transfusion Medicine, Essential Guide to Blood Grouping Geoff Daniels, Imelda Bromilow, AABB, Reports of Australian Blood Bank Services)

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Role of Complements and Immunoglobulins in Type 1 Diabetes Mellitus Shailja Singh1*, Usha2, N K Agrawal3, R G Singh4 1

Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India 2 UGC Advanced Immunodiagnostic Training & Research Centre, Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India 3 Department of Endocrinology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India 4 Department of Nephrology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India Keywords: Diabetes Mellitus, Immunoglobulin, Complement.

ABSTRACT Background: The aim of the present study was to determine the role of complements and immunoglobulins in normal healthy population and patients having Type 1 diabetes mellitus (T1DM). Methods: A total of 26 patients with T1DM and 20 healthy controls were included in the study. The sample was collected from OPD of Department of Endocrinology, SS Hospital, IMS, BHU, Varanasi. Immunoglobulins and complements estimation was done by turbidometry method. Result: A total of 26 patients with T1DM between 8 yrs to 33 yrs age were studied. Out of 26 cases, 19.2% cases had low C4 and 3.9% had elevated serum C4 value. Mean value of serum C4 was significantly reduced in T1DM. The patients having low C4 value belonged to the age group less than 18 years. About 23.1% DM patient had increased serum IgM levels and 7.7% had increased serum IgA levels. But only rise of IgM was statistically significant. C3 and IgG in DM patients were within normal range. There was significant positive correlation of serum C4 with increase BMI. IgA was significantly correlated with rising of BMI and low age of onset of T1DM. Conclusion: Our study concludes that C4 deficiency associated with Type I DM. The study also showed that increased IgM which may be due to recent infection.

*Corresponding author: Shailja Singh, C/O Prof. Usha, Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India Phone: +91 9415455259 E-mail: shailjabhu@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-55

AABS; 3(1): 2016

Introduction Type I Diabetes Mellitus (T1DM) is one of the most common chronic diseases in childhood. The incidence of T1DM in India is 3.0 per 100000 populations (1). It is divided into two subtypes, i.e. autoimmune type-1 (type1A or T1A) diabetes and idiopathic (type-1B or T1B) diabetes (2). Several types of autoantibody to pancreatic islet cells have been recognized as markers of T1A diabetes. These islet-related autoantibodies include islet cell antibodies (ICA), anti-GAD antibodies (GADAb), insulin autoantibodies (IAA), and anti-insulinomaassociated antigen 2 antibodies (IA-2Ab). Patients with T1B diabetes who do not have islet autoantibodies at the time of diagnosis are classified as having idiopathic. Prevalence of T1DM is highest in Finland 57.6 per 100000 population. Contrary to this incidence of TI DM in China and India is 0.6 and 3.0 per 100000 population (3, 4). The incidence of T1DM is increasing, either because of environmental factors accelerating onset of the disease or because of inducement of autoimmune diabetes. A major environmental risk factor disclosed by epidemiological studies is infection in the development of type-1 diabetes (5, 6, 7, 8, 9). The DM patients are very sensitive for infection. The mortality rate of DM patients with an infection and ketoacidosis is 43% (10, 11). Gillani, et al. (2011) conducted the study in Penang, Malaysia and found that every third patient with diabetes mellitus had infectious exposure (11). Although the mechanism of this increased incidence is less well established, studies of the immune cells of DM patients have demonstrated significant defects in both humoral and cellular function (12, 13). Immunoglobulins mediated humoral adaptive immune system and provide protection against infections. Hence, the aim of the present study was to determine the complements and immunoglobulins in normal healthy population and patients with T1DM and also to identify their deficiency and elevation related to the GAD positivity.

Materials and Methods A total of 26 patients with T1DM and 20 healthy controls were included in the study. The sample was collected from OPD of Department of Endocrinology, SS Hospital, IMS, BHU, Varanasi. •

Characteristics of the patients:

Males: Females

Patients (26)

Control (20)

17: 9

12:8

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

Age (years)*

Patients (26)

Control (20)

21.31±6.33

31.45±11.02

Diabetes Mellitus (DM): T1DM Body Mass Index (BMI) (kg/m2)

17.77±3.40 (10.42-23.78)

Disease Duration (Years)

2.06± 3.23 (0.08- 16)

Age at onset (Years)

19.25± 6.37 (6- 30.92)

*Results expressed as mean ± SD

Laboratory Analysis Immunoglobulins and complements estimation was done by turbidometry method. The kit was supplied by Spinrect, Spain through Avadh Scientific, Lucknow. Glutamic acid decarboxylase (GAD) estimation was done by indirect enzyme linked immunosorbent assay (ELISA). The kit was supplied by Medyzyn, through Vishat Diagnostic , Lucknow. Data Analysis All data were analyzed using Statistical Package for Social Sciences (SPSS, Chicago, Illinos, USA version 16),. Statistical tests such as, Student ‘t test’ and one way ANOVA, were done to compare the mean values of different groups. Bivariate correlation was done to compare the multiple groups. P values less than 0.05 was taken as significant for the analysis.

Result

The mean value of complement C4 was significantly decreased in T1DM as compared to controls (17.47±11.59 vs 23.67±5.70 mg/dl; P 0.0336). Although only 19.2% T1DM patient had decreased C4 value as compared to control (5%) (Table-I). All reduced cases of C4 were observed in below 18 years of age (Table-II). IgG in T1DM patients was within normal range (Table-II) and there was no association with age of the patient (TableIV). The mean value of IgM was significantly increased in T1DM patients as compared to controls. Out of 26 cases, 23.1% had increased serum IgM levels and all controls were within normal range (Table V). Correlation between age of T1DM patients and IgM showed that elevated value of IgM was varied in various age group, about 11.11% in below 18 years; 33.33% in between 18-25 years and 20% in patients age greater than 25 years (Table-VI). The mean value of IgA was within normal in majority of cases (92.3%) (Table VII). Correlation between age of T1DM patients with serum IgA showed no significant change (Table-VIII).

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-56

Significant positive correlation was observed among C4 and IgA with increased BMI and serum IgA with age of onset of disease. There was no correlation among IgG and IgM with age of onset, family history, disease duration

and BMI (Table-IX). Mean values of complement and immunoglobulins was decreased in GAD positive patients but only IgA reduction was statistically significant in GAD positive patienrs from GAD negative cases (Table-X).

Table I: Serum C4 level in patients with T1DM and control group Groups

<10 mg/dl No (%)

10-40 mg/dl No (%)

>40 mg/dl No (%)

Mean ± SD mg dl

p-value 0.0336

T1DM (26)

5(19.2)

20(76.9)

1 (3.9)

17.47±11.59

Control (20)

1(5.0)

19(95.0)

0(0.0)

23.67±5.70

Table II: Correlation of Age of T1DM patients and C4 level. Age of the patients

Total (26) No (%)

<10 mg/dl No (%)

10-40 mg/dl No (%)

>40 mg/dl No (%)

Mean±SD mg/dl

<18

5 (55.56)

4 (44.44)

0 (0.0)

10.92±8.89

18-25

0 (0.0)

11 (91.67)

1(8.33)

24.38±12.31

>25

0 (0.0)

5 (100)

0 (0.0)

13.73±4.19

p-value

0.014*

*statistically significant (p<0.05)

Table III: Serum IgG level in patients with T1DM and control group Groups

<600 mg/dl No (%)

600-1600 mg/dl No (%)

>1600 mg/dl No (%)

Mean±SD mg/dl

p-value 0.215

T1DM (26)

0 (0.0)

26 (100)

0 (0.0)

1176.30±206.67

Control (20)

0 (0.0)

20 (100)

0 (0.0)

1216.20±178.21

Table IV: Correlation of Age of T1DM patients and Serum IgG level. Groups

Total (26) No (%)

<700 mg/dl No (%)

600-1600 mg/dl >1600 mg/dl No (%) No (%)

Mean±SD mg/dl

<18

0 (0.0)

9(100)

0 (0.0)

998.43±371.79

18-25

0 (0.0)

12(100)

0 (0.0)

1221.15±168.67

>25

0 (0.0)

5(100)

0 (0.0)

1137.66±205.46

p-value

0.180

*statistically significant (p<0.05)

Table V: Serum IgM level in patients with T1DM and control group Groups

<40 mg/dl No (%)

40-230 mg/dl No (%)

>230 mg/dl No (%)

Mean±SD mg/dl

p-value 0.036*

T1DM (26)

0 (0.0)

20 (76.9)

6 (23.1)

174.59±66.15

Control (20)

0 (0.0)

20 (100.0)

0 (0.0)

135.02±55.25

*statistically significant (p<0.05)

Table VI: Correlation of Age of T1DM patients and Serum IgM level. Groups

Total (26) No (%)

<40 mg/dl No (%)

40-230 mg/dl No (%)

>230 mg/dl No (%)

Mean±SD mg/dl

<18

0 (0.0)

8 (88.89)

1 (11.11)

175.39±53.65

18-25

0 (0.0)

8 (66.67)

4 (33.33)

180.91±75.80

>25

0 (0.0)

4 (80)

1(20.0)

158.01±72.96

p-value

0.821

http://www.pacificejournals.com/aabs

A-57

AABS; 3(1): 2016

Table VII: Serum IgA level in patients with T1DM and control group Groups

<70 mg/dl No (%)

70-400 mg/dl No (%)

>400 mg/dl No (%)

Mean±SD mg/dl

p-value 0.201

T1DM (26)

0 (0.0)

24 (92.3)

2 (7.7)

255.39±105.65

Control (20)

0 (0.0)

20 (100)

0 (0.0)

216.55±93.71

Table VIII: Correlation of Age of T1DM patients and Serum IgA level. Groups

Total (26) No (%)

<70 mg/dl No (%)

70-400 mg/dl No (%)

>400 mg/dl No (%)

Mean±SD mg/dl

<18

0 (0.0)

9 (100)

0 (0.0)

204.99±70.47

18-25

0 (0.0)

11 (91.67)

1 (8.33)

264.22±120.79

>25

0 (0.0)

4 (80.0)

1(20.0)

324.95±87.80

p-value

0.114

Table IX : Correlation of complement and immunoglobulins with clinical detail of T1DM

C4 IgG IgM

IgA

BMI

Disease duration

Age of onset

Family history

0.404*

0.057

0.007

0.067

.040*

.781

.972

0.747

0.039

-0.006

-0.197

-0.189

0.851

0.978

0.335

0.356

0.134

0.002

-0.146

-0.237

0.513

0.991

0.477

0.244

0.584**

0.013

0.525**

0.066

0.002*

0.949

0.006*

0.747

*. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

Table X: Correlation of GAD positivity with complement and immunoglobulins in T1DM Patients. Group

Total (26)

IgG

IgM

IgA

No (%)

(mg/dl)

GAD Ab Positive

13.35±10.81

1114.8±280.85

161.95±54.21

172.74±48.76

GAD Ab Negative

19.65±11.69

1208.85±155.07

181.29±72.32

299.16±101.85

t value

1.3401

1.1090

0.7020

3.4930

p value

0.1928

0.2784

0.4894

0.0019*

*statistically significant (p<0.05)

Discussion The complement proteins play an important role in both innate and adaptive immune responses (14). Dysregulation of the complement system has been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus erythematosus, vasculitides, Sjogren’s syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and rheumatoid arthritis (15). In present study the mean value of C4 was decreased in T1DM patients

as compared to controls (17.47±11.59 vs 23.67±5.70 mg/ dl; P 0.0336). Previous studies were also found the low concentration of serum complement C4 in T1DM (16, 17, 18, 19, 20). We observed that 19.2% cases were below the normal range in T1DM whereas in control 5% below the normal range. Analysis of C4 with reference to age and GAD positivity showed that all C4 reduced cases were present in younger age (below 18 years) and they were GAD positive. Chiarelli et al. (1988) were demonstrated that the mean value of C4 was significantly lower in T1DM

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-58

than in control (27.99 +/- 8.01 vs 32.03 +/- 8.91 mg/dl; P < 0.01). They found 25% low serum levels of C4 in T1DM (20). The lack of serum concentrations may help to identify a subgroup (diabetic microangiopathy) of T1DM patients at high risk of developing diabetic nephropathy (16, 17, 20, 21). There are very few studies on Immunoglobulin in T1DM patients. In our study, we found that the mean value of IgM and IgA was increased in T1DM patients as compared to controls whereas IgG was within normal range. Out of 26 cases of T1DM, about 23.1% had increased serum IgM levels and 7.7% had high concentration of serum IgA. Similar to our study, other researchers also found high concentration of IgM and IgA in T1DM patients than in control subjects or siblings(22). Correlation between age of T1DM patients and immunoglobulin showed that concentration of IgM and IgA were increased with increasing the age of patients. IgA deficiency is relatively common in T1DM patients (23, 24, 25). However, immunoglobulin deficiency was not present in our study. Previously, it has been demonstrated that concentration of IgM increased in 21% cases and IgG decreased in 11% cases in age of onset between 20 and 40 years, whereas in our study IgM was increased in 30.77% and IgG was normal in onset between 20-40 years (22). Svensson et al (2012) found low concentration of IgG and higher concentration of IgA levels, whereas no differences in IgE or IgM value in newly diagnosed T1DM (26). Elevated levels of serum IgMand IgA suggested that diabetic patients suffered from bacterial infections (27, 28, 29). The changes in total Immunoglobulin concentrations at onset were largely reversed under insulin therapy. Rise of immunoglobulin may be due to environmental causes, such as viral infections, or insulinopenia prior to clinical disease onset (22). Patients with T1DM have infections more often than those without DM (10, 11, 12, 30). In our study, we found that serum concentration of complement C4 and serum IgA was increased with rising of BMI and rising of disease onset. There was no correlation among IgG and IgM with age of onset, family history, disease duration and BMI. Mean values of complement and immunoglobulins were decreased in GAD positive patients.

Conclusion

From our study, we conclude that C4 deficiency was present in T1DM and it was related with lower age and GAD positivity. The study also showed that recent infection was the reason for higher concentration of IgM in T1DM. Thus our study concluded that T1DM patients are very sensitive for infection.

Acknowledgements We are thankful to UGC Advanced Immunodiagnostic Training and Research Centre and National Facility For Tribal and Herbal Medicine (NFTHM) Centre for financial support.

Funding We are thankful to UGC Advanced Immunodiagnostic Training and Research Centre and National Facility For Tribal And Herbal Medicine (NFTHM) Centre for financial support.

Competing Interests : No

Reference 1. International Diabetes Federation; IDF Diabetes Atlas; 6th edn.; International Diabetes Federation; 2013; http://www.idf.org/diabetesatlas ISBN: 2-930229-853. 2. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med. 1998; 15:539–553. 3. Maahs DM, West NA, Lawrence JM and Mayer-Davis EJ. Epidemiology of type 1 diabetes. Endocrinol Metab Clin North Am. 2010; 39: 481–97. 4. International Diabetes Federation. IDF Diabetes Atlas. 6th ed. International Diabetes Federation. 2013. 5. Graves PM, Norris JM, Pallansch MA, Gerling IC, Rewers M. The role of enteroviral infections in the development of IDDM: limitations of current approaches. Diabetes. 1997; 46:161–168. 6. Vreugdenhil GR, Schloot NC, Hoorens A et al. Acute onset of type I diabetes mellitus after severe echovirus 9 infection: putative pathogenic pathways. Clin Infect Dis. 2000; 31: 1025– 1031. 7. Yoon JW, Austin M, Onodera T, Notkins AL. Virusinduced diabetes mellitus. N Engl J Med. 1979; 300:1173–1179. 8. Jun HS, Yoon JW. The role of viruses in type I diabetes: two distinct cellular and molecular pathogenic mechanisms of virus-induced diabetes in animals. Diabetologia.2001; 44:271–285. 9. Imagawa A, Hanafusa T, Makino H, Miyagawa J I and Juto P. High titres of IgA antibodies to enterovirus in fulminant type-1 Diabetes. Diabetologia. 2005; 48: 290–293.

http://www.pacificejournals.com/aabs

A-59

AABS; 3(1): 2016

10. Deresinski S. Infections in the diabetic patient: Strategies for the clinician. Infect. Dis. Rep., 1995; 1: 1-12. 11. Gillani S W, Sulaiman SAS, Sundram S, Sari YO, Baig M, and Iqbal MS. Factors associated with infections in diabetic population. African Journal of Pharmacy and Pharmacology, 2011; 5(11): 1414-1421. 12. Geerlings SE, Hoepelman AI. Immune dysfunction in patients with diabetes mellitus (DM). FEMS Immunol Med Microbiol. 1999; 26:259-65. 13. Moutschen MP, Scheen AJ, Lefebvre PJ. Impaired immune responses in diabetes mellitus: analysis of the factors and mechanisms involved. Relevance to the increased susceptibility of diabetic patients to specific infections. Diabete Metab. 1992;18: 187-201. 14. Dunkelberger JR, Song WC. Complement and its role in innate and adaptive immune responses. Cell Res. 2010; 20(1):34-50. 15. Ballanti E, Perricone C, Greco E, Ballanti M, Di Muzio G, Chimenti MS, Et Al. Complement And Autoimmunity. Immunol Res. 2013; 56(2-3):477-91. 16. Mcmillan DE. Elevation Of Complement Components In Diabetes Mellitus. Diabete Metab. 1980; 6(4):265-70. 17. Barnett AH, Mijovic C, Fletcher J, Chesner I, Kulkuska-Langlands BM, Holder R et al. Low plasma C4 concentrations: association with microangiopathy ill insulin-dependent diabetes. Br Med J. 1984; 289 : 943-945. 18. Bertrams J, Hintzen U, Schlicht V, Schoeps S. C41 another marker for type I diabetes. Lancet. 1982; I : 41. 19. Cooper ME, Duff R, Buchanan R, McPherson J, Jerums G. Low serum C4 concentrations and microangiopathy in type I and type II diabetes. Br Med J. 1986; 292 : 801. 20. Chiarelli F, Verrotti A, La Penna G, Morgese G. Low serum C4 concentrations in type-1 diabetes mellitus. Eur J Pediatr. 1988; 147(2):197-8. 21. Flyvbjerg, Allan. Diabetic angiopathy, the complement system and the tumor necrosis factor superfamily. Nature Reviews Endocrinology. 2010; 6: 94-101.

22. Gorus FK, Vandewalle CL, Winnock F, Lebleu F, Keymeulen BV, et al. Increased prevalence of abnormal immunoglobulin M, G, and A concentrations at clinical onset of insulin-dependent diabetes mellitus: A registry-based study. The Belgian Diabetes Registry. Pancreas. 1998; 16:50–9.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

23. Cser A, Ambrus M, Bajtai G, Heim T, Varga F. Juvenile diabetes mellitus and selective IgA deficiency. Ovr Heti. 1974; 1115:803-806. 24. Penny R, Thompson RG, Polmar SM, Schultz RB. Pancreatic malabsorption and IgA deficiency in a child with diabetes. Pediatrics.1971; 78:512-516 25. Silver HKB, Shuster J, Gold P, Hawkins D, Freedman SO. Endocrinopathy and IgA deficiency. Clin Immunol Immunopath. 1973; 1 : 212-219. 26. Svensson J, Eising S, Mortensen HB, Christiansen M, Laursen I, Lernmark A et al. High levels of immunoglobulin E and a continuous increase in immunoglobulin G and immunoglobulin M by age in children with newly diagnosed type 1 diabetes. 2012; 73(1):17–25. 27. Eguchi K, Yagame M, Suzuki D, et al. Significance of high levels of serum IgA and IgA-class circulating immune complexes (IgA-CIC) in patients with noninsulin- dependent diabetes mellitus. J DiabetComplications. 1995; 9: 42–48. 28. Rodriguez-Segade S, Camina MF, Carnero A, et al. High serum IgA concentrations in patients with diabetes mellitus: agewise distribution and relation to chronic complications. Clin Chem. 1996; 42: 1064– 1067. 29. Islam Laila N, Mahmud Hossain and M. Shamim H. Zahid. Complement mediated bactericidal activity and humoral immune response in type 2 diabetes mellitus. Int J Diabetes Metab. 2006; 14: 92-97. 30. Peleg AY, Weerarathna T, McCarthy JS, Davis TM. Common infections in diabetes: pathogenesis, management and relationship to glycaemic control. Diabetes Metab Res Rev. 2007; 23:3-13.

Original Article Clinical Correlation of Coagulopathy in Vivax Malaria Kini Reshma G1, Lobo Veronica2, Lyngdoh Raphael H1, Ms Vedasree R2 1

Department of Pathology, Father Muller Medical College, Kankanady Mangalore, India 2 Undergraduate, Father Muller Medical College, Kankanady Mangalore, India Keywords: PT, aPTT, Malaria, Vivax, coagulopathy

ABSTRACT Background: Malaria is a disease with a great global burden. Understanding the pathogenesis of the disease with particular emphasis on the complications is necessary. The coagulation system plays a key role in the pathogenesis of complicated as well as uncomplicated malaria. Pathogenesis of vivax malaria is less focused upon as compared to that of falciparum malaria and studies on the role of coagulation have yielded conflicting results. Aims: To study the prothrombin time (PT ) and activated partial thromboplastin time (aPTT) in malaria with focus on vivax cases to determine the presence or absence of coagulation abnormalitie and to correlate these with the clinical features. Settings and Design: Hospital based prospective cross-sectional study. Methods: A single citrated blood sample of patients diagnosed with falciparum and vivax malaria was analysed in semi-automated coagulation analyzer at the time of presentation.. The values were compared with healthy controls. Correlations with clinical features and effect of treatment on coagulation profile have been studied. Data was analyzed by mean standard deviation and by ‘t’ test using SPSS software version 16 for windows. ‘p’value is obtained by Mann Whitney U test Results: Prolonged PT and aPTT was noted in vivax malaria as compared to the controls. The difference in the coagulation profile of vivax and falciparum cases was not significant. PT and aPTT were prolonged in 38% and 56% of the malaria patients. The sample obtained at the time of presentation had no significant correlation with the clinical symptoms, antimalarial treatment and complications. Conclusions: Coagulation is involved in the early stages pathogensis of vivax malaria to the same extent as falciparum malaria.

*Corresponding author: Dr. Reshma G Kini, Assistant Professor, Department of Pathology, Father Muller Medical College, Kankanady, Mangalore- 575002, India Phone: +91 9986287395 E-mail: drreshmakini@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

AABS; 3(1): 2016

A-61

Introduction Malaria is one of the most widespread diseases causing nearly 120000 deaths annually. Haematological alterations are the commonest complications encountered in malaria irrespective of the species. Etiology is almost equally attributable to the two major species namely Plasmodium vivax and Plasmodium falciparum except in regions of Africa and the western pacific Falciparum malaria is the cause of majority of deaths and complications and its pathogenesis has been studied extensively.[1,2] Factors contributing to complication in malaria include sequestration of parasitized RBCs in microciculatiom, activation of inflammation with production of cytokine storm, widespread activation of coagulation system accompanied by formation of thrombi in microvascular bed producing multiorgan dysfunction and disseminated intravascular coagulation (DIC).[3] By virtue of PfEMP expressed on the membrane the parasitized RBCs (pRBCs) sequester in different organs adhering to the endothelial cells by using the host receptor adhesion molecules. Sequestration helps the parasite avoid killing by the splenic macrophages and is the root of all major complications. [3,4] Clinical severity of the disease depends also upon the response of the host. The two factors that play a key role in development of complications are a) the release of the inflammatory cytokines- especially TNF and interleukins and b) the activation of coagulation system. [5] Malaria infection is a procoagulant state. Multiple pathways are involved in activating the coagulation systema. The parasitized RBCs become procoagulant by altering the the phospholipid orientation of their cell membrane.[6,7] b. The sequestrated and procoagulant RBCs induce release of tissue factor from activated blood monocytes, endothelial cells and microparticles.TF activates the extrinsic pathway of coagulation after complexing with FVII. The thrombin generated can activate platelets, produce microthrombi and activate the intrinsic pathway of coagulation [9-12] c. In addition to coagulation, TF can also potentiate the inflammatory response. TF thrombin and activated FX together can trigger the inflammatory system by enhancing the production of cytokines. especially interleukins and Tumor Necrosis Factor ( TNF ). [6,10]. d. TNF is responsible for systemic manifestations of malaria such as fever and headache. Additionally TNF also induces expression of adhesion molecules on the

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

surface of endothelial cells which leads to recruitment of inflammatory cells and release of their contents such as elastase. Elastase damages the endothelium which in turn potentiates coagulation.[13,14] e. Platelets when activated not only aggregate at the site of endothelial damage but also release several procaogulant and proinflammatory cytokines contributing significantly to the pathogenesis of complications.[5,7] f. Activation /damage to endothelium by pRBCs causes release of ultra large multimers of Von-Willebrand factor which together with deficiency of ADAMTS13 in affected individuals causes platelet adhesion and aggregation and relase of procoagulant substances. [15] Understanding the role of coagulation system in pathogenesis of malaria and its complications was initiated when prolongation of prothrombin time and aPTT was first recognised in uncomplicated cases. Research into its etiology led to the identification of the interdependence of the coagulation and inflammation and the fundamental role of TF in activation of the two. [16-18] Functional activation of coagulation system in falciparum malaria is undisputed but its activation is still contested in vivax malaria. The aim of this study was to explore this incongruity by doing baseline investigations of PT and aPTT in patients with vivax and falciparum malaria and comparing them with controls and with each other. Correlation with clinical features and complications was done. Research was centred in a region endemic for malaria and hence the cases as well as the controls may be considered as partially immune to malaria. [11,19]

Materials and Methods It was a prospective cross-sectional study done over two years. Patients were included in this study after obtaining the necessary clearance from institutional ethics committee and obtaining consent from participating patients and controls. Inclusion criteria: Patients diagnosed with P vivax, P falciparum and co infection with these two species were included in this study. Exclusion Criteria: • • • •

Patients on anticoagulant therapy Known disease states having abnormal coagulation profiles like hemophilia. Pregnant women Other associated diseases like Diabetes mellitus and renal failure

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-62

A detailed history with emphasis on presenting complaints was obtained. Findings of clinical examination were recorded. The patients were followed up for the duration of stay in hospital.

and spleenomegaly in eighteen patients each and both in three patients. One patient presented with mild right pleural effusion. Six of the patients had icterus associated with fever.

A single citrated sample was obtained at the time of admission. PT and aPTT were performed on semiautomated Sysmex CA-50 using Thromborel S and aPTT kit from Dade Behring. An EDTA sample was also collected simultaneously. Platelet count was estimated in Coulter LH 500.

Complicated malaria was diagnosed in twenty patients and surprisingly twelve patients had infection with P.vivax. and the remaining eight with P falciparum. Complications included septicaemia, acute respiratory distress

A few of the patients already on antimalarial therapy at the time of admission were also included in the study.. PT ratio and aPTT ratio of >1.5 were considered to be prolonged. MNPT and MNaPTT were determined from the samples obtained from the healthy controls. Data was analyzed by mean standard deviation and by ‘t’ test using SPSS software version 16 for windows. ‘p’value is obtained by Mann Whitney U test.

Results

A total of total 300 slide positive malaria patients were included in the study. Two hundred and twenty two were males and 78 were females. The age of the patients ranged from 15 years to 65 years. The distribution of cases by the species was as in Fig.1. The most common symptom was fever. It was seen in almost all patients with malaria. Other common symptoms were headache, bodyache and vomiting. Pain abdomen associated with fever was seen in six patients..One patient was diagnosed with P.vivax malaria incidentally when she presented with loss of consciousness after a accidental fall.

syndrome ( ARDS), multi-organ dysfunction and septic shock. One patient with complicated vivax malaria subsequently died due to septicaemia and ARDS. Haematological profile revealed thrombocytopenia (<1,50,000/cmm) in 96% of the patients. Severe thrombocytopenia with platelet count of 20,000/cmm or less was seen in 42 patients. However none of the patient had any bleeding manifestations.(Fig 2) Comparison of malaria cases vs controls PT and aPTT values were prolonged in 38% and 56% of the malaria patients respectively. The differences in the PT and aPTT between patients with malaria and healthy control was highly significant (p <0.001).(Fig.3 & Fig.4, Table 1) There was no significant differences in aPTT and PT values between genders. Comparison of P vivax vs control The PT and aPTT values of P.vivax, were significantly different from those of the control groups. (p<0.001). Comparison of P falciparum vs control aPTT was significant. (p = 0.025)..

One of the patients had associated acute suppurative otitis media and one patient has had breast engorgement and secretion. Clinical examination revealed hepatomegaly

Comparison of mixed infection vs control The PT and aPTT values of mixed infection groups were significantly different compared to the control groups. (p<0.001).

Fig. 1: Distribution of cases by species

Fig. 2: Distribution of cases by platelet counts

http://www.pacificejournals.com/aabs

A-63

AABS; 3(1): 2016

Fig. 3: Distribution of cases by prothrombin time in malaria

Fig. 4. Distribution of cases by Thromboplastin Time in Malaria

activated

Partial

Table 1: Mann-Whitney U test for coagulation parameters in malaria and controls. Malaria Patients (seconds)

Healthy Control (Seconds)

p Value

12.9

10.5

<0.001

aPTT

35.9

26.2

<0.001

Comparison of P falciparum vs P vivax vs mixed infection There was no significant difference in PT and aPTT values in between patients with P.falciparum ,P.vivax and mixed malaria(p>0.05) Comparison of patients on treatment vs untreated cases Antimalarial treatment did not alter the aPTT and PT values in the patients with malaria. Also among patients who had antimalarial treatment, there was no difference in the values between patients who had started on antimalarials less than 10 hours and more than 10 hours prior to the collection of blood sample Comparisons depending on clinical and laboratory parameters The PT and aPTT values of patients with clinical history and findings of headache, bodyache, cough, pain abdomen, spleenomegaly and hepatomegaly were not significantly different from patients with negative history for these symptoms and signs. However the PT value was significant prolonged in patients who presented with vomiting (‘p’ value:0.033). as compared to those who had no history of vomiting. The correlation between PT and the platelet count was not significant (‘p’value:0.086). However there was a significant correlation between the aPTT values and the platelet count in patients with counts above 50,000/cmm .(‘p’value:0.035).

parameters (Bilirubin, Aspartate amino transferase(AST) and Alanine amino transferase (ALT) Comparison between complicated and uncomplicated cases. There was no difference in PT and PTT values between patients with complicated and uncomplicated malaria. There was also no correlation between the values and duration of hospital stay.

Discussion In our study the difference in the PT and aPTT values between the vivax group the controls was highly significant. This definitely suggests activation of coagulation in vivax malaria. Coagulation factors once activated are used up. Depleted levels of various factors either in the common pathway or both in intrinsic and extrinsic pathway leads to prolonged PT and PTT. There was no significant difference when the values of vivax, falciparum and the mixed infection groups were compared with each other which suggests rather uniform degree of activation of coagulation in all the species.

Interestingly, PT and aPTT values did not differ significantly between the patients with raised and normal liver

PT and aPTT was prolonged in 38% and 56% of the malaria patients respectively The derangement in values is comparable with those of Prasad et al. and Netha et al who noted prolongation of PT in 47.5%, and 22% of cases and deranged aPTT in 35% and 11% of cases, respectively. [20,21] .( Fig.5)

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-64 depletion of anticoagulant proteins and presence of fibrin degradation products in severe disease[5,11,16] Haematological profile revealed thrombocytopenia (<1,50,000/cmm) in 96% of the patients. However none of the patient had any bleeding manifestations supporting the idea that platelets in malaria are hyperresponsive and hence prevent any bleeding.

Fig. 5: PT and aPTT derangements in various studies

The study conducted by Rojanasthein et al. also revealed significantly prolonged PT and aPTT values in patient with P.falciparum malaria but no difference in values in patients with vivax malaria. These coagulation abnormalities were attributed by him to liver dysfunction. However in our study there was no significant difference in the PT and aPTT values between patients who had elevated liver enzymes and those who did not indicating that the changes were unrelated to liver involvement[11] In our study we found no significant differences in the coagulation parameters between patients with complicated and uncomplicated malaria of either species. However derangements of PT and PTT were seen in 80% and 100% of all the complicated cases respectively. These were comparable to the study conducted by Srinivas et al who found no correlation between the coagulation parameters and severity of the disease.Netah et al also did not have correlation of PT, aPTT and thrombocytopenia with mortality[19, 21]. PT and aPTT alone are probably inefficient in predicting course of the disease. Additional information regarding the status of the fibrinolytic and the anticoagulant mechanisms are probably required to recognize the degree of impairement in the homeostasis. DIC is an uncommon state of decompensation in malaria as evidence by the fact that none of the 300 patients included in the study had any bleeding manifestations including those with liver enzyme abnormalities. Most of the cases of malaria remain in a state of compensated DIC wherein the activated factors are kept in check by the anticoagulant mechanisms Decompensated state occurs when the continued presence of the offending agent leads to consumption of the anticoagulants which are then no longer available to manage the activated factors leading to large scale thrombosis.. This hypothesis bears investigation by research which has shown a greater

It has been shown that platelet activation and degranulation occurs early in the disease. As the disease progresses the platelets become exhausted and are cleared from circulation which may be one of the mechanisms of thrombocytopenia in malaria. Immune destruction, sequestration and invasion of platelets by parasite with subsequent platelet destructions are other theories proposed for thrombocytopenia.[12,21] There was no significant differences in PT and aPTT values between genders. Antimalarials do not have effect on the mechanisms that trigger the coagulation system as evidenced by the fact that there was no difference in the PT and PTT values in patients who had received treatment and who had not. Among patients who had antimalarial treatment, there was no difference in the values between patients who had started on antimalarials less than 10 hours and more than 10 hours prior to the collection of blood sample. It has been demonstrated that administering heparin in patients with severe malaria did not affect the outcome of the disease. However it is interesting to know that anti TF antibodies were infact able to bring down the severity of the disease.[6] Correlating the PT and aPTT values with the symptoms the patients came with, showed no significant value except that the PT value was significant for patients who presented with vomiting (â&#x20AC;&#x2DC;pâ&#x20AC;&#x2122; value=0.033). Further studies into this parameter may throw light upon this matter.

Conclusion This study has compared the PT and aPTT values in patients with malaria and normal healthy controls derived from the same population. We conclude that There is a significant difference between the PT and aPTT values of the two groups. This indicates that in patient with malaria there is an activation of intrinsic and extrinsic pathways of coagulation. activation of coagulation cascade is not confined to P falciparum alone but is also seen in significant percentages of patients with P vivax infection most of the cases derangements with or without accompanied thrombocytopenia did not lead to hemorrhagic

http://www.pacificejournals.com/aabs

A-65 manifestations probably because of compensatory anticoagulant/ fibrinolytic activity. Coagulation system is activated even in patients without complications. Since the PT and aPTT values are unaffected by treatment we could hypothesize that antimalarials cannot prevent activation of coagulation however they may contribute in reducing the severity of the disease by clearing off the antigen

Acknowledgements We would like to thank Dr. Sumanth D for his valuable inputs and encouragements during the study.

Funding Part of the study was funded by the Indian Council of Medical Research

Conflicts of interest Nil

References

1. Who.int. Geneva WHO Malaria Report 2012[ Internet]. [ updated in March 2014].available http:// www.who.int/malaria/publications/world_malaria_ report_2014/wmr-2014-annex6a.xls?ua=1 accessed on 02/09/2015 2. Wickramasinghe SN, Abdalla SH. Blood and bone marrow changes in malaria. Ballier’s Clin Hematol. Harcourt Pub Ltd:2000:13:277-299 3. Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis of malaria NATURE 2002; 415: 673674 4. Baruch DI, Pasloske BL, Singh HB, Bi X, Ma XC, Feldman M, Taraschi TF, Howard RJ. Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 1995;82:77–87. 5. Francischetti IM, Seydel KB, Monteiro RQ. Blood coagulation, inflammation, and malaria. Microcirculation 2008 ;15(2):81-107. 6. Francischetti IMB, Seydel KB, Monteiro RQ, Whitten RO, Erexson CR, Noronha ALL, Ostera GR, Kamiza SB, Molyneux ME, Ward JM, Taylor TE. Plasmodium falciparum-infected erythrocytes induce tissue factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes. J Thromb Haemost 2007; 5: 155–65. 7. Ghosh K, Shetty S. Blood coagulation in falciparum malaria. Parasitol Res 2008;102(4):571-576Choia

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 G, Schultza MJ, van der Pollb MLT The relationship between inflammation and the coagulation system Swiss Med Weekly 2006;136:139– 144 8. Choia G, Schultza MJ, van der Pollb MLT The relationship between inflammation and the coagulation system Swiss Med Weekly 2006;136:139– 144 9. Angchaisuksiri P. Coagulopathy in malaria Thrombosis Research 2014; 133: 5–9 10. Levi M, van der Poll T, Büller,HR MD. Bidirectional Relation Between Inflammation and Coagulation Available at http://circ.ahajournals.org/ accessed on August 27, 2015 11. Rojanasthein S, Surakamolleart V, Boonpucknavig S, Isarangkura P. Hematological and Coagulational Studies In Malaria. J Med Assoc Thai 1992;75(1):190194. 12. Mohanty D, Marwaha N, Ghosh K, Sharma S, Garewal G, Shah S, Devi S, Das KC. Functional and ultrastructural changes of platelets in malarial infection. Trans R Soc Trop Med Hyg 1988;82:369. [PubMed: 3068847 13. Clemens R, Pramoolsinsap C, Lorenz R, Pukrittayakamee S, Bock HL, White NJ. Activation of the coagulation cascade in severe falciparum malaria through the intrinsic pathway. Br J Haematol. 1994;87:100–105. Skudowitz RB, Katz J, Lurie A, Levin J, Metz J. Mechanisms of thrombocytopenia in malignant tertian malaria. Br Med J 1973;2:515–518. 14. Pukrittayakamee S, Clemens R, Pramoolsinsap C, Karges HE, Vanijanonta S, Bunnag D, White NJ. . Polymorphonuclear leucocyte elastase in Plasmodium falciparum malaria. Trans. R. Soc. Trop. Med. Hyg. 1992; 86:598-601.. 15. Mast Q , Groot E, Asih PB, Syafruddin D, Oosting M, SebastianS , Ferwerda B, Netea MG, de Groot PG, van der Ven AJAM, Fijnheer R Am. J. Trop. Med. Hyg. 2009; 80(3):492–498 16. Dennis LH, Eichelberger JW, Inman MM, Conrad ME.Depletion of coagulation factors in drug-resistant Plasmodium falciparum malaria..Blood ;1967: 29:713-21 17. Horstmann RD, Dietrich M. Haemostatic alterations in malaria correlate to parasitaemia. Blut.1985;51:329– 335. 18. Mohanty D ,Ghosh K,Nandwani SK,Shetty S ,Phillips C,Rizvi S ,Parmar BD. . Fibrinolysis, inhibitors of blood coagulation, and monocyte derived coagulant activity in acute malaria.American Journal of Hematology1997;54(1):23-29 e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-66

19. Srinivas D,Behera AK, Padhi PK,Barik BK.Coagulation profile in Plasmodium falciparum Malaria Infection. Paper presented at 63rd Annual Conference of Association of Physicians of India January 2008, Kochi, India. Updated on 26th June 2008 Available on :http://www.japi.org/july 2008/ tropical diseases.html. accessed on 23/10/2013 20. Prasad R,Das BK, ,Shukla J, Pengoria R,MishaOP Singh TB. Coagulation status and platelet functions

in children with severe falciparum malaria and their correlation of outcome. J Trop Pediatr 2009 Dec;55(6):374-378 21. Netha SB. Clinical,Hematological and Coagulation Profile in Malaria. Available on http://119.82.96.198:8080/jspui handle/123456789/1237 f. Accessed on 23/10/2013 22. Skudowitz RB, Katz J, Lurie A, Levin J, Metz J. Mechanisms of thrombocytopenia in malignant tertian malaria. Br Med J 1973;2:515â&#x20AC;&#x201C;518.

http://www.pacificejournals.com/aabs

Original Article Diagnostic Value of IL-6 in Neonatal Sepsis Neeraj Kumar1, Manoj Kumar Singh1*, Rajeshwar Dayal1, Shikha Gupta1, Ruchika Garg2 1

Department of Pediatrics, S.N. Medical College, Agra, UP, India. Department of Obstetrics and Gynecology, S.N. Medical College. Agra, UP India

Keywords: Sepsis, IL-6,CRP

ABSTRACT Background: Neonatal sepsis is a serious life-threatening condition with high mortality. The early and accurate diagnosis of sepsis is one of main challenge for prevention of mortality due to sepsis in NICUs. A great effort to reduce the neonatal mortality rate is put into looking for new reliable biomarkers. Among various biomarkers, IL-6 could be promising and reliable biomarker for early diagnosis of neonatal sepsis. Objective: To evaluate the diagnostic value of IL-6 in Neonatal Sepsis. Design: Prospective, observational study. Methodology: By ELISA method the level of serum IL-6 were assessed in 41 neonates with suspected sepsis and 42 healthy neonates with no clinical and laboratory data of infection. Result: The AUC for IL-6 and CRP were 0.87, 0.80 respectively. The cut off value for IL-6 was 181pg/ml at which the sensitivity, specificity, PPV and NPV were 80.1%, 85.7%, 84.6%, 81.8% respectively. The cut off value for CRP is 3.78 mg/dl at which the sensitivity, specificity, PPV and NPV are 61%, 90.5%, 86.2%, 70.3% respectively. In culture positive patients sensitivity of IL-6 and CRP was 90% and 80%, respectively. In culture negative patients sensitivity of IL-6 and CRP was 71.4% and 42.8%, respectively. In EOS sensitivity of IL-6 and CRP was 86.3% and 50%, respectively. In LOS sensitivity of IL-6 and CRP was 73.6% and 72.6%, respectively. Conclusion: IL-6 is a novel biomarker with high sensitivity and good specificity for sepsis. This has better diagnostic value than CRP, especially in Blood culture negative and EOS.

*Corresponding author: Dr. Manoj Kumar Singh, Department of Pediatrics, Assistant Professor, S.N. Medical College, Agra , India,282002. E-mail: drmanuped@yahoo.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-68

Introduction

•

Neonatal sepsis is a global problem and is a significant contributor to morbidity & mortality. Approximately one million deaths a year occurring in the neonatal period (0-28 days) are caused by infection, accounting for over 25% of global neonatal deaths and 10% of all mortality in infants1. The prognosis and outcome of neonatal sepsis depend on early diagnosis and on-time and efficient antibiotic therapy2. The accurate and timely diagnosis of septicemia in the neonatal population is challenging and problematic because of nonspecific clinical presentation and poor diagnostic yield (sensitivity of 50% or less) and delay of the standard blood culture. As such, there is much interest in developing rapid and sensitive diagnostic assays for diagnosis of the infected neonate3. An approach that has gained particular attention is detection of IL-6 level which is a cytokine and produced by monocytes, endothelial cells and fibroblasts. Preliminary studies suggest that this is a novel, early and reliable biomarker for the diagnosis of sepsis. IL-6 is currently under investigation in clinical practice as a reliable marker of neonatal sepsis.

Materials and Methods After taking the ethical clearance from ethical committee, the study was carried out during the period from March 2014 to May 2015 on 83 neonates divided into: Patient group: 41 cases with suspected sepsis admitted to NICU in S.N Medical college & Hospital AGRA.  Control group: 42 healthy neonates with no clinical and laboratory data of infection. An informed consent was taken from parents before enrollment in study. Suspicion of sepsis by the caring neonatologists was based on: a) The presence of one or more clinical signs/symptoms of sepsis4. b) Presence of two or more risk factors (PROM>24 hs, . Low birth weight (<2500 grams) or prematurity, Maternal fever, Foul smelling and/or meconium stained liquor, Single unclean or > 3 sterile vaginal examination(s) during labor, Prolonged labor (sum of 1st and 2nd stage of labor > 24 hrs), Perinatal asphyxia (Apgar score <4 at 1 minute)5,6. Blood samples collection and storage Around 3 ml blood sample was collected by standard techniques. The sample divided into 3 parts: •

0.5-1 ml injected directly into blood culture bottle.

•

1 ml placed into plane tube for CRP.

1 ml placed into plane tube and centrifuged for obtaining serum sample and then stored at -80°C for IL-6 measurement.

Blood culture 0.5-1 ml of blood was injected into the Bactec culture vial under complete aseptic conditions. Positive vials were Gram stained and sub cultured and incubated in appropriate temperature and atmospheres according to established methods. Full identification of organisms was done with standard bacteriological and biochemical methods. IL-6 was measured by using ELISA development kit. It takes around 5-6 hr. Optical density was calculated by using Microplate reader set at 450 nm. Statistical analysis The collected data were tabulated and analyzed using SPSS version 16 software. ROC curve was used to determine cutoff values of IL-6 with optimum sensitivity and specificity in early diagnosis of sepsis. The accepted level of significance in this work was stated at P <0.05.

Result This study was carried out on 83 neonates divided into two main groups:` Patient group: included 41 neonates with suspected sepsis. There were 17 females&24 males; 30 LBW&11 ≥2.5 birth weight; 19 preterm &22 term; 22 EOS&19 LOS.  Control group: included 42 healthy neonates. There were 22 males &20 females; 28 LBW&14 ≥2.5 birth weight; 20 preterm&22 term.

According to the results of blood culture, patient group was subdivided into two subgroups:-

 Proven sepsis group:- included 20 patients who are positive on blood culture.  Probable sepsis group:- included 21 patients who are negative on blood culture. Blood culture was positive in 48.8% of our cases. Staphylococcus aureus was the most common causative organism of sepsis followed by Klebsielia and E. coli (12.2%, 7.3% and 7.3% respectively) (Table 1). Area under the receiver operating characteristics (ROC) curve (AUC) for CRP was 0.80 with sensitivity and specificity of 61% and 90.5%, respectively at the cut off value of 3.7mg/dl. Its PPV and NPV was 86.2% and 70.3%, respectively. The sensitivity of CRP among Blood culture positive, Blood culture negative, EOS and LOS patients were 80%, 42.8%, 50% and 72.6%,respectively.(Table-2)

http://www.pacificejournals.com/aabs

A-69

AABS; 3(1): 2016

Area under the receiver operating characteristics (ROC) curve (AUC) for IL-6 was 0.87 with sensitivity and specificity of 80.1% and 85.7%, respectively at the cut off value of 181pg/ml. Its PPV and NPV was 84.6% and 81.8%, respectively. The sensitivity of IL-6 among Blood culture positive, Blood culture negative, EOS and LOS patients were 90%, 71.4%, 86.3% and 73.6%,respectively.(Table-3)

requisite to improve survival and to improve therapeutic outcome7. As such, there is much interest in developing rapid and sensitive diagnostic assays that can effectively predict and identify patients who are at risk of infection8. Among new biomarker, IL-6 could be one of the most interesting and reliable candidates for sepsis management, specifically for early diagnosis. So, our study was aimed to evaluate the diagnostic value of IL-6 in neonatal sepsis which would help us in early and accurate diagnosis of sepsis. The current study is carry out on 41 neonates with suspected sepsis and 42 healthy controls. According to culture result,the cases were classified into proven sepsis

Discussion

Infections are responsible for significant mortality and longterm morbidity for infants in the neonatal intensive care units. Early diagnosis of neonatal sepsis is essential pre-

Table 1: Frequency distribution of patient group according to result of blood culture. Blood culture result

No.

Staphylococcus aureus

12.2

Pseudomonas aerogenosa

2.4

Klebsiella

7.3

E.Coli

7.3

Candida

4.9

Acenatobacter

4.9

Burkodelia

4.9

Streptococcus

4.9

No growth

51.2

Total

100

Table 2: Result of CRP in Study Population Group

CRP

Positive

Negative

Patients

Controls

Proven sepsis group

Probable sepsis group

EOS group

LOS group

Ď&#x2021;2

p value

24.16

<0.001 (S)

2.25

0.13 (NS)

1.04

0.31 (NS)

Table-3: Result of IL-6 in Study Population Group

No Positive

Negative

Patients

Controls

Proven sepsis group

Probable sepsis group

EOS group

LOS group

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

IL-6

Ď&#x2021;2

p value

36.5

<0.001 (S)

2.25

0.13 (NS)

1.04

0.31 (NS)

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-70

group (+ve blood culture) and probable sepsis group (-ve blood culture). Although blood culture is the gold standard method for diagnosis but have limitation of sensitivity and delayed result9. Hsu et al found 48.8% positive blood culture in his study10. In another study done by Chacko and Sohi found that culture proven sepsis occurred in (41.6%) of cases with sepsis11. The common organism isolates are Staphylococus , Klebseilla and E. coli which was similar to study by Kumhar et al in a tertiary care hospital in New Delhi, India12. This comes in disagreement with the study of Dzwonek et al, in which nearly half of the positive blood cultures grew Klebsiella pneumonia13, also in the study of De Benedetti et al, the isolated pathogens included Klebsiella pneumonia (47.5%) most common14. This variation may be due to differences in the environment, the microbial etiology, type of blood culture and supportive care practice b/w centers. In the current study AUC for CRP was 0.80 with sensitivity and specificity of 61% and 90.5%, respectively. Its PPV and NPV was 86.2% and 70.3%, respectively. Banac et al compared the levels of CRP, PCT and IL-6 in diagnosis of neonatal sepsis in 58 infants. They reported that the sensitivity, specificity, NPV and PPV of CRP at time of diagnosis was 36%, 92%, 43% and 89% respectively using a cut off value of 14 mg/l14. In recent years, chemokines and pro-inflammatory cytokines such as tissue necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-8 and pro-calcitonin (PCT) have been introduced as early markers in infected infants15,16. IL-6 is produced by monocytes, endothelial cells and fibroblasts17. The concentration of IL-6 rises rapidly after the onset of bactaremia, but its half life is short18,19. Previous studies showed that IL-6 may be a valid non-invasive and rapid method for diagnosis of NS18,19. The sensitivity of IL-6 assay ranged from 0.6020 to 0.9621. Martin et al reported the specificity of IL-6 as 0.69, while it was as high as 0.95 in another study by Ng et al22. These differences in sensitivity, specificity and DOR of IL-6 for predicting NS may be due to various factors. Method of study, method of IL-6 assay, cut-off levels for IL-6 assay, neonates characteristics (e.g. birth weight) may be some possible explanations for the discrepancy. The discrepancy in some studies seemed to be due to low sample size 23,24. The pooled values of sensitivity (0.79) and specificity (0.84) showed favorable accuracy of IL-6 for predicting NS25. In the current study, AUC for IL-6 was 0.87 with sensitivity and specificity of 80.1% and 81%, respectively. Its PPV and NPV was 80.5% and 81%, respectively. The result are comparable with the study by Buck et al who reported sensitivity, specificity and PPV of

IL-6 to be 72.7%, 77.8% and 84% respectively26 and with the study conducted by Kuster et al reported sensitivity and specificity of IL-6 to be 85.7% and 85% respectively27. In the current work, we studied the variations in the Sensitivity of IL-6 and CRP among Blood culture positive, Blood culture negative, EOS and LOS patients. In blood culture positive patients sensitivity of IL-6 and CRP was 90% and 80%, respectively. In blood culture negative patients sensitivity of IL-6 and CRP was 71.4% and 42.8%, respectively. In EOS sensitivity of IL-6 and CRP was 86.3% and 50%, respectively. In LOS sensitivity of IL-6 and CRP was 73.6% and 72.6%, respectively. The explanation of this result may need further studying, as there are no available data about other works that study these relations to support or deny our result or to explain it. According to several studies, the authors tend to confirm that IL-6 is a promising biomarker for early diagnosis of sepsis.

Conclusion In light of the result of the present study, IL-6 is a novel biomarker with high sensitivity and good specificity for sepsis. This has better diagnostic value than CRP, especially in Blood culture negative and EOS cases and consequently, we can conclude that measurement of IL-6 can be useful for early diagnosis of neonatal sepsis, especially in Blood culture negative and EOS cases.

Acknowledgements None

Funding None

Competing Interests None declared

Reference 1. Black RE, CousensS, Johnson HL, Lawn JE and Rudan I. Global, regional, and national causes of child mortality in 2008: a systematic analysis.Lancet, 2010; 375(10):1969–1987. 2. Stoll B, Kliegman and Beharman. Infections in neonates: Etiology of fetal &neonatal infection: Kliegman RM, Behrman RE, Jenson HB and Stanton BF (eds). In Nelson text book of pediatrics, 2008; 18th ed;794-811. 3. Mishra UK, Jacobs SE, Doyle LW and Garland SM. Newer approaches to the diagnosis of early onset neonatal sepsis. Arch Dis Child Fetal Neonatal Ed, 2006; 91:F208–12.

http://www.pacificejournals.com/aabs

A-71 4. Rodwell RL, Leslie AL and Tudehope DI. Early diagnosis of neonatal sepsis using a hematologic scoring system. J Pediatr, 1988; 112(5):761-7.

AABS; 3(1): 2016

16. Ng PC, Li K, Leung TF, et al. Early prediction of sepsisinduced disseminated intravascular coagulation with interleukin-10, interleukin-6, and RANTES in preterm infants. Clin Chem 2006; 52(6):1181-9.

17. Stoll BJ. Infection in neonatal infants. In: Kliegman R, Behrman R, Jenson H, et al (eds). Nelson Textbook of Pediaterics. Philadelphia: Saunders. 2007; Pp: 794-811. 18. Kingsmore SF, Kennedy N, Halliday HL, et al. Identification of diagnostic biomarkers for infection in premature neonates. Mol Cell Proteomics 2008;7(10):1863-75. 19. Lam HS, Ng PC. Biochemical markers of neonatal sepsis. Pathology 2008;40(2):141-8. 20. Santana Reyes C, García-Muñoz F, Reyes D, et al. Role of cytokines (interleukin-1beta, 6, 8, tumour necrosis factor-alpha, and soluble receptor of interleukin-2) and C-reactive protein in the diagnosis of neonatal sepsis. Acta Paediatr 2003; 92(2):221-7. 21. Martin H, Olander B, Norman M. Reactive hyperemia and interleukin 6, interleukin 8, and tumor necrosis factor-alpha in the diagnosis of early-onset neonatal sepsis. Pediatrics 2001; 108(4):E61. 22. Ng PC, Cheng SH, Chui KM, et al. Diagnosis of late onset neonatal sepsis with cytokines, adhesion molecule, and C-reactive protein in preterm very low birthweight infants. Arch Dis Child Fetal Neonatal Ed 1997;77(3):F221-7. 23. Berner R, Niemeyer CM, Leititis JU, et al. Plasma levels and gene expression of granulocyte colony stimulating factor, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis. Pediatr Res 1998;44(4):469-77. 24. Messer J, Eyer D, Donato L, et al. Evaluation of interleukin-6 and soluble receptors of tumor necrosis factor for early diagnosis of neonatal infection. J Pediatr 1996;129(4):574-80. 25. Lobat S,MD; Abbasali K,PhD; Arezou M,MD; Ali A,MD and Ghalamreza R,MD. The Role of IL-6 for Predicting Neonatal Sepsis: A Systemic Review and Meta-Analysis. Iran J Pediatr Dec2011;Vol 21(No 4), pp:411-417. 26. Buck C, Bundschu J, Gallati H, Bartmann P, Pohlandt F Pediatrics. 1994Jan;93(1):54-8, Interleukin-6: a sensitive parameter for the early diagnosis of neonatal bacterial infection. 27. Kuster, N Yiho. Immunological basis of neonatal sepsis. Clinical Neonatology. 2000 Vol. No.3.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

5. Singh M, Narang A, Bhakoo ON. Predictive perinatal score in diagnosis of neonatal sepsis. J Trop Pediatr. 1974;11:597-60. 6. Takkar VP, Bhakoo ON, Narang A. Scoring system for the prediction of early neonatal infections. Indian Pediatr. 1974;11:597-60. 7. Birju AS and James FP. Neonatal sepsis. An old problem with new insights. Virulence, 2014; 5(1): 170–178. 8. Ahrens P, Kattner E and Köhler B. Mutations of genes involved in the innate immune system as predictors of sepsis in very low birth weight infants.Pediatr Res, 2004; 55(4):652-6. 9. Kumar Y, Qunibi M and Neal TJ .Time topositivity of neonatal blood cultures. Arch Dis Child Fetal Neonatal Ed, 2001; 85(3): F182-6. 10. Hsu KK, Pelton SI and Shapiro DS. Detection of group B streptococcal bacteremia in simulated intrapartum antimicrobial prophylaxis. Diagn Microbiol Infect Dis, 2003; 45(1):23-7. 11. Chacko B and Sohi I. Early onset neonatal sepsis. Indian Journal of Pediatrics, 2005; 72(1): 23-26. 12. Kumhar G . Ramachandran V.G , Gupta P. Bacteriological Analysis of Blood Culture Isolates from Neonates in a Tertiary Care Hospital in India.J Health PopulNutr 2002:20(4):343-347. 13. Dzwonek AB, Neth OW and Thiebaut R. The role of mannose-binding lectin in susceptibility to infection in preterm neonates. Pediatr Res, 2008;63(6):680-5. 14. BanacBanac B, Dergan CM, Wraber B, Hojiker S. Interleukin 6 and Procalcitonin in early diagnosis of sever bacterial infection in critically ill neonates. Pflugers Arch Eur Physiol 2000;440:72-74. 15. Mehr S, Doyle LW. Cytokines as markers of bacterial sepsis in newborn infants: a review. Pediatr Infect Dis J 2000;19(9):879-87.

Original Article Evaluation of Various Causes of Thrombocytopenia in Patients Attending Hamidia Hospital Maneesh Sulya*, Reeni Malik, Kamlesh Patel Department of Pathology, Gandhi Madical College and Hamidia Hospital, Bhopal. India Keywords: Thrombocytopenia, Peripheral smear, Megaloblastic, Aplastic, Idiopathic thrombocytopenia

ABSTRACT Background: Thrombocytopenia is defined as a platelet count <1.5 lacs/Cumm. Thrombocytopenia usually has no symptoms and is picked up on a routine full blood count (or peripheral blood smear examination). Some individuals experience symptoms and some may not. This study is primarily done to evaluate the various causes of thrombocytopenia. Methods: This study was conducted in central pathology laboratory of Hamidia hospital and associated Gandhi Medical College, Bhopal. Total 100 cases of thrombocytopenia over the period of 3 months from October, 2015 to December, 2015 were included in the study. Result: By evaluating all the 100 cases it was found that the most common cause of thrombocytopenia was megaloblastic anaemia (26%) followed by, Idiopathic thrombocytopenia (22%), Infection (15%) and anaemia of chronic disease (14%) are amongst the common causes. Other causes were hematological malignancies like CML and AML, Malaria, Aplastic anaemia, Iron deficiency anaemia, pregnancy and autoimmune diseases. Out of 100 total patients with thrombocytopenia 45 were male and 55 were females. Megaloblastic anaemia and idiopathic thrombocytopenia were more common in females whereas infections, anaemia of chronic diseases, hematological malignancies and aplastic anaemia were more common in males. Conclusion: The importance of careful examination of peripheral smear is essential to evaluate the causes of thrombocytopenia. Causes of thrombocytopenia are variable with geographical variations in diet and food habits.

*Corresponding author: Dr. Maneesh Sulya, F,83/48, Tulsi Nagar, Bhopal- 462003. Phone : +91 9826609403 E-mail: drmanish1619@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-73

Introduction Thrombocytopenia and thrombopenia refer to a disorder in which there is a relative decrease of thrombocytes, commonly known as platelets, present in the blood.(1) Thrombocytopenia is defined as a platelet count <1.5 lacs/ Cumm.(5) Thrombocytopenia usually has no symptoms and is picked up on a routine full blood count (or peripheral blood smear examination). Some individuals with thrombocytopenia may experience external bleeding such as nosebleeds, and/or bleeding gums. Some women may have heavier or longer periods or breakthrough bleeding. Bruising, particularly purpura in the forearms, may be caused by spontaneous bleeding under the skin. Petechia (pinpoint bleeds in the skin and mucous membranes), may occur on feet and legs.(2,3) In most laboratories, a normal platelet count is between 1.5 to 4.5 lacs/Cumm. No generally accepted definition of mild, moderate or severe thrombocytopenia exists. For cancer patients receiving treatment, the National Cancer Institute (NCI) has developed the Common Toxicity Criteria to describe severity of thrombocytopenia. Platelet counts of 75,000 to 150,000/L are defined as grade 1 thrombocytopenia, 50,000 to 75,000/L as grade 2, 25,000 to 50,000/L as grade 3, and below 25,000/L as grade 4 thrombocytopenia. (4) The relevance of thrombocytopenia in the individual patient is variable and depends on the clinical presentation. Because platelets play an essential role in preserving vessel wall integrity, thrombocytopenia is associated with a defect of primary hemostasis. Clinically significant spontaneous bleeding does not usually occur until the platelet count is less than 10,000-20,000/Cumm. Nutritional deficiencies like vitamin B12 and folic acid, infections, malignancies and pregnancy are amongst the common causes of thrombocytopenia. This study is primarily done to evaluate the various causes of thrombocytopenia.

Materials and Methods

This study was conducted in central pathology laboratory of Hamidia hospital and associated Gandhi Medical College, Bhopal. Total 100 cases of thrombocytopenia over the period of 3 months from October, 2015 to December, 2015 were included in the study and evaluated for various causes of thrombocytopenia by peripheral smear examination. Selection of the cases are based on the platelet count less than 1.5 lac/Cumm on peripheral smear examination. Case were segregated according to the various causes of thrombocytopenia revealed by peripheral blood smear examination. Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016

Result

By evaluating all the 100 cases it was found that the most common cause of thrombocytopenia was megaloblastic anaemia (26%) followed by, Idiopathic thrombocytopenia (22%), Infection (15%) and anaemia of chronic disease (14%) are amongst the common causes. Other causes were hematological malignancies like CML and AML, Malaria, Aplastic anaemia, Iron deficiency anaemia, pregnancy and autoimmune diseases. (table 1) Table 1: Occurrence of various causes of thrombocytopenia: Diagnosis

Number % of patients

Megaloblastic

Idiopathic thrombocytopenia

Infection

Malaria

Hematological malignancies

Aplastic anaemia

Iron deficiency anemia

Anaemia of chronic disease

Pregnancy

Autoimmune disease

Out of 100 total patients with thrombocytopenia 45 were male and 55 were females. Megaloblastic anaemia and idiopathic thrombocytopenia were more common in females whereas infections, anaemia of chronic diseases, hematological malignancies and aplastic anaemia were more common in males. (table 2) Table 2 : Sex wise distribution of causes Diagnosis

Male Female Number of patients

Megaloblastic Idiopathic thrombocytopenia Infection Malaria Hematological malignancies Aplastic anaemia Iron deficiency anemia Anaemia of chronic disease Pregnancy Alcoholism Total

07 09 09 03 04 04 02 09 00 01 45

19 13 06 02 02 01 02 05 02 00 55

26 22 15 05 06 05 04 14 02 01 100

Discussion

The relevance of thrombocytopenia in the individual patient is variable and depends on the clinical presentation. A study by Dr. K.F. Magdalene from Kerala showed that out of the 200 cases studied 55 patients had idiopathic e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-74

thrombocytopenic purpura, 42 cases were due to infections, 2 cases were due to malaria, 21 cases due to hematological malignancies, Megaloblastic anemia was the cause in 19 patient and 16 cases were due to aplastic anemia. Our study revealed that the most common cause of thrombocytopenia was megaloblastic anaemia (26%) which is due to prevalence of nutritional deficiencies in our region followed by Idiopathic thrombocytopenia (22%), Infections (15%) which was very well correlated with the study done by Dr. K.F.Magdalene.

Competing Interests None declared.

Reference

1. Deutschman, Clifford S.; Neligan, Patrick J. (2010). Evidence-based Practice of Critical Care. Elsevier Health Sciences. ISBN 1416054766. Retrieved 201504-30. 2. Bhatia, M.P.S. “B.E. project on platelet count using image processing techniques”(pdf). BTP_Report. Retrieved 30 November 2014.

Table 3: is showing comparison of our study with other studies. Causes

Dr. K.F. Magdalene (5) (200 cases)

W.M. Fowler (6) (160 cases)

Charles A. Doan et al (7) (381 cases)

Present study (100 Cases)

Megaloblastic anaemia

9.5%

Idiopathic thrombocytopenia

27.5%

10.6%

71.1%

Infection

21%

17.5%

16.0%

Idiopathic thrombocytopenia (71.1%) was most common cause of thrombocytopenia followed by infections (16.0%) in the study done by Charles A. Doan et al. (table3) Megaloblastic anaemia and idiopathic thrombocytopenia were more common in females (8) whereas infections, anaemia of chronic diseases, hematological malignancies and aplastic anaemia were more common in males. The study by research in nutritional medicine also suggested that nutritional anaemias and related disorders are more common in females.

Conclusion

The relevance of thrombocytopenia in the individual patient is variable and depends on the clinical presentation and underlying cause. The importance of careful examination of peripheral smear is essential to evaluate the causes of thrombocytopenia. Causes of thrombocytopenia are variable with geographical variations in diet and food habits.

Acknowledgements

I am very much thankful to our head of the department who has given me this opportunity to work on the title.

Funding None

3. Jump up^ Houghton, Andrew R.; Gray, David (201005-28).Chamberlain’s Symptoms and Signs in Clinical Medicine 13th Edition, An Introduction to Medical Diagnosis. CRC Press.ISBN 9780340974254. Retrieved 2015-05-01. 4. CTCAE v3.0; www.ctep.cancer.gov/reporting/ctc. html 5. Dr. K.F.Mangdale; Evaluation of Thrombocytopenia in a Tertiary Care Centre; Ind J App Res; Vol. 6, Issue. 1, JAN- 2016, ISSN - 2249-555X; 73-74. 6. W.M. Fowler. Thrombopenic purpura; an analysis of 160 cases .Ann intern med. 1936;9(11):1475-1487. Doi:10.7326/0003-4819-9-11-1475. 7. Charles A. Doan, Bertha A. Bouroncle, Bruce K. Wiseman, Idiopathic and secondary thrombocytopenic purpura.Clinical study and evaluation of 381 cases over a period of 28 years. Annals of internal medicine 1960;53(5):861-876. doi:10.7326/0003-4819-53-5861. 8. Fiebach, Nicholas H.; Barker, Lee Randol; Burton, John Russell; Zieve, Philip D. (2007). Principles of Ambulatory Medicine. Lippincott Williams & Wilkins. ISBN 9780781762274. Retrieved 2015-04-30.

http://www.pacificejournals.com/aabs

Original Article Clinical Profile of Swine Flu in Children at A Tertiary Care Center Manoj Kumar Singh1, Pankaj Kumar1*, Rajesh Kumar1, Shikha Gupta1, Prabhat Agrawal2 1

Department of Pediatrics, S.N. Medical College, Agra. UP, India Department of Medicine , S.N. Medical College, Agra, UP, India

Keywords: Influenza, Swine Flu, Clinical profile

ABSTRACT Pandemic influenza A (H1N1) 2009 has posed a serious public health challenge world-wide, after that this is circulating as seasonal influenza virus. This study was aimed to analyze clinical profile of the swine flu cases in 2015. This Observational study was conducted in a tertiary care hospital, S. N. Medical College & Hospital Agra in 2015. In this study 28 Swine Flu confirmed cases were included. Mean age of confirmed Swine Flu cases was 3.22Âą3.06 years with age range 1 mo to 14 yrs and Male/Female ratio was 2.1:1. There was no significant difference in mean age in both the sexes, males were significantly higher than females and patient was significantly higher within 1-6 year age group. Fever, cough, coryza were the predominant symptoms. Only one patient (5%) required ICU admission and mortality was nil.

*Corresponding author: Pankaj Kumar, Department of Pediatrics, Assistant Professor , S.N. Medical College, Agra. UP, INDIA 282002 Phone : +91 9557860951 E-mail: drpankaj_peds@hotmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-76

Introduction

Influenza like illness caused by Influenza A [H1N1] was first reported from Mexico on 18th March, 2009 and rapidly spread to neighboring United States and Canada [1]. Subsequently the disease spread to all the continents. On June 11, 2009, the World Health Organisation (WHO) signaled that a global pandemic of novel Influenza A (H1N1) was underway by raising the worldwide pandemic alert level to Phase 6. This was the first of the kind declared by WHO in the past 70 years [2]. While declaring the pandemic to be over in August 2010, WHO conveyed that pandemic influenza A [H1N1] virus that caused pandemic[2009-2010] would circulate as seasonal influenza virus and would continue to do so for years to come [3]. India, first confirmed case of influenza [H1N1] was reported in Hyderabad on May 16, 2009[4]. MOHFW reports total 27236 Swine Flu cases with 981 Swine Flu deaths in year 2009 and 20604 Swine Flu cases with 1763 Swine Flu deaths in year 2010 [5]. Since then small spurt of Swine Flu were there in year 2014 but in year 2015 this H1N1 has affected 15413 people causing 812 deaths .Most affected state was Rajasthan and Gujarat[6]. Influenza spreads through droplets from infected individuals while speaking, coughing or sneezing. Non human influenza spreads from respiratory or gastrointestinal tracts of infected hosts. Flu can occur throughout the year, but peak occurrence is in the winter months [7,8]. Flu epidemics occur every 6 to 10 years, usually due to antigenic shifts which expose the population to strains to which it has not been exposed previously [9,10]. Various definitions: Influenza-like illness (ILI): Fever (temperature of 100°F [37.8°C] or greater) with cough or sore throat in the absence of a known cause other than influenza [7-9]. A probable case of H1N1 influenza A (swine flu): An individual with an influenza-like illness who is positive for influenza A, but unsubtypable for H1 by influenza RT-PCR. A confirmed case of H1N1 influenza A (swine flu): An individual with an influenza-like illness with a laboratoryconfirmed H1N1 influenza A virus detected by RT-PCR or culture [7-9]. All swine flu suspected case are categorize as per guidelines of MOHFW and WHO into 3 category[11]: Category A- Children with mild fever plus cough/sore throat with or without body ache, headache, diarrhea and vomiting.

Category B- If there are high grade fever and sore throat or who in addition to the symptoms of category A with <5 yr age, have chronic systemic viral illness, immunesuppressed conditions like steroid toxicity, nephritic syndrome, HIV/AIDS. Category C- Comprised of children who in addition to the category A and B symptoms, develop breathlessness, chest pain, drowsiness, hypotension, sputum mixed with blood, cyanosis, irritability, refusal to feed and worsening of underlying chronic illness.

Materials and Methods

This observational study was conducted in Department of Pediatrics of S. N. Medical College & Hospital Agra, from february to December 2015 on the basis of avilable patient records. The study had ethical clearance from the institutional ethical committee. Patients presented to our hospital with Influenza like illness (ILI) were subjected to throat swab testing. Throat swabs were sent for RT-PCR. Inclusion criteria: All confirmed cases of H1N1 swine flu were studied. Exclusion criteria: Patients with negative RT-PCR. After taking consent, all these identified swine flu cases were interrogated and if they were not able to communicate then attendant of the patient was interrogated to gather desired information as per-designed proforma. All data thus collected was summarized in MS excel and analyzed using statistical SPSS software and using chi square and ‘t’ test. Qualitative data was expressed in proportions and quantitative as mean and standard deviation.

Result

Total 59 patients were presented to our hospital in pediatric department with ILI during the study period and were tested for H1N1 throat swab with RT-PCR. Among them, 28 (47.6%) confirmed to have H1N1 infection and these cases were further studied. It was revealed from the study that there was no much difference in age range of male and female i.e. 3mo to 14yr for male and 3 mo to 9 yr for female and also there was no significant (p>0.05) difference in mean age in both the sexes.(Table 1) In our study, 19(67.9%) were males, those were significantly(p=<.001) higher than females (32.1%). (Table-1) Most common affected age group year(60.7%, p value <0.05). (Table 2)

was from 1-6

Fever(100%), cough(100%), coryza(71.4%) were the predominant symptoms. Others were vomiting(28.6%), sore throat(14.3%), abdominal pain(10.7%), loose stool(10.7%) and headache(10.7%). Less common symptoms were seizure (3.6%) and respiratory distress(7.1%). (Table 3)

http://www.pacificejournals.com/aabs

A-77

AABS; 3(1): 2016

Table 1: Age and Sex wise Distribution of patients Sex

Number

Mean

Std. Deviation

Minimum

Maximum

(67.9%)

3.27 yr

3.2

1 mo

14 yr

(32.1%)

3.07 yr

2.71

3 mo

9 yr

Total

(100)

3.22 yr

3.06

1 mo

14 yr

Chi-square for Male to Female proportion=12.8 with 1 degree of freedom; P= <0.001 Unpaired t test for male to female mean age= .327 with 26 degree of freedom; P = 0.87

Table 2: Age wise distribution of disease Age

Total

Percentage

0-12 mo 1-6 yr 7-14 yr Total

8 17 3 28

28.6 60.7% 10.7% 100%

Sign/Symptom

Total

Percentage

Fever Cough Coryza Sore throat Vomiting Abdomen pain Loose stool Headache Seizure Respiratory distress

28 28 20 4 8 3 3 3 1 2

100% 100% 71.4% 14.3% 28.6% 10.7% 10.7% 10.7% 3.6% 7.2%

Chi-square for age range = 7.22 with 2 degree of freedom; P=0.02

Table 3: Distribution of signs and symptoms in patients

Out of these, 8(28.6%) were treated on OPD bases and others (71.4%) were hospitalized. Out of these hospitalized patients only one patient (5%) required ICU admission and others admitted in isolation ward. Only two patient required oxygen. No one required ventilator. Mortality was nil.

Discussion

During 2009 swine flu pandemic, India reported more than 2000 confirmed cases equally affecting both sex with 25 deaths. Children and young adults were commonly affected and nearly 40% of those affected have been children less than 14 yrs [12]. Manoj et al found in his study that in 77 swine flu confirmed cases with age range 1.5 to 75 years, male to female ratio 0.51 and no significant mean age difference was found in male and female [13].

according to which males and females were equally affected [12] male preponderance in our study might be because parents seek medical advise more for male children. There was no significant (p>0.05) difference in mean age of both the sexes, similar to study done by Manoj et al [13]. In our study we found that patient were significantly (p<.05) higher within 1-6 year age group(60.7%), till now no one data is available related to this in our knowledge and more data is needed to validate this finding. All patients were presented with fever and cough. Others important presentation was coryza(71.4%), vomiting(28.6%), sore throat(14.3%), abdomen pain(10.7%), loose stool(10.7%), headache(10.7%) and seizure(3.6%). Respiratory distress was presented in only 7.1% cases.

In our study, 59 clinically suspected cases were tested for H1N1. 28 were positive, that accounted for 47.6% positivity. In one study P. Sriram et al founded 30.2% positivity of throat swab in suspected cases [14]. Males 19(67.9%) were significantly (p=<.001) higher than female 9(32.1%) against MOHFW 2009 pandemic report

These results were similar to study done by sriram et al who found that majority was presented with respiratory symptom and only few of them had GI and CNS symptoms. [14] Only two patients required ICU admission, no one required ventilator support and mortality was nil in hospital this was similar to other studies which encountered very low death rate[15,16].

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-78

Limitation of The Study

This study was in a tertiary care institute. Hence this analysis may not reflect the actual distribution of the cases at the population level. Further community based studies are required to analyse the actual impact of H1N1 infection in the community.

Conclusion

7. Centers for Disease Control and Prevention (CDC) Outbreak of swine-origin influenza A (H1N1) virus infection—Mexico, March-April 2009. MMWR Morb Mortal Wkly Rep. 2009;58:467–70. 8. Ministry of Health and Family Welfare, India. Available on http://mohfw.nic.in/press_releases_on_ swine_flu.htm. Accessed. on 04/02/2016

In present study we concluded that most of the patients were having respiratory symptoms. Most of the patients did not required ICU care and mortality was nil. These findings emphasize that panic about the disease should not be created in the community.

9. United States Centers for Disease Control and Prevention. Interim guidance on case definitions to be used for investigations of novel influenza A (H1N1) cases. http://www.cdc.gov/h1n1flu/casedef.htm, June 2, 2009.

Acknowledgements

10. Cunha BA, Syed U, Mickail N, Strollo S. Rapid clinical diagnosis in fatal swine influenza (H1N1) pneumonia in an adult with negative rapid influenza diagnostic tests (RIDTs): Diagnostic swine influenza triad. Heart Lung. 2010;39:78–86.

NONE

Funding None

Competing Interests

11. Ministry of Health and Family Welfare, Government of india. Swine Flu-Clinical management Protocol and Infection control guidelines. http://mohfw.nic.in.

None declared

Reference:

1. Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, et al. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009; 360 : 2605-15. 2. World Health Organization. Human infection with pandemic a (H1N1) 2009 influenza virus clinical observations in hospitalized patients. July 2009-update. and. Weekly epidemiological record. 2009. 3. Ministry of Health and Family Welfare Directorate General of Health Services (Emergency Medical Relief). Seasonal Influenza A (H1N10: Guidelinces for Vaccination with Influenza. Updated on 13 October, 2015. 4. http://mohfw.nic.in/showfile.php?lid=1170 accessed on 04/02/2016 5. Ministry of Health and Family Welfare, Government of india. Consolidated status of Influenza A H1N1. http://mohfw.nic.in/showfile.php?lid=1174 accessed on 04/02/2016 6. Priyanka Pulla. Outbreak of Swine Flu in india is no worse than seasonal flu, say specialists BMJ. 2015 Feb 26;350:h1097. Doi: 10.1136/bmj.h1097

12. Ministry of Health and Family Welfare, Government of india. Influenza A [H1N1]. Status as on 17th August, 2009. http://mohfw.nic.in. 13. Dr. Manoj Verma, Dr. Sanjay Jain, Dr Sbhash Bilonia and Dr. R.K. Manohar. A PointCross-sectional study of Swine Flu Cases admitted at a Tertiary Level Hospital, Jaipur (Rajasthan) India. International Multispecialty Journal of Health (IMJH). Vol-1, Issue-2, April_2015. 14. P. Sriram, Manish Kumar, R. Renitha, Nivedita Mondal, Vishnu B. Bhat. Clinical Profile of Swine Flu in Children at Punucherry. Indian Pediatr (2010) 77:1093-1095. 15. Dilip K. Biswas, Prabhdeep Kaur, Manoj Murhekar & Rama Bhunia. An outbreak of pandemic influenza A (H1N1) in Kolkata, West Bengal, India, 2010. Indian J Med Res 135, April 2012, pp 529-533 16. Writing Committee of the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza, Bautista E, Chotpitayasunondh T, Gao Z, Harper SA, Shaw M, Uyeki TM, et al. Clinical aspects of pandemic 2009 influenza A (H1N1) virus infection. N Engl J Med 2010; 362 :1708-19.

http://www.pacificejournals.com/aabs

Original Article A Retrospective Study to Evaluate Tuberculosis Burden by FNAC of Cervical Lymphnode From the Patients Coming to Hamidia Hospital. Maneesh Sulya*, Sonal Gupta , Kamlesh Patel Department of Pathology, Gandhi Medical College, Bhopal. India Keywords: TB, Cervical lymphadenopathy (CL), FNAC, AFB.

ABSTRACT Background: Tuberculosis is a systemic disease with the possibility of involvement of multiple organs and lymph nodes. This study conducted to evaluate the usefulness of FNAC as a diagnostic tool in cases of cervical lymphadenopathy (CL) and to study the demographic burden. Methods: The present study on 608 patients of CL was conducted in the Department of Pathology Gandhi Medical College and Hamidia Hospital, Bhopal from January 2014 to May 2015. FNAC of the enlarged lymphnodes was performed with informed consent of the patient. Result: Out of total 608 FNAC done, 45 (7.41%) were having granulomatous lesions and 112 (18.42%) were having tubercular lymphadenopathy. Tubercular lesions and granulomatous lesions were found to be more in females, 77.78% as compare to males and that is more in Muslim females, 42.8% and 46.7%, respectively. Conclusion: Tuberculosis is common cause of CL. In contrast to other studies 0ur study showing more incidence in females as compare to males.

*Corresponding author: Dr. Maneesh Sulya, Assistant Professor, Department of Pathology, Gandhi Medical College and Hamidia Hospital, Bhopal. MP. India Phone : +91 9826609403 E-mail: drmanish1619@gmail.com,

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-80

Introduction Fine needle aspiration cytology (FNAC) is a simple and rapid diagnostic technique. Because of early availability of results, simplicity, minimal trauma and complication, the aspiration cytology is now considered as a valuable diagnostic aid and is gaining popularity.(1) Tuberculosis is a systemic disease with the possibility of involvement of multiple organs and lymph nodes. (2, 8) Tuberculosis (TB) remains the number one killer infectious disease affecting adults in developing countries. The 1990 World Health Organization (WHO) report on the Global Burden of Disease ranked TB as the seventh most morbidity-causing disease in the world, and expected it to continue in the same position up to 2020.(3) The present study is undertaken to evaluate the usefulness of FNAC as a diagnostic tool in cases of cervical lymphadenopathy and also to study the demographic distribution of the burden. This is of particular importance in view of the high prevalence of tuberculosis in our country, vivid presentation of tuberculosis and because of the fact that AFB are seen mostly in purulent aspirate smears which do not show granulomas, necrosis or epithelioid cells and which in absence of Ziehl-Neelsen staining can be dismissed as acute suppurative lymphadenitis (4).

Table1: Showing Result of Lymphadenopathy:

FNAC

Lesions Inconclusive Inflammatory smears* Granulomatous lesions Tubercular lymphadenitis Reactive hyperplasia Chronic nonspecific lymphadenitis Metastatic/ Malignant lesions Total

Total

Percentage 19.09 19.24

Patients of all groups with enlarged cervical lymphnodes were taken into the study. In smear the presence of epithelioid cell granuloma with or without necrosis taken as tubercular lesion which was further supported by presence of AFB by Ziehl Neelsenâ&#x20AC;&#x2122;s stain.

Result Out of total 608 FNAC done, 45 (7.41%) were having granulomatous lesions and 112 (18.42%) were having tubercular lymphadenopathy. Other lesions were as shown in table 1.

Male 64 45

Female 52 72

7.41

18.42

2.63

21.21

12.00

277

331

608

100

Tubercular lesions and granulomatous lesions were found to be more in females (77.78%) as compare to males (22.22%). The reason for this is religious variations found in patients undergoing FNAC. Table 2: Showing distribution of tubercular lesions of cervical lymphnode according to sex and religion:

Materials and Methods The present study on 608 patients of cervical lymphadenopathy was conducted in the Department of Pathology, Gandhi Medical College and Hamidia Hospital, Bhopal from January 2014 to May 2015. FNAC of the enlarged lymphnodes was performed with informed consent of the patient; following thorough clinical examination. Palpable nodes (largest one) were aspirated in the minor OT of Hamidia Hospital by a 23-25 G needle and syringe. In all the cases, alcohol fixed smears were made and stained with H & E and Pap stains and one dry smear prepared for Ziehl-Neelsen staining.

Cervical

Sex

Hindu

Muslim

Male

17 (15.2)

15 (13.4)

Female

32 (28.6)

48 (42.8)

42.8% Muslim females were having tubercular lymphadenitis out of total 112 tubercular lymphadenitis cases this was followed by Hindu females (28.6%), Hindu male (15.2%) and Muslim male (13.4%) respectively. (Table 2) Table 3: Showing distribution of granulomatous lesions of cervical lymphnode according to sex and religion: Sex Male

Hindu 8 (17.8)

Muslim 2 (4.4)

Female

14 (31.1)

21 (46.7)

From table 3 it is clear that granulomatous lesions in cervical lymhnode found more in Muslim females (46.7%) followed by Hindu females (31.1%), Hindu male (17.8%) and Muslim male (4.4%) Table 4: Showing distribution of tubercular lesions of cervical lymphnode according to sex and religion: Sex

Hindu

Muslim

Male

25 (15.9)

17 (10.8)

Female

46 (29.3)

69 (44.0)

http://www.pacificejournals.com/aabs

A-81

AABS; 3(1): 2016

P value for this table is 0.0295 (<0.05) so the findings are significant.

department for her kind support throughout the work for this paper.

Another interesting fact was found in the age distribution that almost 60% of the affected patients were in the age group of 41 to 60 yrs followed by age group of 0 to 20yrs (31.20%). No one was found in the age group of above 60yrs.

Funding

Table 5: Age and gender wise distribution:

None

Competing Interests None

Age group Tubercular Granulomatous (yrs) lesion lesion

Total %

0-20

31.20

21-40

59.90

41-60

08.90

Above 60

Total

112

157

100

Discussion Our study reveals tuberculosis and other granulomatous lesions were most frequently diagnosed lesions in cervical lymphnodes, 18.42% and 7.41%, respectively which is supported by the study done by Mazhar Iqbal et al (5) from Jinnah Postgraduate Medical Centre, Karachi they found tubercular and granulomatous lesions were 75% in occurrence. The study done by Paliwal Nidhi et al, New delhi (6) and Sharma et al (7)was showing just higher accurance of tubercular lesions in females which supporting our results female dominance of tubercular lesions in cervical lymphnode FNAC. Female predominance in our study was due to higher occurance of tubercular lesions in Muslims as compare to Hindu females. In present study the maximum occurrence of tuberculosis in cervical lymphnodes were found in age group of 21- 40 yrs which is almost 60% followed by 0-20yrs age group (31.20%). This is very well supported by the study done by Paliwal Nidhi et al, New delhi.

Conclusion Tuberculosis is common cause of CL. In contrast to other studies 0ur study showing more incidence in females as compare to males.

Reference 1. S Shamsad Ahmad, Akhtar Shakeel et al. Study of Fine Aspiration Cytology in Lymphadenopathy with special reference to Acid Fast Staining in cases of Tuberculosis. JK Science 2005;7(1),1-4. 3 2. Rana SS, Sharma V, Sharma R, Bhasin DK. Involvement of mediastinal/intraâ&#x20AC;&#x2018;abdominal lymph nodes, spleen, liver, and left adrenal in presumed isolated pancreatic tuberculosis: An endoscopic ultrasound study. J Dig Endosc 2015;6:15â&#x20AC;&#x2018;8. 3. Quality assurance network in sputum smear microscopy under RNTCP. Revised National Tuberculosis Control Programme. Central Tuberculosis Division. Directorate General of Health Services. Ministry of Health and Family Welfare, New Delhi, 2001. 4. Metre MS, Jayaram G. Acid fast bacilli in aspiration smears from tuberculous lymphnodes. Acta Cytologica 1987; 31: 17-19. 5. Mazhar iqbal, anis subhan, asadullah aslam, frequency of tuberculosis in Cervical lymphadenopathy. J Sur Pakistan (international) 15 (2) april - june 2010; 10709. 6. Paliwal Nidhi1, Thakur Sapna1, Mullick Shalini2 and Gupta Kumud, fnac in tuberculous lymphadenitis: experience from a tertiary level referral centre, Indian J Tuberc 2011; 58: 102-107. 7. Sharma S, Sarin R, Khalid UK, Singla N,Sharma PP, Behera D. Clinical profile and treatment outcome of tuberculous lymphadenitis in children using DOTS strategy. Indian J Tuberc 2010; 57: 4-11.

I am thankful to our head of the institute who have given me free hand to work and also thankful to our head of the

8. Orell SR, Sterret GF, Whitaker D, Heerde PV, Miliaukas J, and Field A. Lymph Node Chapter 5.In Orell SR, Sterret GF, Whitaker D (eds): Fine Needle Aspiration Cytology,4th edition, New Delhi, Elsevier . 2005 ; 83-124.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Acknowledgements

Original Article Antenatal HIV Voluntary Counseling and Testing: Acceptance in Government centre of North India Ruchika Garg*, Shikha Singh, Saroj Singh, Indira Sarin, Manisha Pathak Department of Obstetrics and Gynecology, Sarojini Naidu Medical College and Hospital, Agra, UP, India. Keywords: antenatal women, HIV prevalence, Prevention of Mother To Child Transmission, Tertiary care,Voluntary Counseling Testing

ABSTRACT Background: Routine HIV counseling and testing done as a mandatory part of antenatal care in India has lead all pregnant women comes under the prevention of mother to child transmission of HIV(PMTCT) program. Despite such strategies, the effective execution and uptake of these programs remains a major obstacle. It is thus, important to understand experiences of pregnant women undergoing HIV testing to detect the flaws on the part of the provider and the benefiter and eliminate them to strengthen the PMTCT services. Aim: Westudied the acceptability of HIV voluntary counseling and testing (VCT) in antenatal women attending a tertiary health centre of north India. The impact of sociodemographic factors on HIV prevalence and uptake of PMTCT was also studied and the possible reasons for dropouts were determined. Methods: Firstly we performed pretest counseling and sociodemographic data and blood samples collected from the consenting antenatal pregnant women were also taken. Samples were tested for HIV antibodies as per WHO guidelines. Data was analysed and presented as mean, percentages and tables. Results: Of 30150 pregnant women counseled, 23464 (77.82%) underwent testing.136 / 23464 women tested seropositive. The prevalence of HIV in antenatal women was found to be 0.58%. Majority of these women were young and belonged to the age group 20-24 years (0.23%).22% refused testing, the reasons for which were tried to b sought. Strong associations were found between the HIV seroreactive status and marital status, low education status, low social class, high parity and unemployment. Conclusion: To eliminate pediatric transmission of HIV and to create more awareness regarding HIV infection and parent to child transmission, there is a need to make VCT and PMTCT programs more acceptable to the population. The observations found in the study were consistent with the national projections.

*Corresponding author: Dr Ruchika Garg, Assistant Professor, Department of Obstetrics and Gynecology, Sarojini Naidu Medical College and Hospital, Agra, UP, India E-mail: ruchikagargagra@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-83

Introduction HIV continues to be one of the greatest health challenges in the world with approximately 35 million people living with HIV infection globally in 2013. [1] India has the third largest HIV epidemic in the world. [2] In India major route of HIV transmission is through sexual contact (85.6%). Nearly, 5% of infections are attributable to parent to child transmission. [3] The epidemic disproportionately affects women, who account for 39% of the total infections in the country. [2] Women stand at a higher risk of HIV infection and are a source of transmission to their children, thus forming the focus of most AIDS control programs aimed to meet the goal of achieving virtualelimination of pediatric HIV.Voluntary counseling and testing (VCT) has beenidentified as an effective tool in reducing HIV transmission. So far, very few studies have been conducted citing the acceptance of VCT and Prevention of Mother to Child Transmission programs (PMTCT) from this part of India. Undertaking this study was therefore important to understand the current trend of HIV seroprevalence and its sociodemographic impact, to formulate better strategies for success of PMTCT in India.

Methods Place of study This study is carried out in a tertiary care referral hospital of north India where mostly patients referred from other centers undergo antenatal checkups. The present study sets out to determine the impact of VCT in preventing mother to child transmission of HIV and to simultaneously identify the lacunae in the prevailing PMTCT programs. Patients and period of study Pregnant women consulted at the antenatal clinic of this hospital are routinely advised to undergo HIV screening after pretest counseling done by trained field workers and informed consent. The local pathological laboratory of our department serves these laboratory services to all such patients, and the tests are carried out as per the guidelines laid down by the national aids control organization (NACO), India. [4] The results were collected from all pregnant women tested in this laboratory and no selection bias was observed. The findings were analyzed over 9.5 years from October 2005 to April 2015. Ethical consideration Informed written consent was obtained from each participant after pretest counseling and the participants were free to withdraw from the test any time they wanted. Ethical clearance for the study was obtained from the ethical committee of the institute. Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 Testing procedure If the participant agreed to the testing,she was referred to the laboratory technician who performed a rapid HIV test (SD BIOLINE HIV -1/2 3.0 Rapid Test Procedure; bio standard diagnostics Pvt ltd, India). If the participant tested positive for HIV or had an indeterminate test result, the result was checked using the COMBAIDS â&#x20AC;&#x201C;RS Advantage-ST HIV1 & 2 IMMUNODOT TEST KIT (Span Diagnostics Ltd. India). Rapid test kits were kept under optimal conditions and used before the expiry date. Results were read under good illumination test quality was ascertained by running regular negative and positive control tests. The results were obtained in 5-10 minutes after the first test and in about 20 minutes after the second. The first rapid test kit used was previously evaluated by Consortium of National Reference Laboratories(NRLs) of Govt. of India and was found to have high sensitivity(>=99.5%) and specificity (>=98.0%); and the second test showed 100% sensitivity and specificity and intra and inter run precision. Samples giving positive results were re analyzed using the COMBAIDS- RS Advantage-ST immunodot test kit. Standard biosafety, record keeping and client confidentiality procedures were observed. Statisticalanalysis: Theresults were presented in percentages and means. Simple inferential statistics were used. Qualitative data collected from group discussions were analyzed through detailed content analysis and ethnographic summary.

Result Data was collected and analysis was done from a total of 30,150 pregnant women visiting the antenatal clinic during the period of October 2005 to April 2015. These women were provided voluntary pretest counseling for HIV Testing, out of which 23464 (77.82%) consented for testing. About 22% pregnant women opted out from testing and hence we could not assess the seroreactivity in these women. 136 out of 23464 women, i.e. (0.579%) testedseropositive for HIV antibodies in the double rapid tests (table 1). The age of subjects ranged from 15 to 42 years with a mean age of 26.10 years. Most of these women, (10839) 46.3% were in the age group of 25 - 34 years followed by 20-24 years, (10373) 44.2%, >35 years (5.1%), and least in 15-19 years (4.4%). Among the seropositive women, the majority (38.9%) were aged 20-24 years with the prevalence of this population being 0.23%, followed by the age group of 25-34 years (36%), the prevalence being 0.20%, then > 35 years (18%) the prevalence of 0.076%, and in the age group of 15 â&#x20AC;&#x201C; 19

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-84

years (11.76%) with the prevalence of this category being the lowest of 0.06% as shown in table 2. The mean age of the HIV positive women was found 26.29 years.

unemployed, 15028 subjects (64%) had primary education, and as many as 17840 (76%) were found to be in the low socio economic class.

Table 3 shows the socio demographic characteristics, 21583 women (92%) were married, 21579 (92%) were

102 /136 seroreactive women (75%) accepted to utilize the PMTCT facility.

Table 1: Acceptance of HIV testing among pregnant women who were counseled Percentage (%) 77.82% 0.579%

Number 30150 23464/30150 136/23464

Total no. of pregnant women counseled Total no. of pregnant women tested for HIV Total no. of HIV seropositive pregnant women

Table 1 - The prevalence of HIV among antenatal women visiting our centre during the period of study was found to be 0.58% which is higher than the national prevalence of 0.4% among antenatal women. This data is of concern as this part of India has been categorized as a low prevalence area. Table 2: Age wise distribution of pregnant women Age group 15-19 years 20-24 years 25-34 years >35 years

HIV positive N=136

% positive

Prevalence

HIV negative N=23328(%)

16 53 49 18

11.76% 38.9% 36% 13.2%

0.06% 0.23% 0.20% 0.08%

1018 (4.4%) 10320 (44.2%) 10790 (46.3%) 1200 (5.1%)

Table 2 shows that HIV is fairly prevalent among the antenatal women in the age group of 20-24 years followed by 25-34 years, when the sexual activity is maximal. This is in consensus with the national survey report by NACO. Table 3: Socio Demographic characteristics Of Pregnant Women Characteristics Marital status Single Married Educational attainment No formal education Primary education Secondary education Higher education Occupation Unemployed Employed Social class High Middle Low Parity Primipara Multipara Grandmultipara Gestational Age 1st trimester 2nd trimester 3rd trimester

HIV positive

% positive

HIV negative

15 121

11% 89%

1866 (8%) 21462 (92%)

22 97 15 02

16.4% 71.2% 10.9% 1.5%

4665(20%) 14931 (64%) 2799(12%) 933 (4%)

117 19

86% 14%

21462 (92%) 1866 (8%)

2 23 111

1.5% 17% 81.5%

467 (2%) 5132 (22%) 17729 (76%)

52 74 10

38.2% 54.4% 7.4%

7302 (31.3) 12131 (52%) 3895 (16.7%)

15 20 101

11.0% 14.7% 74.3%

2799 (12%) 6998 (30%) 133531(58%)

Table 3 demonstrates the relationship of HIV prevalence with the socio demographic factors of pregnant women. this clearly suggests that illiteracy, poverty, unemployment, are the factors responsible for the ignorance regarding HIV transmission and hence high prevalence. http://www.pacificejournals.com/aabs

A-85

AABS; 3(1): 2016

Discussion [2]

India has a low HIV prevalence of 0.3%. Yet, in terms of individuals infected, it is home to the third largest number of people living with HIV in the world. Sexual contact is the most important route of HIV transmission in India. Mother to child transmission (MTCT) is by far the most important route of HIV spread in the pediatric population (90%). [5]The transmission of the virus from the mother-to-child during pregnancy, labour and delivery or breastfeeding is called mother to child transmission. [3] According to WHO, globally, anestimated 2.1 million individuals became newly infected with HIV in 2013. This includes over 240,000 children (<15years), and most of them were from developingcountries and were infected by their HIV positive mothers during pregnancy, delivery or breastfeeding. [1]It is estimated that out of 27 million pregnancies every year, nearly 49,000 occur in HIV positive mothers. [3] However, out of these 27 million pregnancies, only about 52.7% attend health services for skilled care during child birth in India. Of those who availed health services, 8.83 million antenatal patients received HIV counseling and testing (March 2013) out of which 12,551 pregnant women were detected to be HIV positive.Of the 12,000 pregnant women found to be living with HIV, 84% were provided antiretroviral drugs (ARVs) to prevent mother to child transmission of HIV. [6] It is well established that MTCT can now be reduced to less than 2 percent from 25–30 percentearlier.[7]With the use of effective antiretroviral treatment (ART) and nonantiretroviral (ARV) strategies, MTCThas been virtually eliminated in developed countries. However, prevention of transmissionthrough breast milk and formulating an effective ARV regimen has remaineda major challenge for developing countries.[8]According to WHO, in 2013, 67% of pregnant women living with HIV in low and middle income countries (970,000) received ART to avoid transmission of HIV to their children. This is up from 47% in 2010. [9] The Indian government is committed to eliminating new HIV infections among children by 2015. India’s PMTCTprogram started in 2002. To date, there are over 15000 sites offering PMTCT servicesbased on 2013 WHO guidelines, the program initiates antiretroviral treatment for all pregnant and breastfeeding women living with HIV regardless of cd4 count or stage of infection.[9] In 20132014, 9.7 million pregnant women accessed HIV testing against a target of 13.2 million ---a coverage of 74%.[9]

of clients significantly facilitates PMTCT programs by reducing travelling time and expenses. The sensitivity and specificity of these tests are greater than or equal to 99% and similar to those of ELISA. The rapid HIV tests are most suitable in developing countries where the majority of pregnant women are attended by traditional birth attendants (TBA), the pregnant women use the formal formal health sector only as a back up arrangement. If pregnancy and labour progress as expected, the pregnant women attends a local formal health post once at booking which mostly may be late in pregnancy. In these circumstances, rapid HIV tests afford the opportunity to screen for HIV in women attending peripheral units and provide results during same visit VCT hasproved to be of promising help in reducing HIV transmission and has shown to provide behaviorchange and emotional support for those who test positive for HIV1. At the same time, it is feasible and acceptable in reducing perinatal transmission of the virus.In order to improve the effectiveness of India’s PMTCTprogram and to meet the goal of achieving the virtualelimination of pediatric HIV in the country, it is importantto devise appropriate evidencebased strategies.Ideally,all women should be screened for HIV before delivery during an initial prenatal care visit so that potent ART can be started in those found to be HIV infected. VCT is recognized as a priority in national HIV programs because it forms the gateway to HIV/AIDS prevention,care, treatment and support interventions but this has still not become embedded in peripheral health facilities in India. It is critical to increase the prenatal detection rate of HIV infected pregnant women so that effective interventions can be delivered. This study recorded the apprehension of pregnant women regarding getting tested. This was found to be attributed to the fear of being tested positive, and the stigma attached to HIV. We found the overall prevalence of HIV amongst pregnant women to be 0.57%. The national prevalence in antenatal women was 0.4%, which is lower than our finding. [9]One reason for this could be that our center being a referral tertiary health center, so most of the seroreactive cases are referred here which could be a reason for the overall higher prevalence.

The availability of affordable, accurate, reliable, simple and rapid HIV tests providing results within the time frame of a single brief antenatal visit for single or a small number

Our study supports that HIV prevalence is higher inthe reproductive age group when there is maximal sexual activity. The age wise distribution showed a higher prevalence of infection in young reproductive population, being maximum in age group of 20-24 years (0.23%) followed by 25 – 34 years (0.20%), >35 years (0.08%) and 15-19 years (0.06%). This observation seen in our study is

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-86

consistent with the national data, where the prevalence of HIV in India among 20-24 years is 0.18% and that among 15-19 years old is 0.04%. [3] Marital status was seen to have a strong relation with the HIV status of the study participants. Also, in Indian patriarchal society, women especially of the lower social classcan not influence their husbandâ&#x20AC;&#x2122;s behavior and demand safe sex. This study also depicts that the infection of HIV is more prevalent among the pregnant women who had no formal education at all or who did not complete secondary school education. This is in agreement with the study that reported that women having higher education have better knowledge of HIV transmission in contrast, the lower levels of female education promotes ignorance about the transmission and prevention of HIV infection especially in the unborn child. Most of the women could not complete secondary education. This may explain their being unemployed and also belonging to the low socio economic class. Unemployment being associated with poverty, and has also been linked with unsafe sex for money, thus, increasing the risk of HIV infection.[10][11] Also, in this study it was found that most women who attended our antenatal OPD were multigravida, while primigravida who might be at higher risk of HIV infection by virtue of relatively younger age and the associated risky sexual behaviors, do not utilize formal antenatal services. Furthermore, those that utilized the services presented late in third trimester, when interventions could be late to address some adverse trends of HIV on pregnancy especially in resource limited settings. Financial constraint, ignorance and lack of awareness were cited as reasons for late and poor utilization of antenatal services. This is in synchronization with other studies.[12][13] The implementation of the governmentâ&#x20AC;&#x2122;s mandatory screening policy, which explicitly states that universal HIV screening should be included as an integral component of routine ANC check up, ensured that the women who are diagnosed with HIV would be linked to HIV services for their own health as well as to ensure prevention of HIV transmission to newborn babies under the PMTCT program. In this study, 22% of the initially counseled women opted out testing after HIV counseling. The reasons for dropping out were the taboo attached to HIV, apart from the ignorance and financial constraints. The factors that influence the acceptance to HIV testing and receiving test results including the counseling technique used, were suspicion of being already infected, fear of having to cope

with the result should it be positive, fear of discrimination, domestic violence or divorce. There is an almost hysterical kind of fear in India regarding the stigma and discrimination of HIV that the parents and in-laws can blame women for infecting their husbands, while children can be denied right to go to school, most women depended on their husbands and in laws to take the decision regarding getting tested. This is in accordance with the findings of other authors too. [9][12]

While comparing group and individual pretest counseling techniques during the study, it was shown that there was a better patient acceptance of HIV testing following individual rather than group counseling. In our study, pretest counseling was partly provided as group counseling because lesser trained personnel are required and can be easily implemented at a public health set up. To maintain confidentiality, post test counseling was always provided individually. At the same time it is important to understand that there is a strong need for couple counseling in order to make them understand the benefits of HIV testing, reducing HIV transmission and helping couples to cope with the HIV seropositive status. Couple counseling and testing will also ensure inclusion of underserved couples, especially those who are less educated and economically disadvantaged.We can thus, identify discordant couples and offer methods to them for reducing risk and transmission within their relationships.The World Health Organization recommends that couple testing be expanded in settingswhere routine HIV testing is offered, with supportfor mutual disclosure to empower couples to makeinformed decisions about HIV prevention and familyplanning.However there is a need to assess the acceptability and impact of this effort among couples.[14] Among the study subjects, VCT was found to behigh so also the utilization of PMTCT services, 77% and 75% respectively. The study shows an upward trend in the uptake of PMTCT program in this part of north India. This is in accordance with the national survey report. [2] Conclusion:For this part of north India the prevalence in our study was found higher than that of the national survey because ours is a referral centre with a higher input of HIV infected pregnant women and the general population of antenatal women is being tested at adjoining district hospital. The acceptance of VCT was high so also was the uptake of PMTCT facility. Social and demographic factors play an important role in the spread of HIV infection, knowledge about the transmission, awareness of self status and protection, awareness

http://www.pacificejournals.com/aabs

A-87 regarding transmission to the baby and its protection, various government and government aided programs and their uptake by pregnant women. The prevailing national PMTCT program is being mostly jeopardized due to large number of women dropping out of the PMTCT cascade. In order to increase this uptake PMTCT programs can be integrated to demonstrate other health benefits to the mother and the child. It is imperative to look into alternative methods for implementation of PMTCT programs in order to maximize the reach of services to HIV infected women in least cost. Involvement of nongovernment organizations and national programs could maximize the reach of these services to infected women.

Reference 1. Report on Global HIV/AIDS epidemic https:// www.aids.gov/hiv-aids-basics/hiv-aids-101/global statistics/index.html 2. National Aids Control Organization (NACO), Government of India, Annual Report (2012-13) department of AIDS Control, ministry of health and family welfare, India 3. New global plan to eliminate HIV infections among children launched at UN http://www.unicef.in/ Story/1123/HIV-AIDS 4. Baveja UK: HIV antibody testing with special reference to HIV-1. In HIV Testing Manual, laboratory diagnosis, Biosafety and Quality control National AIDS Control Organisation: New Delhi:45-67 5. Prevention of mother to child transmission, HIV prevention programs [http://www.avert.org/ prevention-mother-child-transmission-pmtct-hiv.htm] 6. NACO guidelines for the prevention of mother to child transmission of HIV [http://ww.naco.nic.in/ pmtct.html] 7. Cooper ER, Charurat M, Mofenson L, Hanson IC, Pitt J, Diaz C, HayaniK,Handelsman E, Smeriglio V, Hoff R, et al: Combination antiretroviral strategies for

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016

9. 10.

11.

12.

13.

14. 15.

the treatment of pregnant HIV-1-infected women and prevention of perinatal HIV-1 transmission. J Acquir Immune DeficSyndr 2002, 29(5):484–494. ShrinivasDarak, MayuriPanditra, RituParchure, VinayKulkarni, SanjeevaniKulkarni and Fanny Janssen et al: Systematic review of public health research on prevention of mother-to-child transmission of HIV in India with focus on provision and utilization of cascade of PMTCT services: BMC Public Health 2012, 12:320 National Aids Control Organisation (NACO), Government of India, Annual Report (2013-2014). Dandona R, Kumar SG, Kumar GA, Lakshmi V, Dandona L: HIV testing among adults in a high prevalence district in India. Natl Med J India 2009, 22(6):289–293. Panditrao M, Darak S, Kulkarni V, Kulkarni S, Parchure R: Socio-demographic factors associated with loss to follow-up of HIV-infected women attending a private sector PMTCT program in Maharashtra, India. AIDS Care 2011, 23(5):593–600. Rogers A, Meundi A, Amma A, Rao A, Shetty P, Antony J, Sebastian D, Shetty AK: HIV-related knowledge, attitudes, perceived benefits, and risks of HIV testing among pregnant women in rural Southern India. AIDS Patient Care STDS 2006, 20(11):803–811. Samuel NM, Srijayanth P, Dharmarajan S, Bethel J, Van Hook H, Jacob M, Junankar V, Chamberlin J, Collins D, Read JS: Acceptance of HIV-1 education & voluntary counselling/testing by &seroprevalence of HIV-1 among, pregnant women in rural south India. Indian J Med Res 2007, 125 (1):49–64. Orne-Gliemann J, Tchendjou PT, Miric M, Gadgil M, Butsashvili M, Eboko F, Perez-Then E, Darak S, Kulkarni S, Kamkamidze G, et al: Couple-orientedprenatal HIV counseling for HIV primary prevention: an acceptabilitystudy. BMC Publ Health 2010, 10:197.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Clinical Spectrum of non-Hodgkin lymphoma: A Hospital Based Study of 410 Cases. Vijayashree Shivappa Neeravari1*, Doddappa Bannigidad2 1

Department of Pathology, Kasturba Medical College, Manipal, India Department of Biochemistry, Kasturba Medical College, Mangalore, India

Keywords: Clinical Manifestation, “B” Symptoms, Lymph Node, Hepatomegaly, Splenomegaly, Non-Hodgkin Lymphoma.

ABSTRACT Background: Lymphoma can be misdiagnosed due to lack of awareness of various clinical features. There is a rising incidence of lymphomas over the past few years. Several non Hodgkin lymphoma can present with different ‘B’ symptoms, different clinical signs and different sites of involvement. This study was done to evaluate the different clinical features and lymph node involvement at presentation in non Hodgkin lymphoma. Methods: 410 diagnosed and treated cases of non-Hodgkin lymphoma were selected from Kasturba Medical College, Manipal between Jan 2009 and December 2012. Clinical history, ‘B’ symptoms, laboratory investigations, including imaging procedures were noted in all the cases. The diagnosis of lymphoma was based on histology, IHC and WHO2008 classification. Results: Out of 410 cases of non Hodgkin lymphoma, Nodal lymphomas comprised of 57.34% of total cases while extranodal lymphomas accounted for 42.6% of cases. B cell lymphoma accounted for 80.3% and T cell lymphoma were 19.6%. Most of the patients with NHL were in the age group of 50-60 yrs. 69% of cases were males while 31% of cases were females. Anemia was the commonest presenting feature among ‘B’ symptoms and cervical lymph node was most commonly involved lymph node. Conclusion: The clinical spectrum of lymphoma sometimes delays its diagnosis, leading to its eventual presentation in late stages. Awareness is required among the health professionals regarding its varied clinical presentations to diagnose at the earliest.

*Corresponding author: Dr Vijayashree S Neeravari, Department of Pathology, KIMS, Koppal, India 583231. Phone: +919886241910 E-mail: drvijayashreen@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-89

Introduction There is a rise in incidence of lymphomas over the past few years. Non-Hodgkin’s lymphomas (NHL) can arise in lymph nodes, other lymphatic tissues and extranodal organs.1 Lymphoma can occur at any age; however, it has a bimodal presentation with one peak in 0-10 years of life and another peak after 50years. Patients with lymphoma, both Hodgkin and Non-Hodgkin usually present with “B” symptoms or with enlarged lymph nodes. “B” symptoms refer to systemic symptoms of fever, night sweats and weight loss. Symptoms may also develop due to pressure effects of lymph nodes on surrounding structures or due to involvement of extra nodal sites such as gastrointestinal tract, central nervous system, liver, or bone, thus leading to atypical presentations.2 Due to the varied clinical manifestations, many patients are misdiagnosed and mistreated.3 Patients having “B” symptoms show a more severe condition than asymptomatic patients with the same cancer stage, tumour location or size. Onset of “B” symptoms at the time of diagnosis suggests that lymphoma is progressing.4 This study was planned to evaluate the varied clinical presentations of non Hodgkins lymphomas and the patterns of lymph node distribution.

Materials and Methods Diagnosed and treated 410 cases of non Hodgkin lymphoma were selected from the Department of Pathology, KMC, Manipal between Jan 2009 and December 2012(4 years), between the age group of 1–90 years. Clinical history, physical examination and basic laboratory investigations, including imaging procedures and bone marrow examination were done in all the patients. The diagnosis of lymphoma was based on histology, IHC and WHO-2008 classification. Appropriate clinical information regarding age, gender, anatomic location and occurrence of “B” symptoms were noted which included fever (temperature >38°C [>100.4°F] for 1-2 weeks), weight loss of >10% of body weight in ≤6 months and drenching night sweats. Types of NHL, anatomic location and occurrence of “B” symptoms were confirmed with the help of medical records. Data was statistical analysed using SPSS - soft ware.

Result Out of 410 cases, nodal lymphomas comprised of 57.34% of cases while extranodal lymphomas accounted for 42.6% of cases. B cell lymphoma accounted for 80.3% and T cell lymphoma were 19.6%. Most of the patients with NHL were in the age group of 50-60 yrs. There was male preponderance with 69% of cases being males while 31% of cases were females.

AABS; 3(1): 2016 other ‘B’ symptoms like weight loss and fever which was observed in 23.9% and 21.7% of cases respectively. Hepatomegaly and splenomegaly was observed in 23.1% and 20.9% of cases respectively, while effusion was noted in 10.9% of cases. (Table 1) 55.8% of NHL cases presented with cervical lymphadenopathy, followed by supraclavicular lymph node enlargement, which was involved in 39.5% of NHL cases and axillary lymph node involvement was seen in 32.4%. Mesenteric, inguinal and mediastinal lymph node involvement was seen in 28.4%, 25.6% and 12.6% of NHL cases respectively. (Table 2)

Discussion The incidence of the subtypes of non-Hodgkin lymphoma shows geographical variations, differs in history and clinical presentation. These variations are dependent upon environmental influences, such as local common viral infections including Epstein Barr virus and socioeconomic factors.5 Hingorjo et al5, and Hassan et al6 have documented anemia in 100% and 87.5% of NHL cases respectively, which is similar to our present study. However Cunlan et al7, Ghosh et al8 and Idris et al9 have documented much lesser incidence of anemia with 55%, 49% and 65% of NHL cases respectively. (Table 1) Multiple mechanisms contribute to the development of anaemia in NHL patients. These include anaemia of chronic disease (ACD), autoimmune haemolytic anaemia (AIHA), bone marrow infiltration, nutritional deficiencies and blood loss. Cytokines such as IL-6 have been shown to increase hepcidin levels which result in iron restriction and signs of anaemia of chronic disease.10 It is now recognized that anaemia may lead to symptoms that adversely affect physical status and diminish functional capacity and quality of life.11-12 The presence of anaemia is also associated with poorer prognosis and increased mortality.13-14 Anemia in the presence of bone marrow involvement has poor prognosis.5 However, anemia is poorly recognized and under-treated. Hepatomegaly was observed in 83.6% and 45% of cases by Hingorjo et al5 and Hassan K et al6 respectively, which is much higher in comparison with our study. However, Idris et al9 has documented 22% of cases which is similar to our present study. (Table 1)

In the present study anemia was the commonest presenting sign and accounted for 90.4% of the cases followed by

Lymphoma cell infiltration of the liver with hepatomegaly is more common in NHL than in Hodgkin Lymphoma(HL), with 16%–43% of cases showing hepatic involvement. 15-16 Extrahepatic obstruction is also more common in NHL than in HL, and hepatic infiltration is more common in low-grade B-cell lymphomas than in high-grade lymphomas. Acute

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-90

hepatic failure can occur in NHL as seen in HL, which is caused by sudden ischemia related to massive infiltration of the sinusoids or replacement of liver parenchyma by malignant cells.17 Elevated level of serum LDH is also often seen in patients with NHL, especially in highly aggressive type such as Burkitt or lymphoblastic lymphoma, reflecting high tumor burden, extensive infiltration of the liver, and coincident immune-mediated Hemolytic Anemia, which are associated with poor prognosis.15 Spleenomegaly was observed in 44.8% and 47.5% of cases by Hingorjo et al5 and Hassan K et al6 respectively, which is much higher comparison to study by Idris et al with 22% of cases and to our present study.(Table 1) Splenomegaly is an uncommon presenting feature of nonHodgkin lymphoma. B-cell types of NHL involving spleen are predominantly low grade and occur in older individuals whereas the T-cell NHL are predominantly high grade and occur in adolescents and young adults.18

Fever was obsereved in 67.3%, 62.5% and 72% of cases by Hingorjo et al5, Hassan K et al6 and Idris et al9 respectively. These associations were much higher compared with our study (Table 1). However Sra N et al19 obsereved 22% of cases presenting with fever which is similar to our study. In most patients, no particular fever pattern emerges that is pathognomic of cancer. Fever can occur during the day or night, although drenching night sweats are often a manifestation of malignancy and, if persistent, should prompt the clinician to consider neoplastic disease. Cytokines (endogenous pyrogens) induce prostaglandin E2, which in turn causes hypothalamic set point surge, and fever. Interleukin-1 (IL-1), TNF (Tumour Necrotic Factor), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-12 (IL-12), or interferons are elevated with neoplasm or infection20. While the cause of elevated cytokines during infection is precipitated by pathogens, the trigger in cancer is unclear. One study found that activation of IL-1? was

http://www.pacificejournals.com/aabs

A-91

AABS; 3(1): 2016

induced by mutated RAS21. Another possibility includes inflammation secondary to ulceration or necrosis caused by the tumor itself. A key player in this cytokine jumble is IL-6. High levels of IL-6 are seen in Hodgkin Lymphoma, Diffuse Large Cell Lymphoma, and CLL. IL-6 is associated with B-symptoms and has prognostic value in these three cancer types.22-26. In Diffuse-Large Cell Lymphoma, the independent factor that most correlates with complete remission and disease free survival is IL-6 serum level27.

Lymphadenopathy is a common sign of both benign and malignant diseases.30 Krol et al1, Naz et al4, Hassan et al6 and Hahn et al31 have also documented cervical lymphadenopathy to be the most common presenting symptom. (Table 2) Supraclavicular lymph node was the second most common lymph node involved in our study, However Hahn et al31 reported aortic lymph node as second most common lymph node, Naz et al4 and Krol et al1 documented inguinal lymph node as second most common lymph node. Krol et al1 in their study showed mediastinal lymphadenopathy in 11.4%, similar to our study. (Table 2)

Weight loss as presenting symptom was observed with almost similar incidence by most of the study groups. (Table 1) An ‘unexplained’ weight loss means a weight loss of more than a tenth of your total body weight over a period of <6 months. The pathologic mechanism underlying weight loss and cachexia are poorly understood. Soluble factors such as tumor necrosis factor- alpha (TNF-α), interleukin-1 (IL-1), interferon γ (IFN-γ) and interleukin-2 (IL-2) are involved in the development of tumor cachexia28. Weight loss can also occurs because aggressive lymphoma cells place an heavy demand on body and use up its resources to feed the cancerous cells.29

Presence of lymphoid cells in Fine Needle Aspiration Cytology of lymph node are usually considered to be associated with the diagnosis of lymphoma; however, there are other Benign conditions involving lymph nodes like tuberculosis, some metastatic tumours with lymphocytes, that may be misleading.32 Hence these patients with NHL may be misdiagnosed and treated incorrectly with antituberculous drug therapy as this is the most prevalent condition in our population. On the contrary, some patients with benign conditions like Kikuchi’s disease are subjected to unnecessary surgery or chemotherapy because of their clinical and histological resemblance to NHL.5 Clinicopathologic features and lymph node involvement by NHL as observed in our study and other asian studies show variation from NHL encountered in the Western world.4,5 Hence the knowledge of these variations is at most necessary.

The lymph mode involvement of NHL and HD are distinctive, with HD presenting with regional enlargement of single group of peripheral lymph nodes as opposed to disseminated nodal involvement in NHL.

Table 1: Clinical manifestation of NHL at presentation in various studies compared with present study. Symptoms Fever Weight loss Anaemia Hepatomegaly Splenomegaly Effusion

Present study 21.7 23.9 90.4 23.1 20.9 10.9

Hingorjo et al4 67.3 24.8 100 83.6 44.8 28.5

Hahn et al6

Cunlan et al5 55 -

16.7 26.4 -

Hassan K et al7 62.5 7.5 87.5 45.0 47.5 -

Ghosh J et al8 49% -

Idris et al9 72 47 65 22 18 -

Sra N et al10 22.0 25.0 -

Table 2: Frequency of lymph node involvement in NHL in various studies compared with present study. Lymph nodes

Present study

Krol et al1

Hahn et al6

Hassan K et al7 Naz et al4

Sra N et al10

Cervical

55.8

40.7

62.5

32.3

Axillary

32.4

21.8

13.1

9.6

Supra clavicular

39.5

3.2

Mediastinal

12.6

11.4

12.8

Mesenteric

28.4

13.7

Retroperitoneal

11.4

2.5

Aortic

21.2

20.1

Inguinal

25.6

26.1

15.8

14.5

Waldeyers ring

12.1

12.5

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-92

Conclusion Incidence of NHL is on the rise specially extra-nodal NHL with wide variations in the presentation of the disease due to involvement of different sites. The presence or absence of “B” symptoms has prognostic significance and is reflected in the staging of NHL. NHL presents commonly as Cervical lymphadenopathy, Anemia, weight loss and hepatomegaly with a close resemblance to tuberculosis and other disorders. This may cause difficulty to practitioners for early diagnosis. An awareness regarding the clinical manifestations and sites of involvement of NHL may help in early diagnosis and treatment.

Reference 1. Krol ADG, Cessie le, S Snijder et al. Primary extranodal non-Hodgkin’s lymphoma (NHL): the impact of alternative definitions tested in the Comprehensive Cancer Centre West population-based NHL registry. Annals of Oncology 2003; 14: 131–39. 2. Jose BO, Koerner P, Spanos WJ Jr, Paris KJ, Silverman CL, Yashar C, et al. Hodgkin’s lymphoma in adults – clinical features. J Ky Med Assoc 2005;103:15–7. 3. Al-Mobeireek AF, Arafah M, Siddiqui N. An African male with cough, hemoptysis, weight loss and hypercalcemia: TB or not TB? Eru Respir J 2002;20:1060–3. 4. Erum Naz, Talat Mirza, Sina Aziz. Frequency and clinicopathologic correlation of different types of Non Hodgkin’s lymphoma according to WHO classification. J Pak Med Assoc 2011; 61: 260-63. 5. Hingorjo MR, Sadiqa S. Presentation, staging and diagnosis of lymphoma: a clinical perspective. J Ayub Med Coll Abbottabad 2008; 20(4). 6. Hassan K, Ikram N, Bukhari KP, SH Shah. The pattern of Bone Marrow Infiltration in Non-Hodgkins Lymphomas. Journal of Pakistan Medical Association 1995; 45:173. 7. Cunlan MG, Armitage JO, Bast BSM, Weisenburger DD et al. Clinical significance of hematologic parameters in non-Hodgkins lymphoma at diagnosis. Cancer 1991; 67: 1389 -95. 8. Ghosh J, Singh RKB, Saxena R, Gupta R, Vivekanandan S, Sreenivas V et al. Prevalence and aetiology of anaemia in lymphoid malignancies. TNMJI 2013; 26: 2. 9. Idris M, Shah SH, Fareed J, Gul N. An experience with sixty cases of haematological malignancies; a clinicohaematological correlation. J Ayub Med Coll Abbottabad 2004; 16: 51-4.

10. Hohaus S, Massini G, Giachelia M, Vannata B, Bozzoli V, Cuccaro A, et al. Anemia in Hodgkin’s lymphoma: The role of interleukin-6 and hepcidin. J Clin Oncol 2010; 28:2538–43. 11. Ludwig H, Strasser K. Symptomatology of anemia. Semin Oncol 2001;28 (2-8):7–14. 12. Cella D. Factors influencing quality of life in cancer patients: Anemia and fatigue. Semin Oncol 1998;25 (3-7):43–6. 13. Moullet I, Salles G, Ketterer N, Dumontet C, Bouafia F, Neidhart-Berard EM, et al. Frequency and significance of anemia in non-Hodgkin’s lymphoma patients. Ann Oncol 1998; 9:1109–15. 14. Hasenclever D, Diehl V. A prognostic score for advanced Hodgkin’s Disease. International Prognostic Factors Project on Advanced Hodgkin’s Disease. N Engl J Med 1998;339:1506–14. 15. Murakami J, Shimizu Y. Hepatic Manifestations in Hematological Disorders. International Journal of Hepatology 2013;1-13. 16. Biemer JJ. Hepatic manifestations of lymphomas. Ann Clin Lab Sci 1984; 14(4):252-60. 17. Salo J, Nomdedeu B, Bruguera M et al. “Acute liver failure due to non-Hodgkin’s lymphoma,” The American Journal of Gastroenterology 1993; 88;5:774–776, 1993. 18. Nair S, Shukla J, Chandy M. Non-Hodgkin’s lymphoma presenting with prominent splenomegalyclinicopathologic diversity in relationship to immunophenotype. Acta Oncol 1997;36(7):725-7. 19. Sra N, Verma SK, Singh D & Bal MS. Incidence of lymphoma in head and neck region. Indian J Otolaryngol Head Neck Surg 2003; 55(4): 303–305. 20. Kurzrock R. The role of cytokines in cancer-related fatigue. Cancer 2001;92(6 Suppl):1684–1688. 21. Beaupre DM, Talpaz M, Marini FC, Cristiano RJ, Roth JA, Estrov Z, et al. Autocrine Interleukin-1? Production in Leukemia Evidence for the Involvement of Mutated RAS6. Cancer Research 1999;59(12):2971–2980. 22. Seymour JF, Talpaz M, Cabanillas F, Wetzler M, Kurzrock R. Serum interleukin-6 levels correlate with prognosis in diffuse large-cell lymphoma. Journal of Clinical Oncology1995;13(3):575–582. 23. Seymour JF, Talpaz M, Hagemeister FB, Cabanillas F, Kurzrock R. Clinical correlates of elevated serum levels of interleukin-6 in patients with untreated Hodgkin’s disease. The American Journal of Medicine 1997;102(1):21–28.

http://www.pacificejournals.com/aabs

A-93

AABS; 3(1): 2016

24. Fayad L, Keating MJ, Reuben JM, O’Brien S, Lee BN, Lemer S, Kuzrock R. Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia: correlation with phenotypic characteristics and outcome. Blood 2001;97(1):256–263. 25. Blay JY, Negrier S, Combaret V, Attali S, Goillot E, Merrouche Y, et al. Serum level of interleukin 6 as a prognosis factor in metastatic renal cell carcinoma. Cancer Research 1992;52(12):3317–3322. 26. Tran TT, Liu Y, Zurita A, Lin Y, Baker-Neblett KL, et al. Prognostic or predictive plasma cytokines and angiogenic factors for patients treated with pazopanib for metastatic renal-cell cancer: a retrospective analysis of phase 2 and phase 3 trials. Lancet Oncology 2012;13(8):827–837. 27. Preti HA, Cabanillas F, Talpaz M, Tucker SL, Seymour JF, Kurzrock R. Prognostic value of serum interleukin-6 in diffuse large-cell lymphoma. Annals of Internal Medicine 1997;127(3):186–194.

28. Denz H, Orth B, Weiss G, Hermann R, Huber P, Wachter H et al. Weight loss in patients with hematological neoplasias is associated with immune system stimulation. Clinical Investigator 1993;71: 37-41.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

29. Lymphoma Association. Symptoms of Hodgkin lymphoma.1-6. 30. Olu-Eddo AN, Ohanaka CE. Peripheral lymphadenopathy in Nigerian adults. J PakMed Assoc 2006;56:405–8. 31. Hahn JS, Seok K, Young S, Chong et al. Eight- Year experience of malignant Lymphoma-survival and prognostic factors, Yonsei Medical Journal 1997; 38;5:270-284. 32. Song JY, Cheong HJ, Kee SY, Lee J, Sohn JW, Kim MJ, et al. Disease spectrum of cervical lymphadenitis: analysis based on ultrasound-guided core-needle gun biopsy. J Infect 2007;55:310–6.

Original Article Discovery of Novel Gene Biomarker For Acute Myeloid Leukemia Through Differential Gene Expression Analysis Sweta P Tripathi*, Himanshu A Pandya Department of Bioinformatics, Gujarat University, Gujarat, India Keywords: Acute Myeloid Leukemia Disease, Microarray Analysis, Differentially Expressed Genes, Pathway Analysis.

ABSTRACT Acute Myeloid Leukemia (AML) is the major cause of death in developing countries. It is more prevalent in males as compared to females. Various cytogenetic detection methods are currently available, but their results are varied due to chromosomal changes. To overcome this, multiple microarray studies have been conducted on transcriptional profiling of AML patients. In depth reanalysis of microarray studies was performed. It was found that 21 genes involved in the seven pathways. The most significant pathway is antigen presentation and processing and genes are CD4, CD8a, and CD8b. On the basis of this result, we suggested that genes CD4, Neuraminidase (NA), Androgen-dependent TFPIregulating (ADTRP) are helpful in discovering novel biomarkers and also as a therapeutic target of AML.

*Corresponding author: Sweta Tripathi, Department of Bioinformatics, Gujarat University, Gujarat, India Phone: +91 9722641135, 380024 E-mail: sweta.tripathi2012@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-95

Introduction Leukemia is cancer of the blood and bone marrow. It can be either acute or chronic [1]. Further, it is classified on the basis of origin into myeloid and lymphoblast [2]. Acute myeloid leukemia (AML) is the diversified disorder which is caused by the accumulation of abnormal white blood cells in the bone marrow [3] or when the blood cells are not able to produce granulocyte or monocytes [4] resulting in the formation of myeloid cells. These myeloid cells differentiate into haematopoietic progenitor cells clump together and form the blast cells. The accumulated blast cells in the bone marrow lead to diseases like anaemia, blood clotting and severe infection caused the death from AML[5]. In human T-cell three types of oncogenes HTLV-1, HTLV-2 and simian T-cell are responsible for the AML[6].These oncogenes forms Natural killer T cell complex (NKT) which kills the target cells(MHC-I,MHCII) Besides this, genes involved in inhibition of T-cell proliferation are also known to cause AML [7]. The microarray technology based study on AML patients is very useful in identification genomic mutations. Microarray gene expression profiling has identified various upregulated and down regulated genes PML, TNFÎą, JUNB, FOS,TP63,VEGFA and ID2 [8]. Cytogenetic analysis of certain patients with AML led to identification of 133 genes [9]. This study aims to identify biomarkers of AML in differentially expressed genes involved in the different pathways by reanalysis of transcription profile of AML patients available in ArrayExpressdatabase.

Materials and Methods Data mining from the microarray databases: The microarray data is available in the European Molecular Biology Laboratory Array Express database [10]. It consists of 47068 experiments and 1336717 bio assays. There are two types of data: Minimum Information about Microarray Experiments [11] and Minimum Information about a high-throughput SEQuencing Experiment. The data is available for download in two formats viz. CEL (Affymetrix) file and MAGE-ML format. E-GEOD-14924 the transcriptional profiling of human T-cell in the AML CEL files was downloaded. The file contains 41samples which constitute four types viz.: AML with CD4 (n=10) and CD8 (n=10) and two healthy samples with CD4 (n=10), and CD8 (n=11). All these samples are taken from the patients of leukemia and healthy persons.

AABS; 3(1): 2016 were downloaded and installed. It was used to load the downloaded CEL files and further processing of raw data. The RcolourBrewer package was used for the colour of probes, after the loading step we check the quality control of the raw data through box plot and find its standard errors, removed all the variability between each of the sample file. The AffyPLM package [14, 15] was used to generate pseudo CEL file image. The gcrma package was used to normalize the raw data [16, 17, 18]. This step involves three results probe PM and MM background correction, adjusts the binding and non-binding specificity and expression calculation on the basis of GC% content then normalized the sample data. These data is transformed into the log2 based transformation in the excel file. After normalization step a quality control check was carried out using arrayQualityMetrics [19]. Statistical Analysis: The Genefilter [20] package was used to filter the microarray data and limma package [21] for the differential gene expression identification. Annotationdbi package was used for the annotation of those differentially expressed genes. On the basis of these a heat map was generated of differentially expressed genes. Pathway Enrichment Analysis: DAVID functional annotation tool was used to determine the pathways involved [22].

Result and Discussion Normalization and Quality Control Checks: A boxplot was generated for the raw and normalized data and further transformed into log2 based values (Figure 2.A and B). This box plot was also based on the interquartile range (IQR) values which lie between 5.5 to 8.2. Figure 2.A illustrates the raw data boxplot indicating that array 29 has a high level probe intensity value as compared to other arrays[23]. Probe intensity can be adjusted through normalization. Hence the normalized boxplot shows equal probe level intensity for all the arrays (Figure 2.B). Heatmap for the differentially expressed gene

Pre-processing and Processing of Raw Data: The Bioconductor software [12] and its packages [13]

The hierachical clustering based heatmap is a useful method for microarray analysis [25, 26]. The heat map was designed for differentially expressed genes based on their statistical values (Figure.3). The heat map shows probe intensities for each gene probe_id indicating the up regulated (yellow) and down regulated (red) genes. These expressions are based on logFC value and P-value. The logFC value for up regulated genes lies in the range 4 to 8 and for down regulated genes it lies between -4 to -5 whereas the P-value should be between P<0.05 to P<5*108. The probe_ids for up regulated genes are 205758_at, 207795_s_at, 1555691_a_at, 215332_s_at. These genes

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

The steps involved in data mining and processing are summarized in figure1

Original Article

A-96

Fig. 1 : Schematic of microarray analysis

Fig. 1 : Quality control check (A) Raw data box plot (B) Normalized data box plot

http://www.pacificejournals.com/aabs

A-97

AABS; 3(1): 2016

Fig. 3 : Heat map for differentially expressed genes

Fig. 4 : Pathways summary for the Acute my eloid leukemia

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-98

have higher intensity as compared to rest of the genes. The Probe_id for down regulated genes are 232584_at, 203547_ at, 229070_at have low intensity based expression. Limma package is used for differential gene expression analysis. It takes all the different experimental condition for microarray data as input. The result is given in a tabulated format which contains 24 unique differentially expressed genes(Table.1).Amongst the 24, 3 genes are not identified and properties for the remaining 21 (listed above) including gene symbol, name, function and expression have been shown. The gene expression can be identified based on P-value and logFC values. If the P-value is less than P< 0.05 and logFC <1.5 â&#x20AC;&#x201C; 4 or more, it is considered significant [27, 28]. Out of the 21 genes; XCL1,GPR56,GZMB,GZMH,CCL4 have been reported in AML disease gene expression, S1PR5,CD160,SLAMF7,NEG7 have not been reported in AML disease patients. On the basis of gene ontology, 7 genes have been selected for differentiation studies between AML and normal tissues. CD8 B molecule: CD8 molecule is involved in the T cell mediated killing process [29] and was up regulated (logFc 7.10) in the present study. The CD8 T cells have an abnormality that is instead of binding to the target cell, it kills the target cell (MHC class I) molecules. As a result, the antigen specific activation and cytokine production process is stopped. However, reports have been published showing relationship between CD8 and AML [30]. NK2G (natural killer gene family): This family includes KLRK1 (Killer cell lectin-like receptor subfamily K, member1), KLRD1(Killer cell lectin-like receptor subfamily D, member 1), KLRC3(Killer cell lectin-like receptor subfamily C, member 3), KLRC4(Killer cell lectin-like receptor subfamily C, member 4), KLRF1(Killer cell lectin-like receptor subfamily F, member 1) genes. They form the natural killer complex and are present on chromosome 12.In the present study, expression of these genes was shown to be upregulated (logFc 5.40 to 7.45). The KLRK1 gene is involved in increased production of multiple myeloma cells (plasma cells) .Multiple myeloma stage II and III are involved in acute lymphoblastic leukemia [30]. The KLRD1 gene is infected by cytomegalovirus resulting in inhibition of major histocompatibility complex class II by the interuption of the Jak/Stat pathway[31].The KLRC3 gene is involved in the cytolytic process of natural killer T cells.The KLRC4 gene activates or inhibits natural killer T cells. The KLRF1 gene stimulates the natural killer cell mediated cytotoxicity and cytokine release [32]. Hence, the NK2G gene family involved in the NKT celllymphoblastic leukemia, breast cancer disease [33].

CRTAM (Cytotoxic and regulatory T cell molecule): It belongs to nectin family of proteins. It is involved in tumour immunosurveillance and act as a supressor for the NKT cellreceptor surface proteins. In the present study, it was shown to be upregulated (logFc 5.40).Also, reports have been published showing relationship between CRTAM and lung cancer [34], breast cancer [35] , AML and chronic leukemia [36]. CD4 molecule: It is used for lymphocyte count and present on chromosome 7. It form neoplasm in lymphoma and hence has been shown to cause AML. In this study it was downregulated (logFc -4.44). ENPP5 (Ectonucleotide phosphodiesterase5): It is involved in the neuronal cell communication and was upregulated (logFc 4.16) in the present study. Though its pathway is not reported, it has been shown to cause chronic myeloid leukemia disease. NA (Neuraminidase): It is involved in neuroacanthocytosis disorder related to the brain tissue and spiculated blood cells. In the present study it was downregulated (logFc -5.600) and reported in AML disease (T lymphocytes) and carcinoma (B lymphocytes) gene expression [37]. ADTRP (androgen-dependent TFPI-regulating): It is involved in the tissue factor inhibitor pathway, it regulates the cell associated anticoagulating activity of TFPI inhibitor in endothelial cells [38] and was down regulated (logFc -4.30) in this study. Its cancer related study and pathway is not reported yet. Pathway Enrichment Analysis For differentially expressed genes, KEGG pathway based analysis was used which showed 7 differentially regulated pathways.CD4 molecules are involved in antigen processing and presentation, primary immunodeficiency, T-cell receptor signalling pathway, hematopoetic lineage and cell adhesion molecule. CD8 molecules are involved in all CD4 molecule pathways and additionally in GraftVersus-Host disease pathway. A new pathway was derived from these studies i.e.Vascular tissue factor inhibitor pathway. This pathway and the gene (ADTRP) involved in it have not been reported yet. The significance of the pathways is indicated based on FDR (False discovery rate) and P-value (<0.05). It was found that the most significant pathway is antigen processing and presentation pathway (Table 2) which involves 7 genes: CD4, CD8a, CD8b, KLRC1, KLRC3, KLRC4, KLRD. Since CD4, CD8a and CD8b genes are involved in 5 pathways, they are considered the most significant genes. On the basis of this study, a flowchart of the pathways involved in acute myeloid leukemia was made.

http://www.pacificejournals.com/aabs

A-99

Conclusion

AABS; 3(1): 2016

1. Bardelli, A., & Velculescu, V. E. (2005). Mutational analysis of gene families in human cancer. Current opinion in genetics & development, 15(1), 5–12. doi:10.1016/j.gde.2004.12.009 2. Boles, K. S., Barchet, W., Diacovo, T., Cella, M., & Colonna, M. (2005). Plenary paper The tumor suppressor TSLC1 / NECL-2 triggers NK-cell and CD8 ϩ T-cell responses through the cell-surface receptor CRTAM, 106(3), 779–786. doi:10.1182/ blood-2005-02-0817.The 3. Bonnet, D., & Dick, J. E. (1997). Human acute myeloid leukemia is organised as a hierarchy that originates from a primitive hematopoietic cell. Nature, 3, 730–737. doi:10.1038 4. Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns, 95(December), 14863–14868. 5. Brazma, A., Hingamp, P., Quackenbush, J., Sherlock, G., Spellman, P., Stoeckert, C., … Vingron, M. (2001). Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nature genetics, 29(4), 365–371. doi:10.1038/ng1201-365 6. Carbone, E., Neri, P., Mesuraca, M., Fulciniti, M. T., Otsuki, T., Pende, D., … Venuta, S. (2005). IMMUNOBIOLOGY HLA class I , NKG2D , and natural cytotoxicity receptors regulate multiple myeloma cell recognition by natural killer cells, 105(1), 251–258. doi:10.1182/blood-2004-04-1422. Supported 7. Cheng, X., & Kao, H.-Y. (2012). Microarray analysis revealing common and distinct functions of promyelocytic leukemia protein (PML) and tumor

necrosis factor (TNF alpha) signaling in endothelial cells. BMC Genomics. doi:10.1186/1471-2164-13-453 8. Dalman, M. R., Deeter, A., Nimishakavi, G., & Duan, Z.-H. (2012). Fold change and p-value cutoffs significantly alter microarray interpretations. BMC bioinformatics, 13 Suppl 2(Suppl 2), S11. doi:10.1186/1471-2105-13-S2-S11 9. Dudoit, S., & Yang, J. (2003). Bioconductor R packages for exploratory analysis and normalization of cDNA microarray data. The Analysis of Gene Expression Data, 1–35. Retrieved from http://link. springer.com/chapter/10.1007/0-387-21679-0_3 10. Estey, E. H. (2009). Treatment of acute myeloid leukemia. Haematologica, 94(1), 10–6. doi:10.3324/ haematol.2008.001263 11. Gautier, L., Cope, L., Bolstad, B. M., & Irizarry, R. A. (2004). Affy - Analysis of Affymetrix GeneChip data at the probe level. Bioinformatics, 20(3), 307–315. 12. Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, S., … Zhang, J. (2004). Bioconductor : open software development for computational biology and bioinformatics, (10). 13. Gillespie, C. S., Lei, G., Boys, R. J., Greenall, A., & Wilkinson, D. J. (2010). Analysing time course microarray data using Bioconductor : a case study using yeast2 Affymetrix arrays. 14. Glienke, J., Sobanov, Y., Brostjan, C., Steffens, C., Nguyen, C., Lehrach, H., … Francis, F. (1998). The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex. Immunogenetics, 48(3), 163–173. 15. Goswami, R. S., Sukhai, M. A., Thomas, M., Reis, P. P., & Kamel-Reid, S. (2009). Applications of microarray technology to Acute Myelogenous Leukemia. Cancer informatics, 7, 13–28. 16. Grimwade, B. D., Walker, H., Oliver, F., Wheatley, K., Harrison, C., Harrison, G., … Goldstone, A. (1998). The Importance of Diagnostic Cytogenetics on Outcome in AML: Analysis of 1,612 Patients Entered Into the MRC AML 10 Trial, 2322–2333. 17. Harty, J. T., Tvinnereim, A. R., & White, D. W. (2000). CD8+ T cell effector mechanisms in resistance to infection. Annual review of immunology, 18, 275– 308. doi:10.1146/annurev.immunol.18.1.275 18. Heber, S., & Sick, B. (2006). Quality Assessment of Affymetrix GeneChip Data, 10(3), 358–368. 19. Hehlmann, R., Berger, U., Aul, C., Büchner, T., Döhner, H., Ehninger, G., … Hasford, J. (2004). The German competence network “Acute and chronic

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

We analyzed the transcriptional profiling of human T-cell involved in the AML disease. We found the differential expressed genes CD8B, KLRK1, CRTAM, KLRD1, ENPP5, NA, CD8A, KLRC3, KLRC4, KLRF1, S1PR5, XCL1, S1PR5, CD160, SLAMF7, GPR56, ADTRP, GZMB, FGFBP2, NKG7, CCL4, GNLY have varied expression in AML patients. We suggested that CD4, NA, ADTRP genes may be helpful to discover the novel diagnostic biomarker, therapeutic targets and gene therapy for the AML disease.

Funding Source None

Conflict of Interest None

References

Original Article

20.

21.

22.

23.

24.

25.

26.

27.

28.

A-100

leukemias”. Leukemia, 18(4), 665–9. doi:10.1038/ sj.leu.2403317 Hikami, K., Tsuchiya, N., Yabe, T., & Tokunaga, K. (2003). Variations of human killer cell lectin-like receptors: common occurrence of NKG2-C deletion in the general population. Genes and immunity, 4(2), 160–7. doi:10.1038/sj.gene.6363940 Hua, Y. J., Tu, K., Tang, Z. Y., Li, Y. X., & Xiao, H. S. (2008). Comparison of normalization methods with microRNA microarray. Genomics, 92(2), 122–128. Huang, D. W., Sherman, B. T., Tan, Q., Kir, J., Liu, D., Bryant, D., … Lempicki, R. A. (2007). DAVID Bioinformatics Resources: Expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Research, 35(SUPPL.2). Kauffmann, A., Gentleman, R., & Huber, W. (2009). arrayQualityMetrics - A bioconductor package for quality assessment of microarray data. Bioinformatics, 25(3), 415–416. Le Dieu, R., Taussig, D. C., Ramsay, A. G., Mitter, R., Miraki-Moud, F., Fatah, R., … Gribben, J. G. (2009). Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts. Blood, 114(18), 3909–3916. Lung, H. L., Cheung, A. K. L., Xie, D., Cheng, Y., Kwong, F. M., Murakami, Y., … Lung, M. L. (2006). TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma. Cancer research, 66(19), 9385–92. doi:10.1158/0008-5472. CAN-06-0590 Lupu, C., Zhu, H., Popescu, N. I., Wren, J. D., & Lupu, F. (2011). Novel protein ADTRP regulates TFPI expression and function in human endothelial cells in normal conditions and in response to androgen. Blood, 118(16), 4463–4471. Miwa, H., Mizutani, M., Mahmud, N., Yamaguchi, M., Takahashi, T., Shikami, M., … Kita, K. (1998). Biphasic expression of CD4 in acute myelocytic leukemia (AML) cells: AML of monocyte origin and hematopoietic precursor cell origin. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K, 12(1), 44–51. doi:10.1038/sj.leu.2400877 Opin, C., Biol, C., Biol, M. C., & Trans, B. S. (1998). Tandem duplication of the FLT3 gene in acute lymphoblastic leukemia: a marker for, 522–524.

29. Parkinson, H., Kapushesky, M., Shojatalab, M., Abeygunawardena, N., Coulson, R., Farne, a, … Brazma, a. (2007). ArrayExpress--a public database of microarray experiments and gene expression profiles. Nucleic acids research, 35(Database issue), D747–50. doi:10.1093/nar/gkl995 30. Pradervand, S., Weber, J., Thomas, J., Bueno, M., Wirapati, P., Lefort, K., … Harshman, K. (2009). Impact of normalization on miRNA microarray expression profiling. RNA (New York, N.Y.), 15(3), 493–501. 31. Rasi, S., Spina, V., Bruscaggin, A., Vaisitti, T., Tripodo, C., Forconi, F., … Rossi, D. (2011). A variant of the LRP4 gene affects the risk of chronic lymphocytic leukaemia transformation to Richter syndrome. British Journal of Haematology, 152(3), 284–294. 32. Robinson, M. D., & Oshlack, A. (2010). A scaling normalization method for differential expression analysis of RNA-seq data. Genome biology, 11(3), R25. 33. Smyth, G. K. (2005). limma: Linear Models for Microarray Data. In R. Gentleman, V. J. Carey, W. Huber, R. A. Irizarry, & S. Dudoit (Eds.), Bioinformatics and Computational Biology Solutions Using R and Bioconductor (pp. 397–420). SpringerVerlag. doi:10.1007/0-387-29362-0 34. Vilchez, R. A., & Butel, J. S. (2003). SV40 in human brain cancers and non-Hodgkin’s lymphoma. Oncogene, 22(33), 5164–5172. doi:10.1038/ sj.onc.1206547 35. Wu, Z., LeBlanc, R., & Irizarry, R. (2003). Stochastic models based on molecular hybridization theory for short oligonucleotide microarrays. John Hopkins University, Department of Biostatistics Working Paper, 4. Retrieved from http://biostats.bepress.com/ jhubiostat/paper4/ 36. Yu, M. W., Pai, C. I., Yang, S. Y., Hsiao, T. J., Chang, H. C., Lin, S. M., … Chen, C. J. (2000). Role of N-acetyltransferase polymorphisms in hepatitis B related hepatocellular carcinoma: impact of smoking on risk. Gut, 47(5), 703–709. 37. Zhang, H., & Li, S. (2013). Dataset Paper DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells, 2013. 38. Gentleman R,Carey V,Huber W,Hahne F:Genefilter:method for filtering from microarray experiments. R package version1.44.0

http://www.pacificejournals.com/aabs

Original Article Evaluation of Glycosylated Hemoglobin (HbA1c) Levels in Hypothyroid and Hyperthyroid Patents Sumit Sohal1*, Aanchal Wats1, Chittaranjan Vij2 1

Intern, Government Medical College, Patiala, India Department of Biochemistry, Government Medical College, Patiala, India

Keywords: Diabetes Mellitus; Hemoglobin A, Glycosylated; Hyperthyroidism; Hypothyroidism

ABSTRACT INTRODUCTION: Almost one-third of the worldâ&#x20AC;&#x2122;s population lives in areas of iodine deficiency. Thyroid hormone action has long been recognized as an important determinant of glucose homeostasis. Diabetes and thyroid disorders have a propensity to appear together in patients OBJECTIVES: To compare and correlate the glycosylated Hb levels and TSH levels in non diabetic patients. METHODOLOGY: The cross sectional study was conducted among 30 patients of diagnosed thyroid disorders. The biochemical parameters were Hemoglobin, TLC, glycemic control (fasting and post prandial blood glucose levels, glycated haemoglobin A1c), blood urea, serum creatinine and a thyroid profile (TSH, T3, and T4). These parameters were measured and compared with the normal population. RESULTS: Out of 30 patients 26 were diagnosed as hypothyroid and 4 were diagnosed as hyperthyroid. HbA1c levels were found to be elevated in patients of thyroid disorders (hypothyroidism and hypothyroidism) as compared to control. CONCLUSION: Based on this study all the thyroid patients especially hyperthyroid patients should have regular checkup of their glucose levels. Patients should have adequate treatment of the thyroid disorders and thus should prevent themselves from adverse effects of hyperglycemia

*Corresponding author: Dr. Sumit Sohal, 16/34, New Krishna Nagar, Hoshiarpur, Punjab, India, 146001 Phone: +91 9464079194 E-mail: s.sohal1530@yahoo.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-102

Introduction Almost one-third of the world’s population lives in areas of iodine deficiency. The prevalence of goitre in areas of severe iodine deficiency can be as high as 80%. In iodine-replete areas, most persons with thyroid disorders have autoimmune disease, ranging from primary atrophic hypothyroidism, Hashimoto’s thyroiditis to thyrotoxicosis caused by Graves’ disease.[1] Thyroid hormone action has long been recognized as an important determinant of glucose homeostasis[2] The role of hyperthyroidism in diabetes was investigated in 1927, by Coller and Huggins proving the association of hyperthyroidism and worsening of diabetes. It was shown that surgical removal of parts of thyroid gland had an ameliorative effect on the restoration of glucose tolerance in hyperthyroid patients suffering from coexisting diabetes[3] 5′ adenosine monophosphateactivated protein kinase (AMPK) is a central target for modulation of insulin sensitivity and feedback of thyroid hormones associated with appetite and energy expenditure[4] In hypothyroidism, there is a reduction in glucoseinduced insulin secretion by beta cells, and the response of beta cells to glucose or catecholamine is increased in hyperthyroidism due to increased beta cell mass. Moreover, insulin clearance is increased in thyrotoxicosis[5] Possibly, thence, diabetes and thyroid disorders have a propensity to appear together in patients[6] HbA1C is widely used for the assessment of glycemic status of the diabetic patients and the American Diabetes Association (ADA) recommended its use for diagnosing diabetes[7]. The glycated hemoglobin represents the fraction of hemoglobin that undergoes non-enzymatic glycation over the circulatory life span of the erythrocytes (usually 120 days)[8] A positive association between thyroid and diabetes mellitus is well recognized but to

study the effect of thyroid disorders on glucose metabolism in non diabetic patients ((ie patients diagnosed with only thyroid and not diabetes) is an area for extensive research. Our present study was planned to assess correlation between thyroid disorders and non diabetic patients and find out if any association exists between glycosylated haemoglobin levels with those of thyroid hormones in non diabetic patients Methodology The cross sectional study was conducted among 30 patients of diagnosed thyroid disorders in the department of medicine at Rajindra hospital, Patiala, Punjab, India. The patients who presented with clinical symptoms of thyroid disorders were followed up and were subsequently diagnosed based on the laboratory findings. The duration of study was 6 months from January 2014 to June 2014. 30 patients who tested negative for thyroid disorders and who were not using any type of medication, with no mental or physical mutilation and no history of chronic disease were taken as control for the study. Those who agreed to participate were asked to sign a written informed consent form after assurance with regards to confidentiality provided to them. Permission and clearance was taken from the institutional ethics committee. Inclusion Criteria 1. Patient who presented with symptoms of thyroid disorders and were diagnosed with a specific thyroid disorder. 2. Patient with FBS within the normal range 3. Patients who consent to the participation. Exclusion Criteria 1. Patient with previously diagnosed thyroid disorder and were on a specific medication 2. Patients with frank diabetes mellitus. 3. Total/subtotal thyroidectomy 4.

Patients on I131 treatment, lithium, antithyroid drugs.

5. Radiation exposure 6.

Gestational hyperthyroidism

7. Chronic renal failure 8. Abnormal haemoglobinopathy, 9. Hemolytic disorder 10. Recent (< 3 months) blood transfusion

http://www.pacificejournals.com/aabs

A-103

AABS; 3(1): 2016

Study Method All patients were subjected to routine investigations such as:1. Hb -- Acid hematin haemoglobinometer

method

using

TABLE 1: AGE DISTRIBUTION OF STUDY AND CONTROL GROUPS AGE IN YEARS

STUDY GROUP

CONTROL GROUP

21-30

31-40

sahli’s

2. TLC -- Thoma zeiss haemocytometer

41-50

51-60

61-70

TOTAL

3. Blood sugar -- Asatoor and king method, 1954 4. Serum Creatinine -- Method of broad and sirota 5. Blood Urea -- Diacetyl monoxime method(DAM) Special investigations: Glycosylated haemoglobin (Reference range: Non-diabetic—4.5-8.0% , Good control—8.0-9.0%, Fair control—9.0-10.0%, Poor control-- >10.0%) was analysed using commercially available Infinite Glycosylated hemoglobin kits( ACCUREX biomedical private Ltd. Mumbai, India). TSH(reference range 0.44 - 3.45 (microIU/ml)), T3(reference range: 0.49 – 2.02 ng/ml), T4(reference range: 4.7 -12.8(microgram/dl) ) estimation was done using ERBA kits (immmunoenzymometeric assay) Data analysis Data was recorded on a pre designed proforma and managed using Microsoft Excel 2007 (Microsoft Corp, Redmond,WA)All data was analyzed using statistical program SPSS package (Chicago, Illinois). t-test, chi square and pearson’s correlation were applied with significance levels at 95% CI and p < 0.05 accordingly.

Results

The present study was conducted on 30 clinically confirmed cases of thyroid diseases attending the OPD of Department of Medicine of Rajindra Hospital,Patiala. The study also included 30 age and sex matched healthy controls. Subsequent investigations were carried out in Biochemistry department of Government Medical College.

TABLE 2: DISTRIBUTION OF STUDY AND CONTROL GROUPS ACCORDING TO SEX SEX

STUDY GROUP

CONTROL GROUP

No.of cases

%age

No.of cases

%age

MALE

4 26 30

13.33

86.67

100

FEMALE TOTAL

HbA1c levels in hypothyroid patients were found to be in a range of 7.2-7.79% with a mean of 7.41±0.15% against the range of 5.89-7.2% with the mean of 6.69±0.34% in controls. The difference was found to be highly significant (p<0.001). the HbA1c levels in hyperthyroid patients were found to be in a range of 7.23-7.89% with a mean of 7.51±0.23%, which was significantly(p<0.001) greater than the controls. Graphical relation between the levels of HbA1c is shown in Graph 1. Table 5 shows correlation between values of TSH and HbA1c levels in hypothyroid and hyperthyroid patients.

The age in the experimental group varied from 21- 65 with mean age of 42.7 ± 11.05 and that of control varied from 21-64 with a mean of 42.77±10.72 with no significant difference between the two groups(p>0.05). Age and sex distribution of patients are shown in Table 1 and 2. The various parameters of routine tests were within normal limits and were non significantly different between the study group and control group. Out of 30 patients 26 were diagnosed as hypothyroid and 4 were diagnosed as hyperthyroid. The comparison of mean levels of serum T3, T4 and TSH between hypothyroid v/s control and hyperthyroid v/s control is given in table 3 and 4 respectively. Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

Discussion

As Johnson et al. said, “The thyroid hormones, triiodothyronine and tetra- iodothyronine, are insulin

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-104

TABLE 3: COMPARISON OF MEAN LEVELS OF SERUM T3,T4 AND TSH IN HYPOTHYROIDAND CONTROL GROUP HORMONE

NO.OF PATIENTS

RANGE

MEAN±S D

HYPOTHYROID

0.12-1.71

1.01±0.34

14.78

<0.001

CONTROL

0.54-2.01

1.56±0.38

T4(microg/dl)

HYPOTHYROID

6.0-8.81

7.05±1.05

3.72

<0.001

CONTROL

6.2-10.66

8.16±1.14

TSH(microIU/ml)

HYPOTHYROID

5.0-27.38

7.79±4.42

-6.89

<0.001

CONTROL

1.0-3.32

2.21±0.59

T3(ng/ml)

GROUP

TABLE 4: COMPARISON OF MEAN LEVELS OF SERUM T3,T4 AND TSH IN HYPERTHYROID AND CONTROL GROUP HORMONE

GROUP

NO OF PATIENTS

RANGE

MEAN±SD

T3(ng/ml)

Hyperthyroid

2.13-2.67

2.38±0.25

4.32

<0.001

Control

0.54-2.01

1.56±0.38

T4(microg/dl)

Hyerthyroid

15.5-19.97

17.92±1.89

14.78

<0.001

Control

6.2-10.66

8.16±1.14

TSH(microIU ml)

Hyerthyroid

0.03-0.31

0.12±0.13

6.97

<0.001

Control

1.0-3.32

2.21±0.59

TABLE 5: CORRELATION BETWEEN TSH AND HbA1c IN HYPOTHYROID AND HYPERTHYROID PATIENTS PARAMETER

r VALUE

p VALUE

SIGNIFICANCE

HbA1c AND TSH (HYPOTHYROID)

0.51

<0.001

HbA1c AND TSH (HYPERTHYROID)

-0.76

<0.001

antagonists that also potentiate the action of insulin indirectly.”[9] Thyroid hormones exert both insulin agonistic and antagonistic actions in different organs. However, this occurs in a fine balance necessary for normal glucose metabolism. Deficit or excess of thyroid hormones can break this equilibrium leading to alterations of carbohydrate metabolism[10] Our study showed significant increase in HbA1c levels in hypothyroid and hyperthyroid patients when compared to controls. Hypothyroidism Two theories have been put forward to explain the increased levels of Hba1c levels in hypothyroid patients. One of those theories blames dysglycemia to be the cause of increased HbA1c levels. Various reasons have been put forward to explain the dysglycemia caused in hypothyroidism.

1. Altered glucose homeostasis with decreased absorption and conversely decreased utilization also associated with hyper insulinemia and insulin resistance probably causing transient elevations in the glucose concentrations thus contributing to glycation of serum proteins[11] 2. Thyroid hormones have been shown to exert some of their actions synergically with insulin. The upregulation of the expression of genes such as GLUT-4 or phosphoglycerate kinase (PGK) , involved in glucose transport and glycolysis respectively, is a good proof of concept. Therefore in hypothyroidism insulin resistance in peripheral tissue is present. Insulin resistance is the cause of increased glucose levels in hypothyroid levels.[12,13] 3. At the cellular level, thyroid hormones can also increase mitochondrial biogenesis, fatty acid oxidation, and TCA cycle activity . These findings are quite relevant since the role of mitochondrial dysfunction, leading to cellular lipid excess and impaired oxidative

http://www.pacificejournals.com/aabs

A-105 metabolism, has been clearly demonstrated in the pathogenesis of type 2 diabetes. Furthermore, it has been described that in skeletal muscle, the lack of thyroid hormones might dysregulate mitochondrial gene expression. PPAR gamma coactivator-1 alpha (PGC-1 alpha), a key transcriptional regulator of mitochondrial content and function, fatty acid oxidation, and gluconeogenesis, has been involved in the process whereby thyroid hormones regulate mitochondrial function . It has been shown that PGC-1 alpha gene expression is increased by T3, as much as 13-fold 6 hours after T3 treatment . The regulation pattern of T3 on PGC-1 alpha is complex and may occur through nongenomic activation of kinases to induce the expression of PGC-1 alpha or through transcriptional upregulation via the presence of a thyroid responsive element (TRE) in the PGC1 alpha promoter or by genomic upregulation of a transcription factor (via a TRE), which then binds to the PGC-1 alpha promoter and increases PGC1 alpha transcription. It is hypothesized that PGC-1 alpha can be dysregulated by reduced T3 levels, thus contributing to insulin resistance.[10] Other theory cites different other causes for increased HbA1c levels in patients of hypothyroidism. 1. Decreased metabolism leading to decreased turnover of proteins and thus prolonging their half - life. 2. Increased oxidative stress causing increased glycation of proteins. 3. Low grade inflammation adding to the free radical formation and its effects. Raised immunoglobulins in response to inflammation and preferential glycation rates of immunoglobulins. 4.

The tendency of glycated proteins to accumulate in tissues resisting easy proteolysis and being further source of free radicals.[11]

5. data suggest that thyroid hormone replacement is associated with a decrease in A1C level, which is influenced by increased erythropoiesis rather than by changes in glucose level[8,14] Theories almost go against each other in explaining the reason behind increased HbA1c levels in hypothyroid patients. In present study either of theories can be applicable but anemia was excluded by comparable levels between controls and test patients. Patients with Chronic renal failure were also excluded to remove the effects of Erythropoeitin. All patients with any hemoglobin disorders were also excluded to narrow the basis of results. There are a number of factors which play a role in increasing the Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 HbA1c levels and it is difficult to differentiate the causative factors of increase in HbA1c. This elevation of HbA1c was also demonstrated in a study of 45 hypothyroid patients in which HbA1c was higher than that in control subjects (5.54± 0.43% vs. 5.34±0.31% in hypothyroid patients and controls respectively; p < 0.001)[14] Another study by Christy et al selected 30 hypothyroid, non diabetic patients with normocytic normochromic anemia and compared these patients with 30 euthyroid non diabetic patients also with normocytic normochromic anemia. HbA1c in the hypothyroid patients was 6.32 ±0.75 % vs. 5.87±0.46% in the euthyroid group, the difference being statistically significant.[15] The correlation which was seen in this study was also shown by another study which significantly correlated TSH and HbA1c (r =0.46, p <0.05)[16] Hyperthyroidism According to Kim H B et al. (1992), The mean values of FBS and HbAlc in hyperthyroid group are higher than those of normal controls. The higher level of FBS and HbAlc in hyperthyroid group compared to normal controls appeared as changes of carbohydrate metabolism.[17] Permissive influence of T3 is seen in the glycogenolytic and gluconeogenic effects of epinephrine and glucagon. Other hepatic gluconeogenic enzymes that have been found to be positively regulated by thyroid hormones include phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that catalyzes the rate-controlling step of gluconeogenesis [18] Another mechanism, whereby thyroid hormones are known to increase hepatic glucose output, is through increased hepatic expression of the glucose transporter GLUT2[19] In another study significant (p = 0.002) increase in the mean value for hemoglobin A1C were found in the hyperthyroid group which is similar to the finding in our study[20] In another study similar findings were found where HbA1c levels were significantly higher in hyperthyroid group when compared to hypothyroid and euthyroid groups (p<0.001)[21]

Conclusion

From the present study it is concluded that HbA1c levels are increased in both hypothyroid and hyperthyroid patients. Both the disorders have increased levels of HbA1c due to differential actions of thyroid hormones on liver, skeletal muscles and e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-106

adipose tissue. However many of these actions are complex and based on gene expression changes brought about by thyroid hormones. However an imbalance between the two created by abnormal levels of thyroid lead to a state of hyperglycemia in both conditions.

6. Nima V. Thakkar and Sunita M. JainThe Impact of Diabetes on Thyroid Dysfunction and Outcomes in a Native Indian Female PopulationThyroid Science 6(4):1-9, 2011

Based on this study all the thyroid patients especially hyperthyroid patients should have regular checkup of their glucose levels. Patients should have adequate treatment of the thyroid disorders and thus should prevent themselves from adverse effects of hyperglycemia. This study certainly proves the existence

8. Sailaja Anantarapu, Suresh Vaikkakara, Alok Sachan, Bobbidi Venkata Phaneendra, Mustur Manohar Suchitra, Amaresh Ponnala Reddy, Sunil Epuri, Arun Mukka, Dharaneswari Vemvakam. Effects of thyroid hormone replacement on glycated hemoglobin levels in non diabetic subjects with overt hypothyroidism. Arch Endocrinol Metab. 2015;59/6

of aterm called “thyroid diabetes.”

Acknowledgements We would like to extend our gratitude to ICMR short term studentship for extending their support. We would also like to acknowledge the biochemistry department of GMC, Patiala for helping by providing all technical and academic support. Declarations Funding: ICMR(STS)

Conflict of interest

7. American Diabetes Association.Diagnosis and classification of diabetes mellitus. Diabetes Care. 2010; 33:S62–69.

9. Johnson, J.L. and Duick, D.S.: Diabetes and thyroid disease: [16]A likely combination. Diabetes Spectrum, (2002) 15:140-142 10. Gabriela Brenta, “Why Can Insulin Resistance Be a Natural Consequence of Thyroid Dysfunction?,” Journal of Thyroid Research, vol. 2011, Article ID 152850, 9 pages, 2011. 11. Nagraj Soni, G G Kaushik, VandanaYadav TO Study Glycemic Status and Lipid Fractions In Subclinical Hypothyroid, Overt Hypothyroid And Hyperthyroid Subjects. International Journal of Advanced Research (2014), Volume 2, Issue 9, 761-767 12. S. P. Weinstein, E. O’Boyle, and R. S. Haber, “Thyroid hormone increases basal and insulin-stimulated glucose transport in skeletal muscle. The role of GLUT4 glucose transporter expression,”Diabetes, vol. 43, no. 10, pp. 1185–1189, 1994. View at Google Scholar · View at Scopus

NONE

Ethical approval From the IEC, GMC Patiala

References

1. Vanderpump MP. The epidemiology of thyroid disease. Br Med Bull. 2011;99:39-51. 2. Peeyush Yadav, G.G.Kaushik, Sonali Sharma. Importance of Screening Type-II Diabetics for Thyroid Dysfunction and Dyslipidemia International Journal of Biochemistry and Biophysics 2015; 3(2): 7-12 3. Coller FA, Huggins CB. effect of hyperthyroidism upon diabetes mellitus: striking improvement in diabetes mellitus from thyroidectomy. Ann Surg. 1927 Dec;86(6):877-84. 4. Goglia F, Moreno M, Lanni A. Action of thyroid hormones at the cellular level: the mitochondrial target. FEBS Lett. 1999 Jun 11;452(3):115-20. 5. Chaoxun Wang. The Relationship between Type 2 Diabetes Mellitus and Related Thyroid DiseasesJ Diabetes Res. 2013; 2013: 390534.

13. L. C. Moeller, A. M. Dumitrescu, R. L. Walker, P. S. Meltzer, and S. Refetoff, “Thyroid hormone responsive genes in cultured human fibroblasts,” Journal of Clinical Endocrinology & Metabolism, vol. 90, no. 2, pp. 936–943, 2005. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus 14. Kim MK1, Kwon HS, Baek KH, Lee JH, Park WC, Sohn HS, Lee KW, Song KH. Effects of thyroid hormone on A1C and glycated albumin levels in nondiabetic subjects with overt hypothyroidism. Diabetes Care. 2010 Dec;33(12):2546-8. doi: 10.2337/ dc10-0988. Epub 2010 Sep 7. 15. Christy AL, Manjrekar P, Babu RP, M S R, Hegde A. Elevation of HbA1C in non-diabetic hypothyroid individuals: is anaemia the connecting link? – A preliminary study. J Clin Diagn Res.2013;7(11):2442-4.

http://www.pacificejournals.com/aabs

A-107 16. Edina Bilic-Komarica , Amela Beciragic , Dzelaludin Junuzovi The Importance of HbA1c Control in Patients with Subclinical Hypothyroidism Mat Soc Med. 2012 Dec 24(4): 212-219

AABS; 3(1): 2016

18. E. A. Park, D. C. Jerden, and S. W. Bahouth, “Regulation of phosphoenolpyruvate carboxykinase gene transcription by thyroid hormone involves two distinct binding sites in the promoter,” Biochemical Journal, vol. 309, no. 3, part 3, pp. 913–919, 1995

19. S. P. Weinstein, E. O’Boyle, M. Fisher, and R. S. Haber, “Regulation of GLUT2 glucose transporter expression in liver by thyroid hormone: evidence for hormonal regulation of the hepatic glucose transport system,” Endocrinology, vol. 135, no. 2, pp. 649–654, 1994. 20. Henry C. Ford, W.Chew Lim, Michael J. Crooke Hemoglobin A1 and serum fructosamine levels in hyperthyroidism CLINICA CHIMICA ACTA 166(23):317-21 1987 21. Abbas Ali Tam, Hasan Tam, Celil Alper Usluoğulları, İsa Dede, Fevzi Balkan, Cafer Kaya, Rıfkı Üçler, Erdal Eskioğlu. The levels of HbA1c in patients with thyroid dysfunction Gaziantep Med J. 2015; 21(1): 5-8

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

17. Kim H B, Han K H, Lee B W, Kim H, Lee M H and Chung E S. HbA1c and serum fructosamine levels in hyperthyroidism. J Kor. Soc.Endocriol.1992;7:46-51.

Original Article Diagnostic Role of Fine Needle Aspiration Cytology in Thyroiditis along with Thyroid Hormone Assay Prahlad Chandra Agrawal, Reena Naik, Kishori Moni Panda* Department of Pathology, Govt. Medical College (LSLAMMC), Raigarh, Chattisgarh, India Keywords: Thyroiditis, Hashimotos, Hypothyroidism, FNAC, Cytomorphology

ABSTRACT Background: Thyroiditis is the inflammation of the thyroid gland. Hashimotoâ&#x20AC;&#x2122;s thyroiditis (HT) is the most common cause of hypothyroidism in those areas of the world where iodine levels are sufficient. Hashimotoâ&#x20AC;&#x2122;s thyroiditis, a synonym of chronic lymphocytic thyroiditis or autoimmune thyroiditis, is the second most common thyroid lesion diagnosed on FNAC after goiter. FNAC plays a significant role in the diagnosis of thyroid lesions due to its simplicity and low cost. Fine needle aspiration cytology (FNAC) is highly sensitive in diagnosing HT, with a diagnostic accuracy rate of 92%. AIMS AND OBJECTIVE: To correlate FNAC cytologic findings with TFT in different types of thyroiditis. Method: All the patients with thyroid swellings were referred to department of pathology for FNAC as well as thyroid hormone assay. FNAC was performed using nonaspiration or aspiration techniques by 23 G needle with 10 mL syringe. Air-dried smears were stained with Giemsa stain and wet ethanol fixed smears were stained with, PAP hematoxylin and eosin. Cytomorphologic features were reviewed and graded. The level of T4,T3 & T.S.H. is correlated with cytomorphological findings of different types & grades of thyroiditis in our study. Conclusion: Fine needle aspiration cytology (FNAC) is highly sensitive & specific technique in diagnosing HT and other types of thyroiditis. A correct cytological diagnosis was achieved in the majority of cases in this study, thus obviating the need for a surgical intervention & provided life long hormonal therapy in justified cases.

*Corresponding author: Dr. Kishori Moni Panda, Department of Pathology, Govt. Medical College (LSLAMMC), Raigarh, Chattisgarh, India Phone: +91 08763874020, 08225934525 E-mail: drkishorimonipanda@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-109

Introduction

Thyroiditis is the inflammation of the thyroid gland. Causes of thyroiditis include Hashimoto’s thyroiditis (HT), postpartum thyroiditis, subacute thyroiditis (de Quervains thyroiditis), silent thyroiditis, drug-induced thyroiditis ,radiation-induced thyroiditis, acute thyroiditis, and Riedel’s thyroiditis.[1] Hashimoto’s thyroiditis (HT) is the most common presentation of hypothyroidism. In the active phase of thyroiditis there may be transient clinical manifestations of thyrotoxicosis followed by subclinical or overt hypothyroidism in evolution in some cases. Inflammatory destruction of follicular architecture with total replacement by fibrosis seen in long standing thyroiditis. [2] FNAC plays a significant role in the diagnosis of thyroid lesions due to its simplicity and low cost. Hashimoto’s thyroiditis, otherwise called as chronic lymphocytic thyroiditis or autoimmune thyroiditis, is the second most common thyroid lesion diagnosed on FNAC after goiter. [2,3] Hurthle cell change, infiltration of lymphocytes into the follicular cells, epithelioid granuloma with giant cells and lymphoid follicle formation are the cytologic features characteristic of Hashimoto’s thyroiditis.[2] FNAC is considered superior as well as more cost-effective in diagnosing HT than antibody screening.[4] It is important to diagnose HT because patients subsequently become hypothyroid and require life long thyroxin supplementation. As there is an increased risk of extranodal marginal B cell lymphoma in patients with HT, long term follow up is necessary.[4] This is a retrospective study of the thyroiditis cases diagnosed by FNAC.

Material and Method In this study we analysed the cytomorphologic features and thyroid hormone status of all the thyroiditis cases. The study period included from Sept 2014 to sept 2015. FNAC was performed in total thirty cases of thyroid swellings using non aspiration or aspiration techniques by 23 G needle with 10 mL syringe. Thyroid hormone assay was done in all the cases. Out of thirty cases, twelve cases were thyroiditis. Air-dried smears were stained with Giemsa stain and wet ethanol fixed smears were stained with hematoxylin and eosin and PAP stain. Cytomorphologic features were reviewed and graded according to the lymphocyte infiltrate and other parameters, for example, presence of granuloma, Hurthle cells, degree of anisonucleosis, giant cells, and so forth ( Table 1). Blood is drawn for hormonal analysis. The cytomorphological findings of different types & grades of thyroiditis were correlated with thyroid hormone assay. Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016

Results

Out of 30 cases of thyroid swelling, lymphoid infiltrate was seen in 12 cases, in which 9 cases (75%) were Hashimotos, 2 cases (16.6%) lymphocytic thyroiditis and one case (8.3%) is de Quervains thyroiditis (Fig 1). Age ranged from 9–55 years, most common age group affected is (21-35) years, with female predominance. In our study only one case was noted in a nine year old male child. These cases were graded from 0-3 on cytomorphological features (Fig 1) based on findings of different proportion of cells like lymphocytes, plasma cells ,histiocytes, eosinophils, giant cells & hurthle cell changes (Fig 2) , as per the criteria mentioned in (Table 1) and were correlated with thyroid hormone assay. Detail description of cytomorphological features were given in ( Table2) according to which we have segregated the cases to different grade. As per the cytologic criteria for the grading of thyroiditis, majority of the cases belonged to either grade 2 or grade 3 (Table 2). Grade 1 was noted in only one case. No case of grade 0 is recognized in our study. Eosinophils are characteristically noted in grade 2. Hormonal status of all the cases given in (Table-3). T4, T3 & TSH measurements showed mostly hypothyroidism with raised TSH level, which is the usual pattern. In de Quervains disease the patient was euthyroid from beginning. On follow up most patients converted to euthyroid state except two cases, where high TSH level persisted.

Discussion

The first report of chronic thyroiditis or struma lymphomatosa was described by Hakaru Hashimoto in 1912, which bears his name.[2] Prevalence rate of thyroiditis is 1–4% and incidence of 30–60/100000 population per year.[5] Female to male ratio is 10:1 to 20:1. This disease tends to be geographical and seasonal, and is most common in summer and fall.[2] Patients usually present with a diffuse enlargement of the thyroid gland or less frequently with one or two prominent nodules.[6] The average age of onset is between thirty and fifty years of age, while Bhatia et al. observed the commonest age group to be the 3rd to 4th decade.[2] Neelam Sood et al observed, the commonest age group was 21–30 years.[7] In present study most cases fall in between 21-35year, with female predominance, which is similar to other studies.[3,8] Recent reports indicate that the incidence of thyroiditis particularly HT has increased 10 times than in the early 1990s.[9] In our study we found the occurrence of thyroiditis is more common when compared to all cases of thyroid swellings. e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-110

Fig. 1. A - Few lymphoid cells infiltrating the follicles. (Giemsa, 40x) B - Moderate lymphocytic infiltration with occasional hurthle cell change.(Giemsa, 40x) C - Florid lymphocytic inflammation. .(Giemsa, 40x) D - Foreign body giant cell in dequervains thyroiditis. .(Giemsa, 40x) E – Epitheloid granuloma in dequervains thyroiditis. .(PAP, 40x)

Fig. 2 A - Giant cell. (PAP, 40X) B – Epithelioid granuloma. (PAP, 40X) C – Eosinophil.(Giemsa, 100x) D – Lymphoid follicle.(H&E, 40X) E – Hurthle cell.(PAP, 40X)

Table 1 : Grading of Thyroiditis on cytological Material [Bhatia et al] Grade Grade 0

Morphological No lymphoid cells

Grade 1

Few lymphoid cells infiltrating the follicles/increased Number of lymphocytes in the background Moderate lymphocytic infiltration or mild lymphocytic Infiltration with Hurthle cell change/ giant Cells/anisonucleosis Florid lymphocytic inflammation with germinal Centre Formation, very few follicular cells left

Grade 2 Grade 3

No. of Cases (%) 0 1 (8.3%) 6(50%) 5(41.6%)

Table 2: Clinical and cytomorphological findings in thyroiditis.(N= 12 ) Case No. 1 2 3 4 5 6 7 8 9 10 11 12

Age & Sex cellularity 33/F 48/F 38/F 21/F 25/F 24/F 22/F 40/F 45/F 55/F 9/M 30/F

+ +++ +++ ++ ++ +++ +++ ++ + +++ +++ ++

Lymphocytes ++ +++ +++ +++ +++ +++ ++ ++ + ++ ++ ++

Cytological Features Plasma cells Giant cells Eosinophil Histiocyte Hurthle cell + 0 0 + + + + + ++ ++ + 0 0 + + + 0 0 0 0 + 0 0 0 ++ + 0 0 + + + + ++ + ++ + 0 0 + + 0 0 0 0 + + + + + + + ++ 0 + ++ 0 + + + ++

http://www.pacificejournals.com/aabs

Epitheloid Granuloma 0 ++ 0 0 0 0 + + + 0 ++ 0

Grade G-2 G-2 G-3 G-3 G-2 G-3 G-2 G-3 G-1 G-2 G-3 G-2

Dignosiss Hashimotos Hashimotos Hashimotos Hashimotos Lymphocytic Lymphocytic Hashimotos Hashimotos Hashimotos Hashimotos Dequervains Hashimotos

A-111

AABS; 3(1): 2016

Table -3 : Thyroiditis, and Hormonal Status Case No.

Dignosiss

T.S.H.

1 2 3 4 5 6 7 8 9 10 11 12

Hashimotos Hashimotos Hashimotos Hashimotos Lymphocytic Lymphocytic Hashimotos Hashimotos Hashimotos Hashimotos Dequervains Hashimotos

L L N L N L L L N L N L

H H N H N H H H N H N H

Follow up after 2 month (TSH level) N N N N N H N N N N H N

( L-Low level , H- High level , N- Normal level )

This increase in incidence has been linked to excess iodine intake.[10] Fine needle aspiration cytology (FNAC) is highly sensitive in diagnosing HT, by characteristic features of mononuclear cells infiltrate consisting of lymphocytes, plasma cells, histiocytes impinging on follicular cells ,multinucleated giant cells & hurthle cell change. with a diagnostic accuracy rate of 92%. However, diagnosis of HT is likely to be missed in cases of nodular goiter that can be differentiated by the absence of multinucleated giant cells & epitheliod cells and lymphoid cells impinging on follicular cells.[11] Other differential diagnosis include differentiation from de Quervainâ&#x20AC;&#x2122;s thyroiditis, lymphocytic thyroiditis and Graves disease. In de Quervainâ&#x20AC;&#x2122;s thyroiditis, dirty smear background with cell debris, colloid, neutrophil, lymphocytes, macrophage, many granulomatous aggregates of epithelioid cells and many multinucleated giant cells present.[11] Lymphocytic thyroiditis smear mainly includes mixed population of lymphoid cells in form of reactive lymphoid proliferations infiltrating thyroid follicular cells, without prominence of hurthle cell metaplasia. Graves disease is revealed by presence of hypercellular colloid free smear showing columnar type follicular cells in monolayered sheets. Cytoplasm is abundant, vacuolated with larger marginal vacuoles (fire flares). Hurthle cells, lymphocytes and multinucleated giant cells are rarely seen[11] Bhatia et al have done cytological grading on FNAC smears for the first time using predefined sets of criteria, where they tried to correlate the lymphoid density with clinical, radiological, and biochemical parameters.[2] In 1960, Schade et al. analysed the relationship between thyroid autoimmunity and the presence of lymphocytes in Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

the thyroid gland in patients with Gravesâ&#x20AC;&#x2122; disease, toxic adenoma, and nontoxic nodular goitre and found that in all these conditions circulating antibody to thyroglobulin was significantly associated with lymphocytic infiltration. [5] Bipin et al reported the importance of presence of eosinophils & high eosinophil: neutrophil ratio for making diagnosis of HT and to differentiate it from colloid goiter. Commonest type of thyroiditis in our study is HT with female predominance and common hormonal pattern noted is hypothyroidism. It is important to diagnose HT because patients subsequently become hypothyroid and require lifelong thyroxine supplementation. Also there is an increased risk of malignancy like extranodal marginal B cell lymphoma in patients with HT.[4] The frequency of carcinoma in patients with HT varies between 0.5 and 23.5% which emphasizes the need for long-term followup. It is also important not to over-diagnose this entity as neoplasms so that unnecessary surgery can be avoided. [6] Potential pitfalls are cytologic atypia occurring in HT, abundance or scarcity of background inflammation, low cell yield, coexisting toxicity and malignancies. But epithelial preponderance over inflammation, nuclear crowding, severe atypia and cell discohesion should raise the possibility of a neoplasm in spite of other features of HT.[4]

Conclusion Fine needle aspiration cytology (FNAC) is highly sensitive & specific technique in diagnosing HT and other types of thyroiditis. A correct cytological diagnosis was achieved in the majority of cases in this study, thus obviating the need for a surgical intervention & provided life long hormonal therapy in justified cases. A careful and diligent search for various cytological features and accurate sampling can help in reducing the number of indeterminate, false-positive and false-negative diagnoses. However, in difficult situations an integrated approach will minimize potential pitfalls.

Acknowledgements It should include persons who provided technical help, writing assistance and departmental head that only provided general support. Financial and material support and conflict of interests must be written in this section.

Funding None

Competing interests Not declared

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-112

References

principles and dilemmas. 1st ed. Germany: Springer; 2006. pp. 99–101.

1. “Thyroiditis.” www.thyroid.org. 2005. American Thyroid Association. 13 Mar. 2008. 8 Dec. 2015 2. A. Bhatia, A. Rajwanshi, R. J. Dash, B. R. Mittal, and A. K. Saxena, “Lymphocytic Thyroiditis—is cytological grading significant? A correlation of grades with clinical, biochemical, ultrasonographic and radionuclide parameters,” CytoJournal, vol. 4, article 10, 2007. 3. B. N. Gayathri, R. Kalyani, M. L. Harendra Kumar, and K. Krishna Prasad, “Fine needle aspiration cytology of Hashimoto’s thyroiditis—a diagnostic pitfall with review of literature,” Journal of Cytology, vol. 28, no. 4, pp. 210–213, 2011. 4. Kumarasinghe MP, De Silva S. Pitfalls in the cytological diagnosis of autoimmune thyroiditis. Pathology.1999;31:1–7. 5. R. O. K. Schade, S. G. Owen, G. A. Smart, and R. Hall, “The relation of thyroid auto-immunity to roundcelled infiltration of the thyroid gland,” Journal of Clinical Pathology, vol. 13, pp. 499–501, 1960. View at Google Scholar · View at Scopus 6. Kocjan G. Lymphoid Infiltrate. In: Schroder G, editor. Fine needle aspiration cytology: diagnostic

7. Neelam Sood and Jitendra Singh Nigam, “Correlation of Fine Needle Aspiration Cytology Findings with Thyroid Function Test in Cases of Lymphocytic Thyroiditis,” Journal of Thyroid Research; 2014, p. 5. 8. Ekambaram M, Kumar B, Chowdhary N, Siddaraju N, Kumar S. Significance of eosinophils in diagnosing Hashimoto’s thyroiditis on fine needle aspiration cytology. Indian J Pathol Microbiol.2010;53:476–9. 9. Marwaha RK, Tandon N, Karak AK, Gupta N, Verma K, Kochupillai N. Hashimoto’s thyroiditis: countrywide screening of goitrous healthy young girls in postiodization phase of India. J Clin Endocrinol Metab. 2000;85:3798–802. 10. Benvenga S, Trimarchi F. Changed presentation of Hashimoto’s thyroiditis in North-Eastern Sicily and Calabria (Southern Italy) based on a 31-year experience. Thyroid. 2008;18:429–41. 11. Orell SR, Sterrett GF, Darell W. Thyroid. In: Orell SR, Sterrett GF, Darell W, editors. Fine needle aspiration cytology. 4th ed. India: Elsevier Science Ltd; 2005. p. 136–8.

http://www.pacificejournals.com/aabs

Original Article Role of Bronchoscopy in Diagnosing Sputum Smear Negative Pulmonary Tuberculosis Prashant Prakash1*, Pooja Agarwal2, Prabhat Agarwal3, Durga Prasad Singh3, Ashutosh Gupta3 1

Pulmonary Medicine Unit, Department Of Medicine, S.N. Medical College, Agra, India 2 Dept of Pathology, S.N. Medical College, Agra, India 3 Department Of Medicine, S.N. Medical College, Agra, India Keywords: BAL, Bronchoscopy, PBS, SSNPTB, TBLB

ABSTRACT Background: In patients of pulmonary tuberculosis microscopic examination of sputum is the method of choice. Sputum examination for AFB microscopy is usually positive but even in advanced disease may be negative due to immunosuppression, poor quality of the sample collection, deficient preparation, staining or examination of the sputum smear. Clinical and radiological based diagnosis can lead either to over or under diagnosis of tuberculosis and unnecessary antitubercular treatment may cause drug resistance and economic burden. Fiberoptic bronchoscopy can provide excellent material for diagnosis of suspected cases of pulmonary tuberculosis when smears of expectorated sputum do not reveal mycobacteria. Aim: To assess the role of bronchoscopy in diagnosis of pulmonary tuberculosis among patients who have clinicoradiological suspicion but remain negative for AFB on sputum examination. Settings and design: After consent, 50 patients with sputum smear negative for AFB and chest X-ray suggestive of pulmonary tuberculosis, underwent fiberoptic bronchoscopy. Bronchoalveolar lavage (BAL), Bronchial brushings, Transbronchial lung biopsy (TBLB) and Post bronchoscopy sputum (PBS) were done and samples analyzed by smear microscopy, histopathology and or culture. Result: Sensitivity of BAL, Brushing and PBS by smear and culture are 87.5%, 54.17% and 50% respectively. The final diagnosis of PTB was established in 24/50 (48%) cases. Conclusion: Fiberoptic bronchoscopy with post bronchoscopy sputum is a useful tool for diagnosis and can thereby prompt treatment of sputum smear negative pulmonary tuberculosis patients.

*Corresponding author: Dr. Prashant Prakash, 79, Gandhi Nagar, Bye Pass Road, Agra (India) 282003. Phone: +91 9897285457 E-mail: dr.prashantprakash@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-114

Introduction India has the highest burden of tuberculosis in the world and account for nearly one fourth of the global burden of tuberculosis per year.1,2 Globally, the absolute number of incident TB cases per year has been falling since 2006 and the incidence rate (per 100 000 population) has been falling by 1.3% per year since 2002.3 Case fatality rate prior to RNTCP was generally greater than 25%. In RNTCP era, case fatality has remained less than 5% for new cases registered under this programme.3 “Smear negative – culture positive” state has been observed in 22 to 61% of the cases.4 With the advent of flexible fibreoptic bronchoscope , a more aggressive approach of investigation for sputum negative pulmonary TB has been adopted. Bronchoscopy and related procedures such as bronchial brushing, bronchoalveolar lavage, transbronchial biopsy and post bronchoscopy sputum examination may be alternative way of reaching the diagnosis as early as possible. Therefore, this study was undertaken to determine the role of bronchsocopy in the diagnosis of sputum smear negative pulmonary tuberculosis (SSNPTB).

done. Special emphasis was given to the segments of radiographically suspicious areas. BAL fluid was collected in all patients, bronchial brushing and trans-bronchial lung biopsies were done in patients who had some kind of visible pathological lesion on bronchoscopy and in whom biopsy was feasible. Post bronchoscopy sputum sample were taken on next three occasions in all 50 cases (one immediately after bronchoscopy and other two on next two days). These specimens were further examined by Z-N staining and AFB culture by L-J media. Biopsies were examined histopathologically.

Results

60 patients were admitted with clinical and radiological suspicion of pulmonary tuberculosis. Clinical symptoms consisted of evening rise of temperature, night sweats, weight loss, anorexia associated with cough with or without expectoration, hemoptysis and dyspnea. Radiologically chest x-ray showed one or more of the following lesions like parenchymal consolidation, cavitation, miliary tuberculosis, lymphadenopathy, tuberculoma, fibroproductive lesion and pleural effusion. Out of these, six patients had associated cardiac diseases and were excluded from the study. Four patients did not give consent for the bronchoscopy, so they were also excluded from the study. So, finally 50 patients were included in this study. All these patients (18-60 years) had two negative sputum smear examination for Acid Fast Bacilli (AFB). The enrolled patients were subjected to a protocol, which included detailed history regarding mode of onset, duration of illness, history of drug intake and radiological evidence of pulmonary tuberculosis. These 50 patients underwent bronchoscopic examination by using Fiberoptic bronchoscope (Fiberoptic Bronchoscope Model BE-TE2, manufactured by Olympus corporation, Tokyo, Japan was used for the procedure) and post bronchoscopy sputum (PBS) examination.

Out of the 50 cases, 33(66%) were males and 17 (34%) were females. Amongst the males, history of smoking was present in 30/33 (90.90 %) and in the females 6/17 (35.2%) were smokers. 44 cases (88%) were positive for history of contact with TB. Of these, 38 cases (76%) gave history of pulmonary tuberculosis in neighbourhood. Only six subjects reported negative history of contact with pulmonary tuberculosis. Of the six negative history cases, three were females and three were males. Nine patients (18%) had received whole course of anti-tubercular treatment before, 12 patients (24%) were defaulters, 29 patients (58%) were without any prior history of antitubercular treatment. Bronchoscopic examination revealed no pathological lesion in 21 (42%) of 50 patients. Out of 29 patients where bronchoscopy revealed some pathology, 14 cases (28%) had discharge from bronchus, 11 cases (22%) had unhealthy mucosa /granuloma, 5 cases (10%) had external compression and another 5 cases (10%) had bleeding from bronchus, while growth was visible only in 2 cases (4%) (Table-I). BAL was done in all 50 cases where as brushings were taken from the affected segment in 29 cases. Bronchial biopsies were done only in 12 out of 50 patients, where biopsy was feasible. After bronchoscopy, Post Bronchoscopy Sputums (PBS) was also taken on three occasions in all 50 cases (one immediately after bronchoscopy and other two on next two days). Smear examination for AFB was positive in BAL fluid in 19 cases (38%), in bronchial brushings in 12 cases (41.4%), and in post bronchoscopy sputum in 11 cases (22%). Trans bronchial lung biopsy (TBLB) showed caseating epitheloid granuloma in three cases (25%) out of which AFB could be demonstrated in two (16.66%) (Figure 1). BAL culture for M. tuberculosis was positive in 21 patients (42%), Bronchial brushing culture was positive in 13 cases (44.8%), Post bronchoscopy sputum (PBS) culture was positive in 12 patients (24%) (Figure 2).

Fiberoptic bronchoscopy was carried out under local anesthesia and inspection of both bronchial tree was

During our study, we were able to diagnose 24 cases out of 50 (48%) by combining all the bronchoscopic aided

Methods This study was carried out in the Department of Medicine and Department of Pathology, and the duration of study was from August, 2011 to January, 2014.

http://www.pacificejournals.com/aabs

AABS; 3(1): 2016

A-115

Fig.1: Result Of AFB Microscopy Of Bronchial Specimens (N=50)

Fig. 2: Culture Results Of Bronchial Specimens For Mycobacterium Tuberculosis TABLE I: BRONCHOSCOPIC FINDINGS BRONCHOSCOPIC FEATURES

N (%)

Normalppearance Abnormalities seen* : Growth Unhealthy mucosa/granuloma External compression Discharge from bronchus Bleeding from bronchus

21 (42%) 2 (4%) 11(22%) 5 (10%) 14 (28%) 5 (10%)

* Multiple findings in some cases Table II: Total Yield Of Various Bronchoscopic Aided Methods In Our Study (N= 50)

Fig. 3: Sensitivity Of Various Bronchoscopic Aided Methods (%)

procedures and using either smear examination for AFB or culture for AFB or caseating granulomas as the end result of our study. Of these 24 cases diagnosed as tuberculosis, BAL was the most effective method where diagnosis was clinched in 21 cases (21/24) with a sensitivity of 87.5 % followed by brushings where diagnosis was clinched in 13 cases (13/24) with a sensitivity of 54.17%. Using post bronchoscopy sputum (PBS) as the method of diagnosis, we were able to clinch the diagnosis in 12 out of 24 positive patients (12/24) with a sensitivity of 50% and TBLB clinched the diagnosis in 3 out of 24 positive patients (3/24) with a sensitivity of 12.5% (Figure 3). Bronchial brushings had the highest yield (13/29) of 44.8% followed by BAL (21/50) of 42%. TBLB and PBS comparatively had low diagnostic yield of 25% and 24% respectively, in our study (Table II). Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

CASES CASES TOTAL POSITIVE DONE YIELD 21

42 %

BRONCHIAL BRUSHINGS 13

44.8 %

BAL FLUID PBS

24 %

TBLB

25 %

Discussion The WHO Expert Committee on Tuberculosis recommends that patients of pulmonary tuberculosis in whom the disease has not been confirmed bacteriologically should be classified as â&#x20AC;&#x153;suspectsâ&#x20AC;? till the presence of AFB is demonstrated and a patient with persistent symptoms whose sputum does not contain AFB should be followed up and anti-tubercular treatment should be given only if the diagnosis can be confirmed bacteriologically. In our study, we had selected 50 patients with sputum smear negative on two occasions, out of which 33 were males and 17 were females. This was comparable to the study done by Purohit et al5, where in the sample size of e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-116

50 cases, they had 35 males and 15 females. Thus, the data generated in our study is comparable to this study. Out of the 50 patients in our study, 43 patients presented with cough (86%), 40 patients (80%) had cough with expectoration, 30 (60%) patients had fever, 16 patients (32%) had hemoptysis and 12 patients (24%) presented with chest pain. These patients characteristics were similar to the study done by D D S Kulpati et al6. In our study, 18 cases out of 50 (36%) were active smokers, while 18 of them (36%) quitted smoking for more than one year and 14 of them (28%) had never smoked before. This was higher than the study characteristics done by Chan H S et al7. Amongst the 50 cases, when evaluated for history of contact with a case of pulmonary tuberculosis, 44 cases (88%) were positive. Of the six negative cases, three were females and three were males. This was higher than the study done by Kwan Hoi Yee et al8. This high rate could be due to high prevalence of tuberculosis in India. Among 50 patients, signs in the form of creptitations (with or without rhonchi) or bronchial breath sounds were found among 44 patients. Crepitations (with or without rhonchi) were found in 28 patients. Bronchial breath sounds were found in 16 patients. Bronchoscopic examination revealed no pathology in 21 (42%) of these patients. Out of 29 patients where bronchoscopy revealed some pathology, 14 cases (28%) had discharge from bronchus, 11 cases (22%) had unhealthy mucosa / granuloma, 5 cases (10%) had external compression and another 5 cases (10%) had bleeding from bronchus, while growth was visible only in 2 cases (4%). This was comparable to the study done by Panda et al9 where bronchoscopy was normal in 44% cases and 35% cases had discharge from bronchus, 21% cases had unhealthy mucosa / granuloma, 3% cases had external compression and another 3% cases had bleeding from bronchus, while growth was visible in 5% cases. In our study, the BAL fluid smears were taken in all 50 cases and were positive for AFB in 19 (38%) patients. In previous studies, it varied from 7.5% to 57.1% in studies done by S Charoenratanakul et al10 and Malekmohammad M et al11 respectively. BAL culture yieldedÂ M. tuberculosisÂ in 42% patients in our study which was comparable to the 48% positivity (24 cases out of 50) by Wongthim et al12. In our study, bronchial brush smears were done in 29 patients in whom bronchoscopy revealed some visible pathology. Bronchial brushings were positive for acid fast bacilli in 41.4% (12/29) patients; all of these were confirmed by positive culture. Bronchial brushings when

subjected to culture yielded 44.8% positivity in our study. This was also comparable to the studies done by Willcox et al13 (42%) and Wongthim et al 12(51%). In our study, Post bronchoscopy sputums were collected on 3 occasions and subjected to both smear for AFB and culture, yielding 22 %(11/50) and 24% (12/50) respectively. This was comparable to the 23% positivity (7 out of 30 cases) in post bronchoscopy sputum studied by Wongthim et al12. Kulpati et al6 also noted 25% positivity (5 out of 20 cases) by PBS culture and 26% AFB smear positivity was noted by PurohitÂ et al5. In our study, transbronchial lung biopsies were taken from areas of suspicion in 12 cases, 3 cases (25%) showed caseating epithelioid granulomas with AFB positivity in 16.66% (2 cases out of 12). A.K.Jaiswal et al14 reported bronchoscopic guided biopsy for diagnosis of sputum negative tuberculosis to be 10% (2 out of 20 cases) in their study and D.D.S.Kulpati et al6 reported 20 % (4 cases out of 20) in their study. During our study, we were able to diagnose 24 cases out of 50 cases (48%) by combining all the bronchoscopic aided procedures and using either smear examination for AFB, culture for AFB or caseating epithelioid granulomas as the end result of our study. Out of these 24 cases diagnosed as tuberculosis, BAL was the most effective method where diagnosis was clinched in 21 cases (21/24) with a sensitivity of 87.5 % which was comparable to the study by de Gracia et al15 where BAL had a sensitivity of 88% (15/17). Using brushings, diagnosis was clinched in 13 cases (13/24) with a sensitivity of 54.17%. This was comparable to the previous studies by A.A Bachh et al 16 and Chawla et al17where brushings had sensitivity of 48.33% and 62% respectively. Using post bronchoscopy sputum as the method of diagnosis, we were able to clinch the diagnosis in 12 out of 24 positive patients (12/24) with a sensitivity of 50% which was comparable to the study by Panda et al8 where PBS had a sensitivity of 48.5% (17/35). Trans bronchial lung biopsy clinched the diagnosis in 3 out of 24 positive patients (3/24) with a sensitivity of 12.5% which was comparable to the study by S.Y.So et al 18 12% (7/57).

Conclusion

The study concludes that flexible fiberoptic bronchoscopy along with post bronchoscopy sputum examination is a useful tool in early diagnosis of pulmonary tuberculosis in sputum smear negative patients. Bronchoscopy reveals a higher bacteriological confirmation of diagnosis in

http://www.pacificejournals.com/aabs

A-117 patients with strong clinical and radiological evidence suggestive of pulmonary tuberculosis. Thus bronchoscopic aided procedures should be undertaken in sputum smear negative pulmonary tuberculosis patients with a high index of clinico-radiological suspicion.

AABS; 3(1): 2016

9. Panda B.N., Rajan K.E.et al. Diagnostic yield from flexible fibreoptic bronchoscopy in sputum negative pulmonary tuberculosis. Ind. J. Tub 1995; 42: 207-209.

10. Charoenratanakul S., Dejsomritrutai W. et al. Diagnostic role of fibreoptic bronchoscopy in suspected smear negative pulmonary tuberculosis. Respiratory Medicine 1995; 89: 621-623. 11. Malekmohammad M., Marjani M. et al. Diagnostic yield of post-bronchoscopy sputum smear in pulmonary tuberculosis. Scand J Infect Dis 2012; 44(5): 369-373. 12. Wongthim S., Udompanich V. et al. Fibreoptic bronchoscopy in diagnosis of patients with suspected active pulmonary tuberculosis. J Med Assos Thai 1989; 72: 154-59. 13. Willicox P.A., Benatar S.R. et al. Use of fibreoptic bronchoscope in diagnosis of sputum negative pulmonary tuberculosis. Thorax 1982; 37: 598-601. 14. Jaiswal A.K., Kulpati D.D. et al. Role of bronchoscopy in early diagnosis of suspected smear negative cases of pulmonary tuberculosis. Ind. J. Tub 1989; 36: 233. 15. Gracia J.de, Curull V. et al. Diagnostic value of bronchoalveolar lavage in suspected pulmonary tuberculosis. Chest 1988; 93(2): 329-332. 16. Bachh A.A., Gupta R. et al. Diagnosing sputum smear negative pulmonary tuberculosis: does fibreoptic bronchoscopy play a significant role?. Lung India 2010; 27:58-62. 17. Chawla R., Pant K.et al. Fiberoptic bronchoscopy in smear-negative pulmonary tuberculosis. Eur Respir J 1988; 1: 804-806. 18. S.Y.So, W.K. Lam et al. Rapid diagnosis of suspected pulmonary tuberculosis by fibreoptic bronchoscopy. Tubercle 1982; 63(3): 195-200.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

References

1. http://www.who.int/tb/publications/global_report/en/ 2. Park K. Park’s Textbook of preventive and social medicine; 23rd eds. Epidemiology of communicable diseases:Banarsidasbhanot publishers, 2015: 176-202. 3. http://tbcindia.nic.in/index 4. http://www.who.int/tb publications/2010/9789241547833/ en/ 5. Purohit S.D., Sisodia R.S. et al. Fiberoptic Bronchoscopy In Diagnosis of Smear Negative Pulmonary Tuberculosis. Lung India 1983; 1(4): 143-46. 6. Kulpati D.D.S. and Heera H.S. Diagnosis of smear negative pulmonary tuberculosis by flexible fibre optic bronchoscopy. Ind. J. Tub 1986; 33: 179 7. Chan H.S., Sun A.J. et al. Bronchoscopic aspiration and bronchoalveolar lavage in the diagnosis of sputum smear-negative pulmonary tuberculosis. Lung 1990; 168(4): 215-20. 8. Kwan Hoi-Yee. Use of fibreoptic bronchoscopy in diagnosing sputum smear negative pulmonary tuberculosis. http://hdl.handle.net/10722/133487.

Case Report

Diagnosis of primary abdominal wall endometriosis on cytology: An unusual presentation Ankit Kaushik1, Anamika Jaiswal2 1

Department of Pathology, VMMC and Safdarjung Hospital, New Delhi, India 2 Govt. Medical College, Haldwani, India Keywords: FNAC, Endometriosis, Abdominal Wall

ABSTRACT

Abdominal wall endometriosis is a rare type of extra pelvic endometriosis. The abdominal wall endometriosis mostly occurs in the pre existing laprotomy scars in the lower abdominal wall following caesarian section or hysterectomy. We present a case of primary abdominal wall endometriosis in arising in the anterior abdominal wall above umbilicus with no previous history of any surgery or scar, diagnosed on fine needle aspiration cytology. Primary abdominal wall endometriosis is extremely rare and can be clinically challenging, FNAC provides easy and reliable technique for diagnosing endometriosis.

*Corresponding author: Dr Ankit Kaushik; B 19 Mahesh Park, Modinagar, Ghaziabad, UP, India Mobile: +91-9953816240, kaushikankit30@yahoo.co.in

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Case Report

C-2

INTRODUCTION Endometriosis is presence of functioning endometrial tissue outside uterus. The common site of endometriosis is ovary, rectum, and urinary bladder. Abdominal wall is a rare site for endometriosis [1]. It usually arises on preexisting scar of previous caesarian section or hysterectomy. Primary endometriosis occurs with no pre existing scar in abdominal wall and is extremely rare. Sign and symptoms may include cyclical pain and change in color of swelling indicating functional nature of endometrial tissue.

CASE REPORT A 40 year old multiparous female presented with a blue to black nodular swelling over anterior abdominal wall from past 6 months that was slowly increasing in size. There was no history of any previous surgery. On examination the swelling was well defined, 3 x 3 cm, firm, tender and was felt subcutaneously (Figure 1).

Figure 1:Showing bluish nodular swelling over abdominal wall.

Patient was referred for FNAC with a clinical diagnosis of hemangioma. On fine needle aspiration cytology (FNAC), the smears were cellular with monolayered sheets of small, round to oval, polygonal epithelial cells with scant cytoplasm. The nucleoli were round to oval with inconspicuous nucleoli. The stromal fragments consisting of oval to spindle cells arranged around vessels were appreciated. The background was predominantly hemorrhagic with numerous hemosiderin laden macrophages along with few naked nuclei. Based on above findings a diagnosis of endometriosis of anterior abdominal wall was given that was subsequently confirmed by histopathological findings (Figure 2).

DISCUSSION Endometriosis is defined as presence of functioning endometrium outside the uterus. Abdominal wall endometriosis is rare type of extra pelvic endometriosis. Abdominal wall endometriosis occurs in the post operative scars mostly in the lower abdominal wall following hysterectomy, caesarian sections, appendectomy, aminocentesis. Clinical diagnosis of abdominal wall endometriosis may be difficult because of relatively unfamiliar entity among general surgeons. A palpable subcutaneous mass in or around surgical scars with a history of previous cessarian section should lead to a clinical differential of abdominal wall endometriosis.[1] The incidence of scar endometriosis depends on the type of surgery with higher incidence in scars of mid trimester abortion than caesarian section. The incidence may be attributed to pluripotential capability of early deciduas, resulting in cellular replication producing endometriomas.[ 2,3] The more convincing metastatic theory proposes that endometrial cells get transported to other site via direct, hematogenous and lymphatic spread while the according to metaplastic theory, the pleuripotent cells at the site undergoes the metaplastic change to the endometrial cells.[4] Endometrosis arising from previous scarless abdominal wall is documented but rarely found.[5] The abdominal wall endometriosis presents as long standing slowly increasing painful, firm, nodular swelling over a preexisting scar with overlying bluish discolouration of skin and bleeding manifestation. The cyclicity of symptoms with menstruation is pathognomic of endometrisosis. The common clinical differentials of abdominal wall endometriosis are metastatic disease, desmoid tumor, sarcoma, nodular fasciitis, fat necrosis, hematoma or abscess.

Figure 2: Giemsa stained smear showing round to oval, medium size endometrial cells along with macrophages over a hemorrhagic background.

The cytology smears shows epithelial cells, stromal cells in a hemmoraghic background with variable number of hemosiderin laden macrophages. The presence of either

http://www.pacificejournals.com/aabs

C-3 two among epithelial cells, stromal cells and hemosiderin laden macrophages are sufficient for diagnosing endometriosis on cytology.[5] Clinical differential diagnosis of abdominal swelling includes old hematoma, hemangioma, metastatic deposits and other soft tissue swelling like nodular fasciitis and desmoid tumor.[6] The smears from metastatic deposits show obviously malignant cells depending on the primary tumor. The desmoid tumor presents with scant cellularity with occasional spindle shaped cells. The hematoma presents with a history of trauma with smears showing lot of hemorrhagic background with and without hemosidein laden macrophages with absence of characteristic endometrial cells. The nodular fascitis present with pleomorhic cells in myxoid background. Although FNAC provides a rapid and accurate diagnosis of endometriosis, the imaging modalities can define the extent of lesion for resection especially in cases of large and recurrent lesions. Surgical resection is the principal mode of treatment in endometriosis. CONCLUSION Abdominal wall endometriosis is a very rare with most cases arising in a preexisting scar. A strong clinical suspicion is required for diagnosing primary abdominal wall endometriosis. FNAC provides a rapid, inexpensive and definitive diagnostic tool for diagnosing abdominal wall endometriosis.

ACKNOWLEDGEMENTS

AABS; 3(1): 2016

COMPETING INTERESTS None declared.

REFERENCES 1.

Bektaล H, Bilsel Y, Sari YS, Ersรถz F, Koรง O, Deniz M, Boran B, Huq GE. Abdominal wall endometrioma; a 10-year experience and brief review of the literature. J Surg Res. 2010 ;164(1):77-81. Blanco RG, Parithivel VS, Shah AK, Gumbs MA, Schein M, Gerst PH. Abdominal wall endometriomas. Am J Surg 2003;185:596-8. Goel P, Sood SS, Romilla, Dalal A. Cesarean section endometriosis-Report of two cases. Indian J Med Sci 2005;59:495-8. A Thapa, A Kumar, S Gupta. Abdominal Wall Endometriosis: Report Of A Case And How Much We Know About It?. The Internet Journal of Surgery. 2006 Volume 9 Number 2.Ideyi SC, Schein M, Niazi M, Gerst PH. Spontaneous endometriosis of the abdominal wall. Dig Sur 2003; 20:246-8. Simsir A, Thorner K, Waisman J, Cangiarella J. Endometriosis in abdominal scars: a report of three cases diagnosed by fine-needle aspiration biopsy. Am Surg. 2001 Oct;67(10):984-6. Cozzolino M, Magnolfi S, Corioni S, Moncini D, Mattei A. Abdominal Wall Endometriosis on the Right Port Site After Laparoscopy: Case Report and Literature Review. The Ochsner Journal. 2015;15(3):251-5.

None.

FUNDING None.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report A Diagnostic Dilemma of Nasal Meningo-Encephalocele â&#x20AC;&#x201C; A Case Report Ritu Sharma1, Vivek Gupta1, Gaurav Kumar2 1

Department of Pathology, Hind Institute of Medical Sciences, Lucknow (U.P.) INDIA. Department of E.N.T. Head & Neck Surgery, TSM Medical College, Lucknow (U.P.) INDIA.

Keywords: Meningo-encephalocele, FNAC, Encephalocele, Meningocele

ABSTRACT Background: Nasal Meningo-encephalocele present at birth as congenital deformity with characteristic swelling over the nose. Atypical nasal encephaloceles are usually missed at birth due to absence of any external swelling and they present with diagnostic difficulties. We describe a case of congenital nasal swelling present at the root of nose in a 1 year old male child who underwent FNAC procedure followed by radio diagnostic approach and diagnosed as a case of Meningo-encephalocele in a Pathology department. An encephalocele is a rare disorder, caused by failure of the neural tube to close completely during fetal development. A meningocele is an encephalocele that contains only the meninges and cerebral spinal fluid (CSF).

*Corresponding author: Dr. Ritu Sharma, H.N. 1404, HIG, Sector-I, LDA Colony, Kanpur Road, Lucknow, UP, India Phone: 91 522 9455149391 E-mail: drritzsharma@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-5

Introduction Encephalocele, sometimes known by the Latin name â&#x20AC;&#x153;cranium bifidaâ&#x20AC;?, an ence-phalocoele is a congenital deformity in which intracranial contents herniate through a defect in the skull 1. The most common location for encephaloceles is the occipital region. They occur usually in the mid sagittal plane anywhere from frontonasal region to the occiput but rarely they are found in the parietal region too 2. They are usually found at the sites of fontanelles but they can also appear from the defects in the cribriform plate of ethmoid, or from the foramen caecum, foramen magnum or through a suture line. These defects are caused by failure of the neural tube to close completely during fetal development 3. If the bulging portion contains only cerebrospinal fluid and the overlaying membrane, it may be called a meningocele. If brain tissue is present, it may be referred to as a meningoencephalocele 4. Encephaloceles are often accompanied by craniofacial abnormalities or other brain malformations. Symptoms may include neurologic problems, hydrocephalus, spastic quadriplegia, microcephaly, ataxia, developmental delay, vision problems, mental and growth retardation, and seizures 5.

Case Report

We are reporting a case a one year old male child presenting with firm to cystic swelling at the root of nose presented in ENT department referred to pathology department for Fine needle aspiration cytology (FNAC) procedure. Clinical Presentation: A child presented with swelling at the root of nose that is discovered by parents. On Examination: General examination: Normal milestones of a child. Systemic examination: Firm to cystic swelling present at the root of nose which was gradually progressive, measuring 2.0 x 2.5 cm. Fine needle aspiration cytology (FNAC) was done without sedation. Patient was an infant, highly uncooperative. The moment

AABS; 3(1): 2016 the needle entered the swelling, clear fluid was noticed on hub of the needle, immediately the negative pressure released and the needle was withdrawn from swelling. On aspiration clear fluid was collected in test tube which was subjected to centrifugation and slide was prepared from the sediment and stained with H&E. Smears on microscopic examination reveals hypocellularity, occasional astrocytic cell clusters with fibrillary processes present (Figure 1, 2, 3, 4, 5 & 6). Occasional mature lymphocytes and RBCs also seen. Clear fluid aspirated was suspected to be CSF. Cytological Impression reveals cerebral spinal fluid (CSF) with brain tissue. Work up: Child was adviced Computed Tomography (CT) Scan for further evaluation of case. CT scan Paranasal Sinus (PNS) (Figure 7, 8, 9, 10 & 11) reveals huge Meningo-encephalocoele from left temporo-frontal lobe reaching to root of nose through widened crista gali. Mass is displacing and compressing left fronto ethmoidal sinus. There is prominent cistrna magna. Fetuses with an encephalocele are likely to die before birth. Approximately 21 percent, or 1 in 5, are born alive. Of those born alive, only 50 percent will survive. Fetuses with a front type encephalocele are much more likely to survive than those who have an encephalocele on the back of the head. The absence of brain tissue within the sac is the single most favorable prognostic indicator. Presence of associated malformations is another indicator of prognosis 6.

Discussion: An encephalocele is a rare disorder in which the bones of the skull do not close completely. This creates a gap through which cerebral spinal fluid, brain tissue and the meninges can protrude into a sac like formation. Encephaloceles occur rarely, at a rate of one per 5000 live births worldwide 7. The presence of an encephalocele is associated with an increased incidence of death in utero. Experts estimate that only half of the children with

Fig. 1: and 2: (10x, 40x H&E) Photograph showing Astrocytic cell clusters with fibrillary processes.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

Fig. 3, 4, 5 & 6:

C-6

(40x H & E) Photomicrograph showing clusters and individual cells of astrocytes with fibrillay process.

Fig. 7, 8 & 9: Shows CECT of PNS axial cuts showing Meningo-encephalocoele.

encephaloceles survive to birth. They are seen more commonly in females than males6. Encephaloceles of the back of the head are more common in Europe and North America, while encephaloceles on the front of the head more frequently occur in Southeast Asia, Africa, Malaysia, and Russia. Ethnic, genetic, and environmental factors, as well as parental age, can all affect the likelihood of encephaloceles. The condition can occur in families with a family history of spina bifida 7. Usually encephaloceles are

noticeable deformities and are diagnosed immediately after birth, but a small encephalocele in the nasal or forehead region can go undetected 8.

Conclusion Congenital midline masses of the face are uncommon. Epidermoid cysts, dermoids, gliomas are the most important differential diagnosis in congenital nasofrontal masses. Since they arise from an abnormal fusion during fetal development, intracranial extension of the

http://www.pacificejournals.com/aabs

C-7

AABS; 3(1): 2016

Fig. 10 & 11: Shows huge Meningo-encephalocoele from left temporo-frontal lobe reaching to root of nose.

lesion has to be ruled out.9 Meningo-encephalocele is a medical emergency it will required proper treatment. Fine needle aspiration cytology is advocated by some for ruling out malignancy before any therapy is conducted10. Encephaloceles are frequently associated with other cranial (head, skull, or brain) and/or facial abnormalities. Diagnostic examinations use the MRI and CT method. The location of the encephalocele greatly affects the prognosis. Those located in the front have a 100 percent survival rate, while those located in the back have a 55 percent survival rate.11 This study is not funded by person or organization. Author’s have no conflict of interest.

References 1. NINDS Encephalocele information page. (http://www. ninds.nih.gov/disorders encephaloceles.htm), NINDS, February 12, 2007. 2. Ramachandra C. R. S., Phelps P. D. (1977) Sept. 1991 : Nasal encephalocoeles associated with unilateral absence of cochlea. Journal of Laryngology & Otology, (9) 813-817. 3. Mood G. F. (1938) : Congenital anterior herniations of brain.Annals of Otology, 4. Rhinology and Laryngology, 47 ; 391-401. 5. Encephalocele imaging (http:// emedicine. Medscape. com/ article/ 403308- overview) at eMedicine.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

6. Holmes, Anthony D.; Meara, John G.; Kolker, Adam R.; Rosenfeld, Jeffrey V.;Klug ,Geoffrey L. (2001). “Frontoethmoidal Encephaloceles: Reconstruction and Refinements”. The Journal of Craniofacial Surgery 12 (1): 6–18. PMID 11314190. 7. Encephalocele Children’s Hospital of Wisconsin information page (http; www.org /medical-case/ fetalconcerns-center/ conditions/ infant-complication/ encephalocele) 12/9/2015. 8. “Conditions + Treatments | Boston Children’s Hospital”. Childrenshospital.org. Retrieved 20150626. 9. Nasal Encephalocoele -An Atypical Case. Indian Journal of Otolaryngology and Head and Neck Surgery Vol. 56 No. 1, January - March 2004. 10. A.C. Volck, G.A. Suarez and A.J. Tasman.Management of congenital midline nasofrontal masses: Case report and review of literature.Journal of otolaryngology. Vol 2015, Article ID 159647. 11. M. Dutta, J. saha, G. Biswas et al.” Epidermoid cysts in head & neck: Our experience & review of literature”. Indian J. of otolaryngology and Head & Neck Suregery , vol.65, supplementent 1, pp S14-S21. 12. Turgect M., Ozean O. E. Benti K., Ozen T. (1995) : Congenital nasal encephalocoele : a review of 35 cases. Journal of Cranio Maxillofacial Surgery. 23 ; 1-5.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report Peutz Jeghers Syndrome: A Diagnosis Clinched on Histopathology Somshamkar Chowdhury, Neha Tyagi*, Leelawathi Dawson, Ashish Kumar Mandal Department of Pathology, VMMC and Safdarjung Hospital, Delhi, India

Keywords: Peutz- Jeghers, Autosomal, Mucocutaneous, Malignancies

ABSTRACT Peutz-Jeghers syndrome (PJS) is an inherited, autosomal dominant disorder characterized by hamartomatous polyps in the gastrointestinal tract and pigmented mucocutaneous lesions. Data on prevalence of PJS in India is not available. PJS predisposes sufferers to various malignancies (gastrointestinal, pancreatic, lung, breast, uterine, ovarian and testicular tumors). We report here a case of 18 years old male with Peutz Jeghers Syndrome.

*Corresponding author: Dr Neha Tyagi, E-60 Anand Niketan New Delhi, 110021 Phone: +91 9873696631 E-mail: drnehatayagi@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-9

Introduction Peutz-Jeghers syndrome (PJS) is an autosomal dominant inherited condition determined by a mutation localized at 19p13.3; characterized by the occurrence of gastrointestinal hamartomatous polyps in association with mucocutaneous hyperpigmentation. The diagnosis of PJS is based on clinical findings and histopathological patterns of polyps. The disease has variable penetrance, even within families; some members will only manifest hyperpigmentation, while others may manifest pigmentations and hamartomatous polyps. The estimation of population prevalence of PJS differs between studies. The widest estimated range is from 1 in 8300 to 1 in 280 000 individuals [1,2]. Probable prevalence is around 1 in 100 000 people. Peutz Jeghers syndrome is associated with significant morbidity, variable clinical course and considerable predisposition to gastrointestinal & non-gastrointestinal malignancies. An overall recommendation for PJS patients includes not only gastrointestinal multiple polyps resolution, but also regular lifelong cancer screening. Early detection and proper surveillance are vital to minimize the risk of carcinoma. We report here a case of Peutz-Jeghers syndrome which had an interesting clinical presentation.

Case Report 18 years old male presented with complains of pain abdomen, vomiting and constipation since 3 days. The pain was periumbilical in location, insidious in onset, gradually progressive and colicky in nature. He also had multiple episodes of feculent vomiting. Physical examination revealed a tender lump palpable over the right iliac fossa and right lumbar region. No distension or guarding was present. The bowel sounds were exaggerated. Digital rectal

AABS; 3(1): 2016 examination showed fecal matter that was blood stained. The other systems were within normal limits. Laboratory investigations revealed low haemoglobin concentration: 10.1 g/dL (N: 12.5 to 16.5 g/dL). Blood indices & peripheral blood smear examination suggested microcytic hypochromic anemia. Routine blood chemistry reports were within normal range.Ultrasonography revealed features suggestive of subacute intestinal obstruction due to ileo-colic/ileo-ileal intussuception.The patient underwent emergency exploratory laparotomy with ileal resection and end to end anastomosis. The specimen was sent for histopathology examination. Post-operative course was uneventful. We received a specimen of small intestine measuring 6 cm in length. External surface was congested. A polypoidal growth with a stalk was identified measuring 2x2x1 cm, 2.3 cm from one resected end (Fig 1a). Microscopically Sections from the polyp showed a lining of proliferating mucinous glands with a core consisting of bundles of smooth muscles that showed a characteristic arborizing pattern. Some of the glands were cystically dilated and few areas had pools of mucin(Fig 1b). No atypia or mitotic activity was seen.On basis of above findings, a diagnosis of hamartomatous polyp, ileum, was made. Keeping in mind the age of the patient, clinical presentation and pathological findings, a diagnosis of Peutz Jeghers Syndrome was suspected. The patient was thus re-examined, and found to have multiple, black coloured pigmented lesions on the lips and buccal mucosa, which the patient reported having had since childhood (Fig 2).The histological findings of hamartomatous polyp in ileum along with

Fig. 1a: A polypoidal growth (â&#x2020;&#x2019;) projecting into the lumen of intestine. Fig1b: Arborising pattern of mucinous glands and smooth muscle H&E stain 40X Fig 1a: A polypoidal growth (â&#x2020;&#x2019;) projecting into the lumen of intestine. Fig1b: Arborising pattern of mucinous glands and smooth muscle H&E stain 40X

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-10

Fig. 2: Mucocutaneous pigmentation over the lips

hyperpigmentation of lips and oral mucosa were suggestive of Peutz Jeghers Syndrome (WHO criteria).

Discussion

PJS was first reported in a pair of identical twins with melanotic macules described by Connor in 1895 and illustrated by Hutchinson in 1896[3,4]. The primary description of PJS was published by Peutz in 1921 in one Dutch family (the Harrisburg family) as a gastrointestinal familial polyposis with pigmentations [5]. Jeghers specified the description in 10 cases from different families in his work in 1949 and defined the relations between pigmented lesions, gastrointestinal polyposis and increased risk of carcinoma; approximately half of his patients suffered from gastrointestinal malignancy[6]. PJS, as with the other hamartomatous syndromes, has an autosomal dominant pattern of inheritance with both familial and sporadic transmission. Nowadays, the only identifiable mutations causing PJS affect the STK11 (serine/threonine-protein kinase 11 alias LKB1) gene, located on chromosome 19p13.3 [7]. This gene was identified in 1998. It encodes for a multifunctional serine-threonine kinase, important in second messenger signal transduction. The serinethreonine kinase modulates cellular proliferation, controls cell polarity, and seems to have an important role in responding to low cellular energy levels [8]. This gene has been reported in 80% of patients with PJS. Up to 25% of recorded cases of PJS do not have family history. Those sporadic cases probably arise due to new mutation of STK11 gene or low penetration [9]. In the present case,

there was no positive family history of Peutz-Jeghers syndrome. The Peutz-Jeghers syndrome consists of two major components: hamartomatous polyposis of the gastrointestinal tract and mucocutaneous pigmentation [10]. Mucocutaneous pigmentation is a characteristic finding of PJS and is present in most, but not all, patients who have the disease. The hyperpigmented lesions contain melanotic deposits and commonly manifest in infancy and childhood. Pigmented lesions could fade during puberty and adulthood [9]. The pigmented lesions are often seen on the lips, around the mouth, eyes, nostrils, on the buccal mucosa; and sparsely on the fingers, soles of the feet, palms, anal area and intestinal mucosa [10]. The mucocutaneous lesions of PJS are considered to be hamartomatous in origin and without potential of becoming malignant. Gastrointestinal hamartomatous polyps are another classic finding of Peutz-Jeghers syndrome. Although these polyps are most commonly found in the small intestine, they can occur anywhere from stomach to rectum. The median time to first presentation with polyps is about 11-13 years of age and approximately 50% will have experienced symptoms by the age of 20 years [10]. Patients with PJS often present with a history of intermittent abdominal pain due to small bowel intussusception caused by the polyps. Some intussusceptions spontaneously reduce; others lead to development of small bowel obstruction. Peutz-Jeghers polyps can also ulcerate, leading to acute blood loss or chronic anemia. Although Peutz-Jeghers polyps are most commonly found in the gastrointestinal system, they can also occur in extraintestinal sites such as kidney, ureter, gallbladder, bronchial tree, nasal passages etc. Individuals with PJS are at risk for the development of gastrointestinal & non-gastrointestinal malignancies. Among the non-gastrointestinal type; pancreas, lung, breast, uterus, cervix, ovary, testis & thyroid being the major sites of malignancies[11]. In a study of Hearle N et al, 96 cancers were found among 419 PJS patients [12]. This study reported the risks of developing gastrointestinal cancer (31%), breast cancer (31%), gynecologic cancer (18%), pancreatic cancer (7%), and lung cancer (13%) by 60 years of age. Individuals with PJS are also at risk for developing rare sex cord tumors. Women are at risk for sex cord tumors with annular tubules and men are at risk for developing Sertoli cell tumors. The Peutz-Jeghers polyp varies in size from <1 cm to >3.5 cm in diameter, and may be pedunculated or sessile. Because it appears to be composed of non-neoplastic tissue normally found at the site, the Peutz-Jeghers polyp is generally considered a hamartomatous polyp but with an abnormal growth pattern. The most characteristic feature of a Peutz-Jeghers polyp is a central core of smooth muscle that extends into the polyp

http://www.pacificejournals.com/aabs

C-11

AABS; 3(1): 2016

in an arborizing fashion (Christmas tree like appearance) and that is covered by either normal or hyperplastic mucosa native to the involved site. Adenomatous & carcinomatous changes have been described in PeutzJeghers polyps [13]. Epithelial misplacement, also referred to as pseudoinvasion, is another feature seen in some PeutzJeghers polyps. It is characterized by cystically dilated benign glands and supporting lamina propria within the submucosa, muscularis propria, or subserosal layers of the gut adjacent to a polyp and extravasated mucin pools.

the patients is recommended. It is necessary to investigate all first-degree relatives of the patient. This case report was presented because of the rarity of this condition and the interesting clinical presentation.

This feature can mimic the appearance of invasive adenocarcinoma. However, noting the lack of epithelial dysplasia, the presence of supporting lamina propria and the absence of a desmoplastic stromal reaction can help avoid this interpretive error. The diagnosis of PJS is established by the presence of histopathologically confirmed hamartomatous polyps and at least two of the following clinical criteria: a family history of PJS, the presence of mucocutaneous pigmentation and the presence of small-bowel polyps.

NONE DECLARED

Typical imaging features of Peutz-Jeghers syndrome consist of multiple polypoid lesions involving the stomach, small bowel and colon. Although the polyps are often detected with barium studies, they can also be identified with US or CT. Some authors have suggested using US or magnetic resonance (MR) imaging for follow-up imaging to reduce the lifetime radiation burden. Another important imaging finding in Peutz- Jeghers syndrome is intussusception (10). Over the years, the standard therapy for Peutz-Jeghers syndrome has been laparotomy and bowel resection to remove symptomatic gastrointestinal polyps that cause persistent or recurrent intussusceptions. However, some patients require multiple surgical resections, which can lead to short gut syndrome. Because of this, it has been recommended that endoscopy be performed to remove all polyps. During each laparotomy, the small bowel should be examined by means of intraoperative enteroscopy (IOE). Nowadays, double balloon enteroscopy (DBE) in combination with capsule enteroscopy are the gold standard for the diagnosis and treatment of the small bowel hamartomatous polyps(12).

Conclusion Peutz-Jeghers syndrome is a rare, autosomal dominant disorder characterized by mucocutaneous pigmentation & gastrointestinal hamartomatous polyps. Because of the increased risk of both gastrointestinal and nongastrointestinal malignancies in PJS, careful screening of Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

Acknowledgements NONE

Funding NONE

Competing Interests Reference 1. Chen HM, Fang JY. Genetics of the hamartomatous polyposis syndromes: a molecular review. Int J Colorectal Dis.2009;24:865–874. 2. Gammon A, Jasperson K, Kohlmann W, Burt RW. Hamartomatous polyposis syndromes. Best Pract Res Clin Gastroenterol. 2009;23:219–231. 3. Connor JT. Aesculapian society of London. Lancet. 1895;2:1169. 4. Hutchinson J. Pigmentation of lips and mouth. Arch Surg. 1896;7:290. 5. Peutz JLA. Over een zeer merkwaardige, gecombineerde familiaire pollyposis van de sligmliezen van den tractus intestinalis met die van de neuskeelholte en gepaard met eigenaardige pigmentaties van huiden slijmvliezen (Very remarkable case of familial polyposis of mucous membrane of intestinal tract and nasopharynx accompanied by peculiar pigmentations of skin and mucous membrane; in Dutch). Nederl Maandschr v Geneesk 1921; 10: 134-146. 6. Jeghers H, McKusick VA, Katz KH. Generalized intestinal polyposis and melanin spots of the oral mucosa, lips and digits; a syndrome of diagnostic significance. N Engl J Med 1949;24:1031-1036. 7. Mehenni H, Blouin JL, Radhakrishna U, Bhardwaj SS, Bhardwaj K. Peutz-Jeghers syndrome: confirmation of linkage to chromosome 19p13.3 and identification of a potential second locus, on 19q13.4.Am J Hum Genet.1997;61:1327-1334. 8. Hamartomatous polyposis syndromes. Zbuk KM, Eng C. Nat Clin Pract Gastroenterol Hepatol.2007 Sep;4(9):492-502. 9. Vesna Živković et al. Hereditary Hamartomatous Gastrointestinal Polyposis Syndrome. Scientific Journal of the Faculty of Medicine in Niš 2010;27(2):93-103. e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-12

10. Marcela Kopacova, Ilja Tacheci, Stanislav Rejchrt, Jan Bures. Peutz-Jeghers syndrome: Diagnostic and therapeutic approach. World J Gastroenterol 2009;15(43):5397-5408. 11. Michael Manfredi. Hereditary Hamartomatous Polyposis Syndromes: Understanding the Disease Risks As Children Reach Adulthood. Gastroenterology & Hepatology. 2010;6:185-196.

12. Hearle N, Schumacher V, Menko FH, Olschwang S, Boardman LA. Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res. 2006;12:3209-3215. 13. Karl H. Perzin, Mary F. Bridge. Adenomatous and Carcinomatous Changes in Hamartomatous Polyps of the Small Intestine (PeutzJeghers Syndrome): Cancer 1982;49: 971-983.

http://www.pacificejournals.com/aabs

Case Report Ruptured Ovarian Pregnancy in A Young Primigravida Beant Singh, Balwinder Kaur, Parneet Kaur, Ramiti Gupta* Dept of Obs and Gynae, Government Medical College, Patiala, India Keywords: Ectopic Pregnancy, Histopathological Examination, Laparotomy, Ovarian Pregnancy, Rupture.

ABSTRACT Primary ovarian pregnancy is one of the rarest variety of ectopic pregnancy accounting for 0.15-3% of all ectopic gestations. Heartig estimated that ovarian pregnancy occur in 1 in 25000-40000 pregnancies. Pre operative diagnosis is challenging and needs high index of suspicion. In any case of ruptured ectopic pregnancy where tubes are found to be normal on laparotomy an ovarian pregnancy must be ruled out. We report such a rare case of ruptured ovarian pregnancy diagnosed on laparotomy which was later on confirmed by histopathological examination.

*Corresponding author: Dr Ramiti Gupta, Dept of obs and gynae, Government Medical College, Patiala â&#x20AC;&#x201C; 147001, India Phone: +91 9876635837 E-mail: gupta.ramiti@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Case Report

C-14

Introduction Ectopic pregnancy is one of the most common gynaecological emergencies and accounts for 10% of all maternal mortality [3] and most common cause of maternal mortality in first trimester[4]. Most common location of ectopic pregnancy is fallopian tube but in some cases it can be ovarian. The diagnosis of an ovarian ectopic pregnancy is often made at surgery and requires histological confirmation as it is difficult to differentiate it from a hemorrhagic corpus luteum intraoperatively[5]. Ovarian pregnancy is a rare diagnosis of exclusion made after laparotomy followed by histopathological examination as it was in our case.

Case Report A 21 yr old young primigravida married for 4 months came to emergency of Rajindra Hospital Patiala with complaint of pain abdomen with vomiting and spotting per vaginum. There was no h/o preceeding amenorrhoea but there was h/o scanty periods during last menses which was 15 days back. Her previous cycles were regular with 3-4 days of bleeding every 28-30 days with average flow and no dysmenorrohea. There was no h/o any contraceptive use or ovulation induction. On examination her BP was 90/60 mm of Hg, Pulse 110/min, feeble, Pallor was +++, on P/A there was tenderness in right iliac fossa with rigidity and guarding all over abdomen. Her UPT was positive. Paracentesis done and there was frank hemoperitoneum. Patient was taken for immediate laparotomy with a preoperative provisional diagnosis of right ruptured tubal pregnancy. Emergency laparotomy was performed . Intra operative there was massive hemoperitoneum, about 2.5 litres of blood drained from peritoneal cavity. Both the tubes were found to be normal. Left ovary was normal. The right ovary was enlarged ms about 4.5cm x 4cm, there was one cyst and some irregular mass from the surface of which blood was oozing. Right fallopian tube was found completely normal and separate from the ovary. Wedge resection of bleeding tissue was done. Stitches applied, complete hemostasis obtained. Tissue was sent for histopathological examination . Histopathological examination done wide no. H-710/15, which showed chorionic villi with decidualised ovarian stroma suggestive of ovarian pregnancy.

Discussion Primary ovarian pregnancy is one of the rarest type of extrauterine pregnancies[6]. Some of the cases are associated with factors such as IUCD[7], ART, endometriosis and PID[8]. Our patient did not have any of the risk factors and present pregnancy had occurred in a spontaneous cycle.

The proposed hypothesis for ovarian ectopic are non release of ovum from ruptured follicle, tubal malfunction and inflammatory thickening of tunica albuginea. There is often a delay in diagnosis as gestation sac mimics corpus luteum, haemorrhagic cyst and endometriotic cyst of ovary[7]. Patient was mislead by scanty periods so could not seek advise for her 6 weeks amennorhoea before she presented in emergency with ruptured ectopic pregnancy. Clinicians should be aware of difficulties with clinical, radiological and intra operative diagnosis. With a few exceptions initial diagnosis is made on operating table and final diagnosis only on histopathology on basis of four Spiegelbergâ&#x20AC;&#x2122;s criterias[9] 1. The fallopian tube on affected side must be intact. 2. Fetal sac should occupy the position of ovary. 3. Ovary must be connected to uterus by ovarian ligament. 4. Ovarian tissue must be located in sac wall. Early diagnosis of unruptured ovarian pregnancy with TVS which is usually rare allows for conservative management with methotrexate. However if the diagnosis is made later in case with ruptured ovarian pregnancy local resection of bleeding mass with conservation of ovary is usually done. Even if the last trophoblastic villi cannot be removed it will usually degenerate spontaneously or respond to post operative methotrexate therapy producing no long standing clinical problem[10] No case of recurrent ovarian pregnancy has been reported in contrast to 15% risk of recurrence of tubal pregnancy[11]. Fertility after ovarian pregnancy remain unmodified[12].

Conclusion Primary ovarian pregnancy may occur without presence of any of classical risk factors or symptoms/signs. Early diagnosis and treatment can help in conservative management and retain the future fertility of the patient.

Acknowledgements We like to thank casualty nursing staff, OT staff, blood bank staff for helping in emergency care and pathology department for accurate diagnosis of ovarian pregnancy of the patient.

Funding None

Competing Interests None declared

http://www.pacificejournals.com/aabs

C-15

Reference 1. Nwanodi O, Khulpateea N. The preoperative diagnosis of primary ovarian pregnancy. J Natl Med Assoc. 2006 May;98(5):796-8. 2. Hertig AT. Discussion of Gerin â&#x20AC;&#x201C; Lojoie L. Ovarian pregnancy. Am J Obstet and Gynecol. 1951;62:920. 3. Das S, Kalyani R, Lakshmi M et al. Ovarian Pregnancy. Indian J Path Microbiol 2008;51(1):37-8. 4. Gary C, Kenneth J, Steven L, John C, Catherine Y, et al. Ectopic pregnancy. Williams Obstetrics. 24th ed. New York:McGraw Hill; 2014:377. 5. Ciortea R, Costin N, Chiroiu B, Malutan A, Mocan R, et al. Ovarian pregnancy associated with pelvic adhesions. Clujul Med. 2013;86:77-9. 6. Dhorepatil B, Rapol A. A rare case of unruptured viable secondary ovarian pregnancy after IVF/ICSI treated by conservative laparoscopic surgery. J Hum Reprod Sci. 2012;5:61-3.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

AABS; 3(1): 2016 7. Mehmood SA, Thomas JA. Primary ectopic ovarian pregnancy (report of three case). J Postgrad Med 1985;31:219-22. 8. De Seta F, Baraggino E, Strazzanti C, De Santo D, Tracanzan G, Guaschino S. Ovarian pregnancy: A case report. Acta Obstet Gynecol Scand 2001;43:095-6. 9. Spiegelberg O. Zur casuistic der ovarialschwanger schaft. Arch Gynaekol 1878;13:73-5. 10. Howard WJ, John AR. Ectopic pregnancy. Te Lindeâ&#x20AC;&#x2122;s Operative Gynecology. 11th ed. South Asian Edition: Wolters Kluwer; 2015:790-2. 11. Sergent F, Mauger-Tinlot F, Gravier A, Verspyck E, Marpeau L. Ovarian pregnancies: Revaluation of diagnostic criteria. J Gynecol Obstet Biol Reprod. 2002; 31(8):741-6. 12. Portundo JA, Ochoa C, Gomez BJ, Uribaron A. Fertility and conception of 8 patients with ovarian pregnancy. Int J Fertil. 1984;29:254.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report Pure Mucinous Carcinoma of Male Breast: Case Report of a Rare Histological Subtype of Male Breast Carcinoma. Riju Rani Deka1*, Shiraj Ahmed2, Amitabh Handique1 1

Dept. of pathology, Tezpur Medical College, Assam, India Dept. of pathology, Dr. B. Barooah cancer Institute, Assam, India

Keywords: Biological Characteristics, Hormonal Receptor Status, Male Breast Carcinoma, Pure Mucinous Carcinoma

ABSTRACT Introduction: Male breast carcinoma is are rare malignancies occurring in older males and are of aggressive in nature, presenting in an advanced stage. Rarity and deviation from usual reported biological characteristics led us to report this case of pure mucinous carcinoma of male breast. Case report: A 52 years old male presented with a mass below areola found adhered to overlying skin. Resected mass was lobular 5cm x 3cm x 2.5cm which on cut section was multiloculated filled with mucinous fluid. H&E stain from sections from different area showed features of pure mucinous carcinoma of breast with clear cut margins. Immunohistochemical examination showed nuclear positivity for ER receptor and negative for PR receptor, and membrane did not stain for HER2/neu receptor. Conclusion: More of this rare subtype of male breast carcinoma with its varied biological characteristics should be reported to know more about its biological course so that improvement in the treatment options and steps for its prevention can be taken.

*Corresponding author: Dr. Riju Rani Deka, c/o P.C. Deka, Bishnu Nagar, P.O-Tezpur, Dist-Sonitpur, Assam, Pin-784001, India, Phone: +91 9435180795 E-mail: dr.rijurdeka82@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-17

Introduction Carcinoma of male breast is a rare occurrence. Male breast cancer represents 0.6% of all breast carcinomas and accounts for less than 1% of all malignancies in male. [1] Pure mucinous breast carcinoma is rarer histological subtype of breast carcinoma. About 30 cases of mucinous breast carcinoma of the male breast have been documented in the English literature with only 10 cases of pure mucinous carcinoma.[2] Mucinous carcinoma of breast is histologically sub divided into pure and mixed form. Mucinous carcinoma is defined as pure when more than 90% of tumor mass is composed of mucinous component and infiltration duct carcinoma comprises of less than10% of tumor mass. Histopathologic diagnosis is sufficient for the diagnosis of pure mucinous carcinoma. In general, mucinous carcinoma has a better prognosis than NOSinfiltrating duct carcinoma but in males, breast carcinoma has an aggressive course. Male breast carcinomas present at an older age and with an advanced stage as compared to its female counterpart. Our purpose behind the reporting of this case of pure mucinous carcinoma of male breast is its rarity as a histological subtype, relatively earlier age at presentation and only local involvement at the time of diagnosis.

Case Report A 52 years old male presented with swelling in left breast beneath areola for last four months and suddenly increasing in size for last one month. On clinical examination, the swelling was in size, non tender and was adhered to the overlying skin. The overlying skin was otherwise normal looking. Resected specimen sent for histopathological examination measured , globular mass with smooth surface. On cut section, mass was multiloculated filled with mucinous fluid? H&E stain of the sections taken from different parts of soft tissue mass showed groups of small hyperchromatic ductular epithelial cells floating in pools of mucin. The cells were small with high nuclear cytoplasmic ratio, scanty cytoplasm, hyperchromatic nuclei and inconspicuous nucleoli. The epidermis was free from tumor cells. A diagnosis of pure mucinous carcinoma of breast was made on morphological ground. Cut margins are free from tumor cells. Immunohistochemical analysis on sections showed nuclear positivity for estrogen receptor(ER) and negative for progesterone receptor (PR), HER2/neu receptor was not amplified.

Discussion

AABS; 3(1): 2016 of male breast is rare representing about 0.6% of all breast carcinomas. [2] But recent study has shown that the incidence of male breast carcinoma is steadily increasing. [4] Raajul jain et al in India found male breast lesions to be 6.34% of all breast lesions, malignant lesions being 23.34% of which most common being infiltrating duct carcinoma, NOS.[5] Mucinous carcinoma of breast is a relatively rare histological subtype , more so in males. Shah et al in his study found mucinous carcinoma to constitute 2.38% of all male breast carcinomas. [6] Men tend to be older at the time of diagnosis with a median age of 67 years and also they have more advanced disease at the time of presentation.[1] On the contrary, our patient presented at an early age of 51 years and with a localized disease. Genetic risk factors include BRCA2 mutations. High temperature environments and exhaust fumes were considered as occupational risk factors. Hormonal imbalances, such as gonadal dysfunction, obesity, and radiation exposure also contribute to the occurrence of male breast cancer. [7] Immunohistochemical staining pattern varies from case to case. Our case was positive for ER but was negative for PR and HER2/neu receptor. This finding was similar to the case report in Ishida M at el where the mucinous carcinoma was ER positive and was negative for both PR and HER2/neu receptor.[8] Arslan et al in his study found majority of cases to be positive for ER receptor and also few triple negative tumors.[9] Friedman et al in his study found the incidence of ER positivity to be more in men than that in female.[10] On the contrary, Shah et al found majority of cases to be positive for both ER and PR receptors and few cases to be only positive for ER receptors.[7] Research showed the majority of cancers arising in the male breast to be ER positive although this finding does not correlate with a better prognosis, as it in cases of women.[11] Cases of pure mucinous carcinoma of male breast have been reported metastasizing to axillary lymph nodes, [12, 13] lung [14] and as Paget’s disease of nipple [12]. One case of occult breast cancer in 40 years male first manifesting as axillary lymph node metastasis with part of metastatic mucinous carcinoma similar to our case of early age at diagnosis.[16] On the contrary our case had only localized involvement. For pure mucinous carcinoma male breast in early stage, conservative surgery is the optimal treatment. [16]

Breast carcinoma by far is the most common cancer diagnosed in females in world. On the contrary, carcinoma

Chen et al described nine independent prognostic factors contributing to the male breast cancer lethality like grade; mucinous, medullary, tubular, and scirrhous

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-18

Fig. 1: photomicrograph of gross specimen of tumor mass from left breast (A) showing outer smooth lobulated surface (B) cut section showing multiloculated cystic mass filled with mucinous fluid.

Fig. 2: photomicrograph showing histopathological feature of tumor mass showing groups of malignant ductal epithelial cells floating in pools of mucin. Haematoxylin &

eosin stain magnification,

Fig. 3: Immunohistochemical stain showing (A) diffuse ER expression (B) PR not expressed (C) HER2/neu not amplified

http://www.pacificejournals.com/aabs

C-19 adenocarcinoma; male sex; inflammatory disease; Paget disease; and lymph node status.[17] Research found tumor staging and lymph node status and not the hormone receptor status as the only significant prognostic factors effecting overall survival in male breast cancers but improved survival is being stated in hormone receptor positive disease.[1] Morand et al in his study on DNA flow cytometry of mucinous breast carcinomas established DNA ploidy to be of prognostic significance and that large aneuploid tumors have poor prognosis.[18]

AABS; 3(1): 2016

1. Sharon H. Giordano M.D.,Deborah S. Cohen M.S.,Aman U. Buzdar M.D.,George Perkins M.D. and Gabriel N. Hortobagyi M.D. breast cancer in men: a population based study.cancer2004;vol101(1):51-57. 2. Sawssen Dhambri , Mona Mlika , Aïda AyadiKaddour , Abdelfattah Zeddini , Faouzi El Mezni. An uncommon subtype of breast carcinoma in a man: The pure mucinous carcinoma. Int J Cur Biomed Phar Res. 2011; 1(2): 45 – 47. 3. Bussolati G and Sapino A: Mucinous carcinoma and carcinomas with signet-ring cell differentiation. In : World Health Organization Classification of Tumors of the Breast.Lakhani SR, Ellis IO, Schnitt SJ, Tan OH and van de vijver MJ(eds).IARC Press, France,pp60-61, 2012. 4. Giordano SH, Cohen DS, Buzdar AU, Perkins G, Hortobagyi GN. Breast carcinoma in men: a population-based study. Cancer 2004;101:51–57

5. Jain raajul, Shah Smita A, Kadam Tarang B,Gonsai R,N. Vala karan. Male breast lesion profile in a tertiary care hospital in western india on fine needle aspiration . Int J Cur Res Rev, May 2014/ Vol 06 (10) 6. Shah S, Bhattacharyya S, Gupta A, Ghosh A,Basak S.Male breast cancers: A clinicopathologic study of 42 patients in eastern India.Indian J Surg Oncol.2012 Sept;3(3):245-9. 7. Xue Y, Guo XT, Liu WC. Clinical research advancement on male breast cancer. Ai Zheng. 2007 Oct;26(10):1148-52. 8. Ishida M, Umeda T, Kawai Y, Mori T,Kubota Y, Abe H, Iwai M, Yoshida K,Kagotani A, Tani T, Okabe H. Mucinous carcinoma occurring in the male breast. Oncol Lett. 2014 Feb;7(2):378-380. 9. Arslan UY,Oksuzoglu B,Ozdemir N, Aksoy S, Alkis N, GokA, Kaplan MA, Gumus M,Berk V, Uncu D, Baykara M,Colak D, Uyeturk U, Turker I, Isikdogan A. Outcome of non-metastatic male breast cancer: 118 patients. Med Oncol. 2012 Jun;29(2):554-60 10. Friedman MA, Hoffman PG Jr, Dandolos EM, Lagios MD, Johnston WH, Siiteri PK.Estrogen receptors in male breast cancer: clinical and pathologic correlations. Cancer. 1981 Jan 1;47(1):134-7. 11. Dimitrios Peschos, Elena Tsanou, Pavlos Dallas Konstantinos Charalabopoulos, Christos Kanaris and Anna Batistatou. Mucinous breast carcinoma presenting as Paget’s disease of the nipple in a man: A case report. Diagnostic Pathology 2008, 3:42. 12. Ingle AP, Kulkarni AS, Patil SP, Kumbhakarna NR, Bindu RS. Mucinous carcinoma of the male breast with axillary lymph node metastasis: Report of a case based on fine needle aspiration cytology. J Cytol. 2012 Jan;29(1):72-4. 13. Hammedi F, Trabelsi A, Abdelkrim SB, Abid LB, Jomaa W, Bdioui A, Beizig N, Mokni M. Mucinous carcinoma with axillary lymph node metastasis in a male breast: A case report. N Am J Med Sci. 2010 Feb;2(2):111-3. 14. Kertmen N, Dogan E, Altundag K. Pure mucinous breast carcinoma with lung metastasis in a young male patient. Am Surg. 2010 Aug;76(8):E146. 15. HeMengna , Liu He, and Jiang Yuxin . A Case Report of Male Occult Breast Cancer First Manifesting as Axillary Lymph Node Metastasis With Part of Metastatic Mucinous Carcinoma. Medicine,2015 June:94(25). 16. Cao AY, He M, Liu ZB, Di GH, Wu J, Lu JS, Liu GY, Shen ZZ, Shao ZM. Outcome

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Conclusion Though mucinous carcinoma in general is a subtype of breast carcinoma with better prognosis, male breast carcinomas are more aggressive and different biological characteristics than its female counterpart. Paucity of reported cases of male breast carcinomas, especially of its rare subtypes like pure mucinous breast carcinomas limit the understanding of its carcinogenesis. Reporting of more cases are required to understand more about male breast carcinomas and its histological subtypes for better treatment options and understanding its prognosis. Reporting of this case is considered for rarity of its occurrence and deviation from its other reported biological characteristics.

Acknowledgements None

Funding None

Competing Interests None declared

Reference

Case Report

C-20

of pure mucinous breast carcinoma compared to infiltrating ductal carcinoma: a population-based study from China. Ann Surg Oncol. 2012 Sep;19(9):3019-27. 17. Chen LL, Nolan ME, Silverstein MJ, Mihm MC Jr, Sober AJ, Tanabe KK, Smith BL, Younger J, Michaelson JS. The impact of primary tumor size, lymph node status,

and other prognostic factors on the risk of cancer death. Cancer. 2009 Nov 1;115(21):5071-83. 18. Morand C, Verriele V,Valo I,Remoue P,Paillecher N, Chassevent A. Pure mucinous carcinoma of breast: prognostic study including DNA flow cytometry. Cytometry Part B 2009; 76B: 56-62.

http://www.pacificejournals.com/aabs

Case Report Early Gastric Carcinoma: A Rare Variant of a Common Entity Monal Trisal*, Kangana Sengar , Sumedha Kotwal, Sanjay Deb, Ramesh Dawar Dharamshila Cancer Hospital and research centre, New Delhi, India

Keywords: Early Gastric Carcinoma , Endoscopic Mucosal Resection, Endoscopic Submucosal Resection.

ABSTRACT Gastric Carcinoma is the leading cause of cancer death worldwide. Gastric cancer confined to the mucosa and submucosa is regarded as early gastric cancer due to its overallfavourable prognosis. Although common in Japan, early gastric cancer is an infrequent occurrence in India.Endoscopist should be more suspicious about these lesions as these can be easily neglected.

*Corresponding author: Dr Monal Trisal, Dharamshila Cancer Hospital and research centre, New Delhi, India Phone: +91 9622226333 E-mail: monaltrisal@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Case Report

C-22

Introduction

Gastric Carcinoma is the fifth most common cause of cancer death worldwide.1 Treatment results are favourable when the disease is detected in its early stage. Gastric cancer confined to the mucosa and submucosa is regarded as ‘early’ gastric cancer (EGC) due to its overall favourable prognosis. Although common in Japan due to their aggressive surveillance, EGC is infrequently diagnosed in India. Its important to recognise these lesions on endoscopic biopsies as they have good prognosis as compared to more common types of gastric adenocarcinoma.

Case Report

A 68 year old male presented with complaint of black tarry stools for two months duration. Upper GI Endoscopy revealed an ulcer in the antrum measuring about 1 cm diameterwith thickened folds in the lesser curvature (Figure-1,2). Histopathology of the endoscopic biopsy revealed a moderately differentiated adenocarcinoma. Patient underwent distal radical gastrectomy with perigastriclymphadenectomy. On gross examination, anterior wall and lesser curvature showed a flat rough lesion measuring 1.5 X 1.4cm with loss of rugae in the antrum. Histopathological examination showed a well differentiated adenocarcinoma , superficial spreading type infiltrating uptomuscularismucosae with desmoplasia and chronic inflammation and no involvement of perigastric lymph nodes. Features were consistent with early gastric carcinoma.(Figure 3,4). Pathological stage : (pT1b N0 MO)

Discussion

Early Gastric Carcinoma was originally used in the Japanese literature to describe infiltratingadenocarcinomas in which the primary growth is confined to the mucosa or submucosa of the stomach regardless of regional lymph nodes status. It is more common in males. Most tumors are less than 2cm and seen in distal stomach mostly along the lesser curvature.2 EGC has to be differentiated from carcinoma in situ or gastric dysplasia- conditions in which tumor cells have not penetrated the basement membrane and have no metastatic potential.Rarely cases of EGC may have isolated regional lymph nodes metastasis or even hepatic metastases, but most cases are still potentially curable bysurgery.Asubclassification based on gross appearances of EGC was devised by the Japanese Gastroenterological Endoscopy Society. Lesions are categorised as Type I protruded , Type II (superficial type) : IIa – elevated , Type IIb –flat, Type IIc- depressed , Type III- excavated.3In our case the EGC was type IIb lesion. (Figure 5). The importance of correctly identifying EGC lies in the excellent results of interventional treatment. For the intramucosal cancer, the cure rate is quoted as 93% when no regional lymph nodes metastasis are present and 91% when they are present. For early cancers with submucosal involvement, the overall cure rate is 89%which decreases to 80% in cases with lymph node metastasis.2 A study in series of 10,000 consecutive cases in japan, the 5-year survival rates were 46% for advanced carcinoma and 89% for early carcinoma.4

Fig.1,2: Upper GI endoscopy show linear ulcer with everted margins in antrum .

http://www.pacificejournals.com/aabs

AABS; 3(1): 2016

C-23

Fig. 3 : Photomicrograph of section from stomach shows a well differentiated adenocarcinoma infiltrating uptomuscularis mucosa. (H&E, 4x)

Fig. 4 :Photomicrograph of section from stomach shows a well differentiated adenocarcinoma with desmoplasia and chronic inflammation. (H&E, 10x)

Fig. 5 : Japanese Gastroenterological Endoscopy Society. Lesions are categorised as Type I protruded , Type II (superficial type) : IIa â&#x20AC;&#x201C; elevated , Type IIb â&#x20AC;&#x201C;flat, Type IIcdepressed , Type III- excavated.

Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-24

Early gastric cancers are very amenable to interventional endoscopy. Both endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have resulted in sucessful endoscopic resections of lesions.5,6

Conclusion

The endoscopist must be more suspicious with these lesions that can be easily ignored and multiple sites of biopsies should be taken for correct assesment of the tumor depth since EGC and advanced gastric carcinoma has completely different survival rate , treatment modality and prognosis. In conclusion, endoscopic resection of EGC is well established as a standard therapy in Japan and is increasingly becoming accepted and regularly used in other countries. The indications, pathological assessment, and techniques of endoscopic resection employed in the treatment of EGC are demanding

Funding Source None

Conflict of Interest None

References

1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.1, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11[Internet]. Lyon, France: International Agency for Research on Cancer; 2014. 2. Johansen AA. Early Gastric Cancer.Curr Top Pathol 1976;63:1-47. 3. Green PH, Oâ&#x20AC;&#x2122;Toole KM, Weinberg LM , et al. Early gastric Cancer. Gastroenterology1981;81:247-56. 4. Nakamura K, Ueyama T, Yao T, Xuan ZX, et al. Pthology and prognosis of gastric carcinoma. Findings in 10,000 patients who underwent gastrectomy. Cancer 1992,70:1030-37. 5. Oka S, Tanaka S, Kaneko I, et al. Advantage of endoscopic submucosal dissection compared with EMR for early gastric cancer. GastrointestEndosc 2006;64:877-83 6. Hoteya S, Iizuka T, Kikuchi D, Yahangi N. Benefits of endoscopic submucosal dissection according to size and locationn of gastric neoplasm, compared with conventional mucosal resection. J GastroenterolHeoatol 2009;24(6):1102-6.

http://www.pacificejournals.com/aabs