AABS 4.1 (2017) by PaGe

eISSN: 2349-6991 pISSN: 2455-0396

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

Jan-Mar. 2017; Vol. 4, Issue 1

Cover design: Dr Prashant

DOI : 10.21276/aabs

Pacific Group of eJournals

Annals of Applied Bio-Sciences Co-Editor in Chief

Prof. M H Fulekar Professor & Dean, School of Environment and Sustainable Development, Central University Gujarat, India Dr Igor Iuco Castro-Silva Professor, Clinica Odontologica, Faculdade de Ciencias do Tocantins, Brazil Kapil Agarwal Engineer, Nagoya City, Aichi, Japan Dr Devesh Palharya Consultant Pathologist, Bhopal, Madhya Pradesh, 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, Uttar Pradesh, India Dr Manav Kapoor Assistant Professor Neuroscience, Icahn School of Medicine, Mount Sinai, New York, NY, 10029, United States

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

Co-Editor 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 Vijayabhaskar Varadarajalu Chennai, Tamil Nadu, India Dr B K Guha Associate Professor (Anatomy), NSCB Medical College, Jabalpur, Madhya Pradesh, India Dr Hariom Gupta Associate Professor, SSMC and SGMH, Rewa, Madhya Pradesh, India Dr Shashikant Agarwal Associate Prof. JMC, Jhalawar, Rajastjhan, India

Managing Editor

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

Editorial Board

Dr Francesco Merolla Advanced Biomedical Sciences, University of Naples, Federico II, Italy Dr Chandrassegar Saravanan Novartis Institutes for Biomedical Research, Technology Square, Cambridge, MA, United States Dr Nandhakumar Balakrishnan College of Veterinary Medicine, North Carolina State University, Raleigh, United States

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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.

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Table of Contents Review Articles Titanium mesh in orbital wall reconstruction: a short review

Surya Rao Rao Venkata Mahipathy, Dhivyalakshmi Manavazhagan, Alagar Raja Durairaj, Narayanamurthy Sundaramurthy, James Solomon Jesudasan The most frequent prescription in Obstetrics: Bed Rest! Harkiran Kaur Khaira, Smita Sinha Does Interleukin 22 have a role in periodontal pathogenesis? A short Review Sreenivas Nagarakanti, Arun KV Dhiraj Nikumbh, Roopali D Nikumbh

R1-R3

R4-R7 R9-R11

Gastrointestinal Stromal Tumor (GIST): An Update on Its Uncommon and Complex Presentations: A Short Review Dhiraj Nikumbh, Roopali D Nikumbh

R12-R13

Indigenous Flaps in ENT: Our Experience Manish Munjal, Archana Arora, Gopika Talwar, Amanjot Kaur, Tejinder Singh

R14-R15

Cryptococcal meningitis in HIV (AIDS) patients: A Mini Review Manodeep Sen, Tanushri Chatterji, Vivek Singh, Pushpa Yadav, Anupam Das, Vineeta Mittal

R16-R17

Utility of Interactive Teaching Tools in Classroom Teaching-A Review of literature Dhananjay Shrikant Kotasthane, Vaishali Dhananjay Kotasthane

R18-R21

Giant Cell Lesion and Central Giant Cell Granuloma of Jaw: A Brief Review Nilesh Kumar, Aaditee V Vande, Shivsagar Tewary, Sameer Anil Zope

R22-R29

Clusterin: It’s Implication in Health and Diseases. Sheikh Ishaq, Harnam Kaur, Sonam Bhatia

R30-R34

Original Article Association between the bone turnover markers and the gynecologic factors among Moroccan women Basma Nejjar, Abdellatif Bour, Anis Sfendla, Adil Najdi, Olivier Bruyère, Etienne Cavalier Hepatitis B Versus Hepatitis C in Blood Donors Ravi Jain, Ashok Yadav

A1-A7

A8-A13

A14-A18

Exposure to video games shortens simple visual reaction time – a study in Indian school children Paramita Bhattacharyyia, Subhasis Das, Ashwin R

A19-A23

Client satisfaction in Thalassemia Control Unit , North Bengal Medical College and Hospital, Darjeeling district, West Bengal Nilanjana Ghosh, Indranil Chakrabarti

A24-A30

A study on prevalence and causative factors of megaloblastic anaemia in Hadoti region. Lakshmi Agarwal, Jayant Ramawat, Manmohan Agrawala, Naresh Rai

A31-A34

A comparative study of clinicopathological characteristics and expression of basal markers (CK5/6 & EGFR) in triple negative and non triple negative breast carcinomas in Kashmir valley. Subuh Parvez Khan, Syed Besina Yasin, Fiza Parvez Khan

A35-A40

Cytological Diagnosis of Salivary Gland Lesions with Histopathological Correlation Ranbeer Singh, Pravin Pawane

A41-A45

Evaluation of sympathetic reactivity by Hand Grip Exercise test in children of hypertensive parents. Ajay Kumar Saluja, Sonu Ajmani

A46-A49

III

Case Reports

Characterization and activity of antimicrobial polypeptide of Bacillus spp from wastelands of Kathmandu valley Masna Rai, Sagar Aryal, Pramila Parajuli

A50-A57

Effectiveness of Teaching Aids in Medical Education Deepa Sanjeev Nair, Madhura Y Bedekar, Meena J Agrawal, Anagha M Gupte

A58-A61

Evaluation of Metastatic Cutaneous Lesions on Fine Needle Aspiration Cytology: A Five Year Study Syed Besina Yasin, Naheena Bashir, Subuh Parvez Khan

A61-A66

Observational study of blood groups distribution among medical students in central India Ajay Kumar Saluja, Sonu Ajmani, Priyanka Chouhan

A67-A70

Fine Needle Aspiration Cytology of Parapharyngeal tumors Kuldeep Singh, Gousia Rahim Rather

A71-A73

MicroRNA-155 in patients with Chronic Stable Angina Mohamed M Elshafae, Jehan H Sabry, Mohamed A Salem, Hanan M Elshafee

A74-A82

Pigmented Dermatofibrosarcoma Protuberans (Bednar Tumor) masquerading clinically as malignant melanoma - A Case Report Ira Mondal, Arnab Chaudhuri, Aparajita Samaddar, Nandini Das, Dipanwita Nag

C1-C3

Two point fixation in zygomatic compex fractures Sourav Sharma, Vandana D

C4-C6

Osteolipoma of knee: a rare case report Arnab Chaudhuri, Ira Mondal, Aparajita Samaddar, Nandini Das, Dipanwita Nag

C7-C9

Schistosomiasis of appendix with review of literature. Omaia Mahdy, Nazima Haider, Sohaila Fatima

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Website: www.pacificejournals.com www.pacificejournals.com/aabs

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C10-C12

Review Article DOI: 10.21276/AABS.2017.1307

Titanium Mesh in Orbital Wall Reconstruction: A Short Review Surya Rao Rao Venkata Mahipathy1, Dhivyalakshmi Manavazhagan2, Alagar Raja Durairaj1 Narayanamurthy Sundaramurthy1 and James Solomon Jesudasan3 Dept. of Plastic & Reconstructive Surgery, Saveetha Medical College & Hospital, Thandalam, Tamilnadu, India 2 Dept. of Dentistry, Saveetha Medical College & Hospital, Thandalam, Tamilnadu, India 3 Dept. of Oral & Maxillofacial Surgery, Saveetha Medical College & Hospital, Thandalam, Tamilnadu, India

ABSTRACT Orbital wall fractures are the more common fractures of the mid-face. Though surgical timing and approach are two important factors in management of orbital wall fracture, a third important factor is the choice of material used for reconstruction. There are numerous materials that are available for reconstruction of orbital wall defect. One among which is Titanium. Titanium is a metallic alloplast that has received much attention in the area of craniofacial reconstruction (Mok D et al). Keywords: Orbital Wall, Reconstruction, Titanium, Alloplast

Introduction

Orbital wall fractures are quite common consequence of maxillofacial trauma1. The consequences of an orbital injury are dramatic. They vary from loss of vision, diplopia, loss of an eye, epiphora, a disturbing loss of facial sensation to an unsightly and unacceptable appearance of the eye and the hard and soft tissues around it 2. Damage to the walls themselves can cause disorders such as diplopia, enophthalmos, and much less frequently vertical dystopia. It is therefore mandatory to reconstruct the orbital walls with the same care one invests in repair of orbital rims3. The ideal material for orbital reconstruction remains controversial. It should be cheap, biocompatible, readily available, easy to manipulate and insert in the operating room and it should allow fixation to the host bone by screws, wires, or sutures4. Two main options for volume augmentation are autologous grafts and alloplastic implants. But autologous options are associated with potential donor site morbidity, difficult contouring, and graft resorption. Titanium, high – density porous polyethylene, and many other materials have been used as bone substitutes. They are relatively inert, provide reliable support without donor site morbidity, and are easily adapted to the contour of the orbit 5.

Choice of Materials:

Autografts were the first major class of materials used in the reconstruction of orbital floor defects. Commonly used autologous materials are bone and cartilage6. Autologous bone grafts remain the “gold standard” for orbital floor

reconstruction7. But these grafts have the disadvantages of donor site morbidity related to surrounding anatomy 6. Lyophilized dura mater and banked demineralized bone are the most commonly used allografts for orbital floor fractures. But evidence from past reports has shown that allografts are associated with an increased risk of disease transmission from donor to patient6. Webster proposed the use of lyophilized porcine dermis and found the material to be suitable for orbital floor repair8. Alloplastic materials include polymers, porous polyethylene, titanium, bioactive glass, silicone, nylon, teflon, seprafilm. In many cases, the use of alloplastic materials is less expensive than the increased operative time required to harvest bone5.

Literature Supporting use of Titanium Mesh in Orbital Reconstruction

Gabrielli et al evaluated the efficacy and safety of traumatic orbital defect reconstruction with titanium mesh. After a minimum post – operative follow up of 12 months they documented that titanium mesh is a reliable option for orbital reconstruction9. In the study conducted by Degala et al on 10 patients with symptomatic zygomatico-orbital fractures requiring orbital wall reconstruction, it was documented that titanium mesh could be considered to be the ideal orbital floor repair material. They also stated that their use leads to less morbidity as no donor site operation is needed. Also it provides favourable healing as it is biocompatible10. Kozakiewicz et al compared the functional results of individual reconstructions of orbital wall using either

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

Review Article

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titanium mesh or ultra high molecular weight polyethylene. This study of 6 months functional result assessment of pre bent individual implants and CNC milled ultra-high molecular weight polyethylene of the orbital wall has shown it to be a predictable reconstruction method1. In the study conducted by Reich et al on 10 patients, the authors recommended the prefabricated titanium plate in the treatment of isolated orbital wall fractures or in combination with the medial wall despite the material costs11. Woo et al evaluated the use of titanium mesh plates in 17 patients with large medial orbital wall defects and they proposed that titanium mesh may be a valuable material for the reconstruction of severe medial orbital wall fractures12.

specific orbital reconstructions. Head and Face medicine 2013;9:32.doi: 10.1186/1746-160X-9-32. 2.

Leo FA, Cyrus JK (2007) Peri- and intraorbital trauma and orbital reconstruction. In: Booth PW, Schendel SA, Hausamen JE (ed) Maxillofacial surgery, Vol 1, 2ndedn. Churchill Livingstone, Edinburg, pp 205-222.

Metzger MC, Schon R, Schulze D, Carvalho C, Gutwald R, Schmelzeisen R. Individual preformed titanium meshes for orbital fractures. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:442-447.

Saikrishna Degala, Sujith Kumar Shetty, Lakshith Biddappa. Reconstruction of Post-traumatic Internal Orbutal Wall Defects with Titanium Mesh. J Maxillofac. Oral Surg 2013;12(4):418-423.

Forrest S. Roth, John C. Koshy, Jonathan S. Goldberg, Charles N.S. Soparkar. Pearls of Orbital Trauma Management. Seminars in Plastic Surgery 2010;24(4):398-409.

Yash J. Avashia BS, Ananth Sastry, BS, Kenneth L. Fan, Haaris S. Mir, Seth R. Thaller. Materials used for reconstruction after Orbital Floor Fracture. The Journal of Craniofacial Surgery 2012;23(1):49-55.

Schlickewei W, Schlickewei C. The use of bone substitutes in the treatment of bone defects – the clinical view and history. Macromol Symp 2007;253:10-23.

Discussion

Studies validating titanium as an alloplastic material for orbital floor repair began in the early 1990s 13, 14. It has been used for the repair of large orbital floor defects and correction of globe malposition by restoration of orbital structure and prevention of late complications. By closely reproducing the natural orbital volume and shape, titanium mesh can prevent traumatic enophthalmos from developing 6. Titanium is a metallic alloplast that has received much attention in the area of craniofacial reconstruction 15. An attractive feature of titanium is its ability to osseointegrate16, 17 . Review of literature suggests that titanium may be an ideal material for orbital wall reconstruction 1, 9 - 12. Advantages of titanium mesh plates include availability, biocompatibility, ease of intra operative contouring, and rigid fixation18. Unfortunately, these implants are not easily positioned. Irregular edges of the mesh may catch prolapsed orbital fat19. Typically, titanium mesh plates should be placed 2 mm away from the orbital rim to avoid adherence syndrome20. Custom made titanium implants using computer assisted designs have enabled surgeons to achieve optimal reconstruction in areas of limited visibility and protection of vital structures21.

Conclusion

The various properties of titanium make it an ideal material for orbital wall reconstruction. Ultimately, the goal of reconstruction is to restore the form and function. As biotechnology advances, newer materials will emerge. But finally it is only the surgeon’s decision to choose the best material.

Reference 1.

Marcin Kozakiewicz, Piota Szymor. Comparison of prebent titanium mesh versus polyethylene implants in patient

8. Webster K. Orbital floor repair with lyophilized porcine dermis. Oral Surg Oral Med Oral Pathology 1988;65:161-164. 9.

Gabrielli MF, Monnazzi MS, Passeri LA, Carvalho WR, Gabrielli M, Hochuli – Vieira E. Orbital wall reconstruction with titanium mesh: retrospective study of 24 patients. Craniomaxillofac Trauma Reconstr 2011;4(3):151-6.

10. Degala S, Shetty SK, Biddappa L. Reconstruction of Posttraumatic Internal Orbital Wall Defects with Titanium Mesh. J Maxillofac Oral Surg 2013;12(4):418-23 11. Reich W, Seidel D, Bredehorn Mayr T, Eckert AW. Reconstruction of isolated orbital floor fractures with a pre fabricated titanium mesh. Kin MonblAugenheilkd 2014;231(3):246-55. 12. Woo KS, Cho PD, Lee SH. Reconstruction of severe medial orbital wall fractures using titanium mesh plates by the pericaruncular approach. J Plast Surg Hand Surg 2014;48(4):248-53 13. Glassman RD, Manson PN, Vanderkolk CA, et al. Rigid fixation of internal orbital fractures. Plast Reconstr Surg 1990;86:1103-1109. 14. Sargent LA, Fulks KD. Reconstruction of internal orbital fractures with Vitallium mesh. Plast Reconstr Surg 1991;88:31-38. 15. David Mok, Lucie Lessard, Carlos Cordoba, Patrick G Harris, Andreas Nikolis. A review of materials currently used in orbital floor reconstruction. Can J Plast Surg 2004; 12(3): 134-140.

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R-3 16. Mofid MM, Thompson RC, Pardo CA, Manson PN, Vander Kolk CA. Biocompatability of fixation materials in the brain. Plast Recconstr Surg 1997; 100:14-22. 17. Ungersbock A, Pohler O, Perren SM. Evaluation of soft tissue interface at titanium implants with different surface treatments: Experimental study on rabbits. Biomed Mater Eng 1994; 4:317-25. 18. Ellis E 3rd, Tan YH. Assessment of internal orbital reconstructions for pure blowout fractures: cranial bone grafts versus titanium mesh. J Oral Maxillofac Surg 2003; 61:442-453.

AABS; 4(1): 2017 19. Tuncer S, Yavuzer R, Kandal S, et al. Reconstruction of traumatic orbital floor fractures with resorbable mesh plate. J Craniofac Surg 2007; 18:598-605. 20. Lee HB, Nunery WR. Orbital adherence syndrome secondary to titanium implant material. Ophthal Plast Reconstr Surg 2009; 25:33-36. 21. Lieger O, Richards R, Liu M, et al. Computer â&#x20AC;&#x201C; assisted design and manufacture of implants in the late reconstruction of extensive orbital fractures. Arch Facial Plast Surg 2010;12:186-191.

*Corresponding author: Dr. Surya Rao R.V.M., Flat 1B, Kameshwari Apts,, 23/35, Desika Road, Mylapore, Chennai â&#x20AC;&#x201C; 600004, Tamilnadu, India Email: surya_3@hotmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 04.02.2017 Date of Acceptance : 10.02.2017 Date of Publication : 21.02.2017

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

Review Article DOI: 10.21276/AABS.2017.1310

The Most Frequent Prescription in Obstetrics: Bed Rest! Harkiran Kaur Khaira and Smita Sinha* Dept of OBG, Adesh Institute of Medical Sciences and Research (AIMSR). Bathinda, India

ABSTRACT The most frequent prescription across all medical disciplines and specially in Obstetrics has been bed rest. Even in this age of evidence based medicine, bed rest remains as the most prescribed, although evidence does not support it. The usage is strong because there are no substantial randomised trials to either refute or support bed rest, beginning with lack of definition. In this article we are reviewing bed rest in early miscarriage, hypertensive disorders of pregnancy, multiple pregnancy, preterm, reduced AFI, impaired fetal growth and in pregnancy after artificial reproduction techniques. We found that while women with moderate risk to preeclampsia, low AFI or multiple gestation may demonstrate some beneficial effects, it largely remains a tool for enforcing psychological benefit. It also entails adverse effects like muscle atrophy, stress and economic loss. However till more evidence can be made available, it will retain its numero uno position. Keywords: Bed Rest, Preeclampsia, Preterm, Miscarriage, Reduced AFI, Multiple Pregnancy, ART

Introduction

Bed rest in pregnancy has been one of the most consistent and frequent prescriptions by Obstetricians the world over for the management of almost any deviation from normal gestation. In the common parlance it is advised by some doctors, even in a normal setting. Antepartum bed rest is advised based on the assumption that it might be both effective and safe for the mother and the fetus[1]. On many occasions, it is prescribed without sound evidence to its utility and sometimes just to allow the benefit of doubt to the expectant mother.

Defining Bed Rest

Across medical disciplines, bed rest would easily sum up to be the most popular prescription. However the term has never been defined accurately. Another term used often interchangeably is activity restriction, which leads to some confusion. Aerospace studies use the term bed rest to mean muscle disuse. Maloni JA[1] in her study defined antepartum bed rest as confinement to bed in hospital with activity limited to toileting. With this definition they observed that a pregnant woman spends about twenty-two hours in bed per day. In Obstetric practice, bed rest has often also involved instructions to use bed pan facility by the patient. The practice has been variant depending on clinical situation. Certainly a woman with minimal spotting per vagina in early pregnancy will be advised â&#x20AC;&#x2DC;activity restrictionâ&#x20AC;&#x2122; mostly but not completely limited to allowing toileting only; while a threatened preterm patient with 2 cm cervical dilatation and bag of membranes bulging will be ordered to use bed pan. There is a lack of randomised controlled trials to define what constitutes optimum antepartum bed rest.

Bed rest in early miscarriage

One in five pregnancies is complicated by vaginal bleeding before 20 weeks gestation and bed rest is routinely recommended[2]. It is prescribed based on the idea that as hard work and hard physical activity during pregnancy are associated with miscarriage, bed rest might reduce the risk[3]. However, this hypothesis is limited by the fact that most of the causes of miscarriage are not related to physical activity. A survey among practitioners was conducted in Wessex, UK, 1985-86 and results published on what doctors thought about bed rest. It was thought to be usually ineffective by 413 (32%) of the respondents; 623 (48%) said that it was usually useful, while only 220(17%) thought that it was mandatory[4]. A resonating thought published in this study was that, many practitionersâ&#x20AC;&#x2122; believed that if they did not recommend bed rest, they might be held responsible for a subsequent miscarriage by the patient. In a Cochrane review in 2010, only two studies including total 84 women were identified. There was no statistically significant difference in the risk of miscarriage in the bed rest group versus the no bed rest group (placebo or other treatment) (risk ratio RR 1.54, 95% confidence interval CI 0.92 to 2.58)[5]. There was a higher risk of miscarriage in those women in the bed rest group than in those in the human chorionic gonadotrophin therapy group with no bed rest (RR 2.50, 95% CI 1.22 to 5.11). However the analysis remains inconclusive due to small number of participants. Although there is no definite evidence that bed rest can affect the course of pregnancy, abstinence from active environment for a couple of days may help women feel safer, thus providing emotional relief[6].

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Bed rest in hypertensive disorders of pregnancy

It is common practice to advice bed rest to women with preeclampsia. Restriction of activity and rest has even been advocated for prevention of hypertension in pregnancy[7]. Two small trials (145 women total) compared bed rest with ambulation in women with preeclampsia and found no conclusive evidence to accept or refute it[8]. Randomised trials by Spinapolice 1983 and Herrera 1993, evaluated risk reduction in preeclampsia of women who rested for four to six hours per day compared to women who carried out normal activity. One trial showed that there was a statistically significant reduction in the relative risk of pre-eclampsia with rest (relative risk RR 0.05, 95% confidence interval, CI 0.00 to 0.83), however no risk reduction was observed for gestational hypertension[9]. In the second trial, rest of 30 minutes per day plus nutritional supplementation was associated with a reduction in the risk of pre-eclampsia (74 women; RR 0.13, 95% CI 0.03 to 0.51) and also of gestational hypertension (RR 0.15, 95% CI 0.04 to 0.63)[10]. While these studies reflect a positive impact of bed rest on preventing preeclampsia, there is no information regarding perinatal or maternal outcome, as also bias in these studies cannot be completely ruled out. While literature does not support bed rest as part of management protocol for preeclampsia, there appears to be a small but indefinite role in prevention of preeclampsia in women who are at moderate risk.

Bed rest in multiple pregnancy

AABS; 4(1): 2017 women with an uncomplicated twin pregnancy the results of this review show no benefit from routine hospitalisation for bed rest. Evidence does not support routine prescription of bed rest in all women with multi fetal gestation, as it does not seem to improve preterm birth or perinatal mortality. There is no advantage in prevention of maternal complications as well. But with the slight advantage of improving birth weight of foetus, it may be beneficial in reducing the problems of preterm baby. Further trials are still warranted to accept or refute the small evidence.

Bed rest in preterm

Preterm birth can be due to a multitude of factors including chronic maternal illness, vaginal infections, stress, socio economic factors etc. Extremely few trials have been conducted evaluating bed rest in prevention of preterm birth. Elliot JP et al[16] conducted a randomised trial assessing the efficacy of activity restriction (AR) in preventing preterm birth in women who were negative for fetal fibronectin (fFN). A total of 73 women with negative fFN were randomised (36 with AR, 37 without AR). The overall preterm birth rate was 40%, with 44.4% of patients with AR and 35.1% of patients without AR delivering preterm, p=0.47816. In an earlier study by Hobel CJ et al bed rest was addressed as a secondary outcome and no benefit was reported. However this study lacked in proper randomisation information and several important details like characteristics of women included in the study[17].

Women and infants of a multiple pregnancy are recognised to be at increased risk of adverse outcome when compared with singletons. The greatest risk to infants of a multiple pregnancy is being born preterm[11]. The preterm birth rate less than 37 weeks for women with a singleton pregnancy is 6.3% and 97% for women with a triplet pregnancy[12]. Multiple pregnancies account for 9% to 12% of all perinatal deaths[13]. In addition, multiple births are associated with maternal complications such as preeclampsia, gestational diabetes, caesarean delivery, and postpartum haemorrhage[14]. The common prescription of bed rest is justified with the assumption that it effectively prevents preterm birth by reducing uterine activity and that bed rest is safe for mother and infants[1]. A Cochrane review on bed rest published in 2010, identified seven trials involving 713 women with multiple pregnancy. It was reported that routine bed rest in hospital did not improve the rates of preterm birth or perinatal mortality. There was a suggestion of a decreased number of low birthweight infants (less than 2500 g) born to women in the routinely hospitalised group (RR 0.92; 95% CI 0.85 to 1.00)[15]. For

Thus evidence does not support activity restriction or bed rest as a valid intervention in preterm birth prevention. Again a safe conclusion is fugitive in the absence of trials. Hence though not evidenced, it cannot yet be refuted with a certainty.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Bed rest in impaired fetal growth and reduced AFI

Bed rest during pregnancy is one of the numerous approaches apart from nutritional supplementation, oxygen therapy, plasma volume expansion, beta mimetic drugs suggested to treat impaired fetal growth. We came across a single trial by Laurin in 1987, comparing birth weight among women on bed rest with ambulant but off work women. It was a small study involving 107 pregnant women, which reported that no differences were detected between bed rest and ambulatory management for fetal growth parameters (relative risk 0.43, 95% confidence interval: 0.15 to 1.27) and neonatal outcomes[18]. The study was too small to be accepted as concrete evidence for medical advice.

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In a prospective study conducted in 2011, maternal rest in the left lateral decubitus position was shown to increase the estimated fetal urine production rate and the estimated amniotic fluid volume[19]. In another publication 2012, the principle finding reported was that the estimated amniotic fluid volume increase with maternal rest in the left lateral decubitus position follows the pattern of a saturation curve. The increase is maximal at the beginning; however, it is saturated as the position is maintained. Finally, the amniotic fluid volume increase stops at approximately at the second hour of rest. The maximum increase is in the first fifteen minutes[20]. It may be because the maternal left lateral position, having the least disturbing effect on fetal hemodynamics compared with any other position,[21,22] may increase uterine and placental blood flow. Therefore an apt prescription of bed rest in low AFI must include the instructions for rest in left lateral with rest limited to a short periods. Longer durations of rest is neither warranted nor effective.

Bed rest after ART

Women who have conceived with artificial reproductive techniques come tagged with an indefinite prescription for best rest, lest the ‘precious’ pregnancy be lost. While those who have undergone IUI (intra uterine insemination) may be less prone to resign to complete bed rest, those who have undergone IVF (in vitro fertilisation) are usually greatly stressed and resort to bed rest almost during the entire duration of gestation. Psychological it gives them a leverage in fighting the odds to a successful pregnancy outcome. Clinically it is one aspect that is still not well established; whether or not there are ways to decrease the movement or expulsion of embryos from the uterus following transfer. There are several studies evaluating effectiveness of post-ET (embryo transfer) techniques for women undergoing IVF and intracytoplasmic sperm injection (ICSI). The ongoing pregnancy rate was reported in one trial that compared immediate ambulation after embryo transfer to 30-minute bed rest. The results showed no evidence of an effect of bed rest in improving the rate of ongoing pregnancies (OR 1.00; 95% CI 0.54 to 1.85)[23].

Adverse effects of bed rest

Prolonged bed rest is fraught with potential problems. Bed rest may increase the likelihood of thromboembolic events[24], muscle atrophy and symptoms of musculoskeletal and cardiovascular de conditioning[25,26] may be stressful and costly for women and their family[27], may induce self blame feelings in case of failure to comply with the prescription[28] and may increase costs for the health services[29]. A study, conducted under highly standardised conditions, provides prospective evidence that 60 days

of bed rest accelerated fat accumulation in the vertebral bone marrow of 24 healthy premenopausal women. The accumulation was not reversed 1 yr after a return to normal activities and was associated with normalised haemoglobin with decreased erythropoietin levels and with persistently elevated leukocyte numbers[30].

Conclusion

Bed rest even though is the most popular and maximally used prescription, lacks evidence to its utility. While women with moderate risk to preeclampsia, low AFI or multiple gestation may demonstrate some beneficial effects, it largely remains a tool for enforcing psychological benefit. However it demands to be practised with caution and appropriate titration as prolonged bed rest can lead to several complications.

References 1.

Maloni JA. Lack of evidence for prescription of antepartum bed rest. Expert Rev Obstet Gynecol. 2011 July 1; 6(4): 385–393.

Sotiriadis A, Papatheodorou S, Makrydimas G. Threatened miscarriage: evaluation and management, BMJ 2004;329:152–5.

Lapple M. Occupational factors in spontaneous miscarriage. Zentralblatt fur Gynakologie 1990;112(8):457–66.

Everett C, Ashurst H, Chalmers I. Reported management of threatened miscarriage by general practitioners in Wessex. BMJ 1987; 295: 583-6

Aleman A, Althabe F, Belizán J, Bergel E. Bed rest during pregnancy for preventing miscarriage. Cochrane Database Syst Rev. 2005 Apr 18;(2):CD003576. [PMID: 15846669]

al-Sebai MA, Diver M, Hipkin LJ. The role of a single free beta-human chorionic gonadotrophin measurement in the diagnosis of early pregnancy failure and the prognosis of fetal viability. Hum Reprod 1996;11:881-8

Goldenberg R, Cliver S, Bronstein J, Cutter G, Andrews W, Mennemeyer S. Bed rest in pregnancy. Obstetrics & Gynecology 1994;84:131–6.

Duley L. Strict bed rest for proteinuric hypertension in pregnancy. In: The Cochrane Library, Issue 2, 1995

Spinapolice RX, Feld S, Harrigan JT. Effective prevention of gestational hypertension in nulliparous women at high risk as identified by the rollover test. American Journal of Obstetrics and Gynecology 1983;146:166–8.

10. Herrera JA. Nutritional factors and rest reduce pregnancy induced hypertension and pre-eclampsia in positive rollover test primigravidas. International Journal of Gynecology & Obstetrics 1993;41:31–5 11. Dodd JM, Crowther CA. Hospitalisation for bed rest for women with a triplet pregnancy: an abandoned randomised controlled trial and meta-analysis, BMC Pregnancy and Childbirth 2005, 5:8

http://www.pacificejournals.com/aabs

R-7 12. Australian Institute of Health and Welfare, National Perinatal Statis- tics Unit: Australia’s mothers and babies 2000. Canberra 2003. 13. Norwitz ER, Edusa V, Park JS. Maternal physiologyand complications of multiple pregnancy. Seminars in Perinatology 2005;29(5):338–48. [PUBMED: 16360493] 14. Senat MV, Ancel PY, Bouvier-Colle MH, Breart G. How does multiple pregnancy affect maternal mortality and morbidity?. Clinical Obstetrics and Gynecology 1998;41(1): 78–83. [PUBMED: 9504226] 15. Crowther CA, Han S. Hospitalisation and bed rest for multiple pregnancy. Cochrane Database of Systematic Reviews 2010, Issue 7. Art. No.: CD000110. DOI: 10.1002/14651858. CD000110.pub2 [PMID:20614420] 16. Elliott JP, Miller HS, Coleman S, Rhea D, Abril D, Hallbauer K, Istwan NB, Stanziano GJ. A randomized multicenter study to determine the efficacy of activity restriction for preterm labor management in patients testing negative for fetal fibronectin. J Perinatol. 2005 Oct;25(10):626-30 17. Hobel CJ1, Ross MG, Bemis RL, Bragonier JR, Nessim S, Sandhu M, Bear MB, Mori B. The West Los Angeles Preterm Birth Prevention Project. I. Program impact on high-risk women. Am J Obstet Gynecol. 1994 Jan;170(1 Pt 1):54-62.

AABS; 4(1): 2017 maternal positions and with epidural analgesia. Obstet Gynecol 1986; 68:61–64 22. Ryo E, Okai T, Takagi K, et al. Comparison of umbilical artery Doppler velocimetry between maternal supine position and complete left lateral position in predicting obstetric complications. Ultrasound Obstet Gynecol 1998; 11:415–418. 23. Purcell KJ, Schembri M, Telles TL, Fujimoto VY, Cedars MI. Bed rest after embryo transfer: a randomized controlled trial. Fertility and Sterility 2007;87(6):1322–6. [PUBMED: 17362946] 24. Kovacevich GJ, Gaich SA, Lavin JP, Hopkins MP, Crane SS, Stewart J, et al.The prevalence of thromboembolic events among women with extended bed rest prescribed as part of the treatment for premature labor or preterm premature rupture of membranes. American Journal of Obstetrics and Gynecology 2000;182(5):1089–92. 25. Maloni JA, Chance B, Zhang C, Cohen AW, Betts D, Gange SJ. Physical and psychosocial side effects of antepartum hospital bed rest. Nursing Research 1993;42(4):197–203. 26. Maloni JA, Kane JH, Suen LJ, Wang KK. Dysphoria among high-risk pregnant hospitalized women on bed rest: a longitudinal study. Nursing Research 2002;51(2):92–9.

18. Laurin J, Persson PH. The effect of bedrest in hospital on fetal outcome in pregnancies complicated by intra-uterine growth retardation. Acta Obstetricia et Gynecologica Scandinavica 1987;66: 407–11.

27. Gupton A, Heaman M, Ashcroft T. Bed rest from the perspective of the high-risk pregnant woman. Journal of Obstetric, Gynecologic and Neonatal Nursing 1997;26(4): 423–30.

19. Ülker K, Çeçen K, Temur İ, Gül A, Karaca M. Effects of the maternal position and rest on the fetal urine production rate: a prospective study conducted by 3-dimensional sonography using the rotational technique (virtual organ computer-aided analysis). J Ultrasound Med 2011; 30:481– 486.

28. Schroeder C. Women ́ s experience of bed rest in high-risk pregnancy. Image - The Journal of Nursing Scholarship 1996; 28(3):253–8.

20. Ülker K, Gül A, Çiçek M. Correlation Between the Duration of Maternal Rest in the Left Lateral Decubitus Position and the Amniotic Fluid Volume Increase. J Ultrasound Med 2012; 31:705–709

30. Trudel G, Payne M, Ma ̈dler B, Ramachandran N, Lecompte M, Wade C et al. Bone marrow fat accumulation after 60 days of bed rest persisted 1 year after activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study, J Appl Physiol 2009;107: 540–8

21. Marx GF, Patel S, Berman JA, Farmakides G, Schulman H. Umbilical blood flow velocity waveforms in different

29. Allen C, Glasziou P, Del Mar C. Bed rest: a potentially harmful treatment needing more careful evaluation. Lancet 1999;354:1229–33.

*Corresponding author: Dr Smita Sinha, Associate Professor, Dept of Obstetrics & Gynaecology, AIMSR, Barnala Road, Bhucho. Bathinda. 151101. Punjab, India Email: obsusg@gmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 05.02.2017 Date of Acceptance : 14.02.2017 Date of Publication : 21.02.2017

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Review Article DOI: 10.21276/AABS.2017.1328

Does Interleukin 22 Have a Role in Periodontal Pathogenesis? A Short Review SreenivasNagarakanti1* and ArunKV2 Department of Periodontology, Narayana Dental College & Hospital, Nellore, INDIA 2 Department of Periodontology, Ragas Dental College & Hospital, Chennai, INDIA.

ABSTRACT Many studies from recent years have shown that Interleukin 22 (IL-22) plays a major role in both the defence against certain microbes and the development and maintenance of chronic inflammatory diseases.Periodontal disease, including gingivitis and periodontitis, is caused by the interaction between pathogenic bacteria and the host immune system. Further animal and human studies are needed to investigate the role of IL-22 in periodontal health and disease. Keywords: IL-22, Periodontitis, Host Defence, Inflammation

Introduction

Interleukin 22 (IL-22) is a member of the IL-10 family of cytokines (1). The cytokines of this family (IL-10, IL-19, IL-20, IL-24, IL-26, IL- 28 (a and b), and IL-29) display homologous secondary structures and 20-30% amino acid identity. IL-22 was first described in 2000 as a gene differentially expressed in IL-9-treated BW5147 murine T lymphoma cells (2). IL-22 is mainly produced by activated T and NK cells. Highest IL-22 expression has been detected in CD4+ memory cells (3). IL-22 acts on non-hematopoietic cells in epithelial tissues, which express a functional IL-22 receptor composed of an heterodimer of IL-22R1 and IL-10R2 (4,5,6). IL-22 signalling triggers the production of antimicrobial peptides and pro-inflammatory molecules, and it also promotes tissue repair by inducing epithelial cell proliferation and survival (1-7). IL-22 has a role in the control of host defence against bacterial infections (8) and also limits tissue destruction in hepatitis (9), colitis and graft versus host disease (10) and drives endogenous thymic regeneration (11). Dysregulation of IL-22 signalling contributes to the pathogenesis of psoriasis (12), arthritis (13), and colon cancer (14).Generally, IL-22 acts to strengthen epithelial barrier functions and is involved both in tissue homeostasis as well as tissue repair and wound healing. However, uncontrolled excessive or prolonged production of IL-22 can cause pathology, such as psoriasis-like skin inflammation (15). In general, IL-22 functions to sustain the integrity and barrier functions of many tissues and prevent damage caused by either invading pathogens or by the inflammatory response itself (6,7). In this process, IL-22 can directly elicit proinflammatory epithelial defense mechanisms

that are essential for host protection. However, if not tightly controlled, this bears the risk of IL-22 amplifying inflammation either alone or in synergy with other cytokines, and causing abnormal epithelial proliferation and differentiation, as seen in some infectious and inflammatory conditions (15).

Functions of IL-22

IL-22 promotes epithelial proliferation and helps to maintain and restore the integrity of the epithelial barrier function during the invasion of pathogens. It synergistically with other cytokines (IL-17/Tumor necrosis factor Îą) induces the expression of antimicrobial proteins involved in host defense in the skin, the airways, and the intestine (16,17). IL-22 also has a role in promoting the production of inflammatory mediators, such as IL-6, granulocyte colony-stimulating factor, IL-1Î˛, serum amyloid and lipopolysaccharide-binding protein (18-20). In addition to its antimicrobial and pro-inflammatory functions, IL22 contributes to tissue regeneration and promotes wound healing (21-23). Due to lack of IL-22R expression on leukocytes, IL-22 does not induce anti-inflammatory response. Studies showed, forceful expression of IL-22 receptor on leukocytes results in pro-inflammatory symptoms in vivo (24). The anti-inflammatory activity of IL-22 is probably an indirect consequence of its tissue protective functions. In fact, IL-22 acts as a pro-inflammatory cytokine in many cases, and an uncontrolled expression of IL-22 can lead to pathology (15). The double-headed role of IL-22 as pro and antiinflammatory function depends on many factors such as concentration and duration of local IL-22, the target tissues, and the cytokine environment (16,25).

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Discussion

Many studies proved that the initiation of periodontal diseases is due to colonization of the gingival sulcus by pathogenic bacteria followed by a host response consisting of pro-inflammatory mediators and the infiltration of immune cells in an effort to rid the host tissue of the bacterial insult. Research showed that IL-22 elicits various innate immune responses from epithelial cells and is essential for host defense against several invading pathogens and also protects tissue integrity and maintains the mucosal homeostasis. On the other handit acts as a proinflammatory cytokine that might result in tissue damage. Till date there is no literature on the role of IL-22 in periodontal health and disease. However, further investigations are required to evaluate the effects IL-22 on periodontal tissues.

References 1.

3. 4.

5. 6. 7. 8.

Ouyang W, Rutz S, CrellinNK, Valdez PA, HymowitzSG. Regulation and Functions of IL-10 Family Cytokines in Inflammation and Diseases. Annu Rev Immunol 2011;29:71–109. Dumoutier, L., Louahed, J., and Renauld, J.C. (2000a). Cloning and characterization of IL-10-related T cell-derived inducible factor (IL- TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. J. Immunol. 164, 1814–1819. Wolk, K., Kunz, S., Asadullah, K., and Sabat, R. (2002). Cutting edge: immune cells as sources and targets of the IL10 family members? J. Immunol. 168, 5397–5402. Dumoutier L, Van Roost E, Colau D, RenauldJC. Human interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as an hepatocyte- stimulating factor. ProcNatlAcadSci USA 2000;97:10144–10149. Xie MH, et al. Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R. J BiolChem 2000;275:31335–31339. ZenewiczLA1, Flavell RA. Recent advances in IL22 biology. IntImmunol. 2011 Mar;23(3):159-63. Sonnenberg, G. F., Fouser, L. A. &Artis, D. Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22. Nat. Immunol. 2011. 12, 383–390. Aujla, S. J. et al. IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat. Med. 14, 275–281 (2008).

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22. 23. 24. 25.

Zenewicz, LA. et al. Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation. Immunity 27,647–659 (2007). Hanash, AM. et al. Interleukin-22 protects intestinal stem cells from immune- mediated tissue damage and regulates sensitivity to graft versus host disease. Immunity 37, 339–350 (2012). Dudakov, J. A. et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science 336, 91–95 (2012). Zheng, Y. et al. Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 445, 648–651 (2007). Sherlock, J. P. et al. IL-23 induces spondyloarthropathy by acting on ROR-gammat þ CD3 þ CD4-CD8- entheseal resident T cells. Nat. Med. 18, 1069–1076 (2012). Huber, S. et al. IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine. Nature 491, 259–263 (2012). Rutz S, Eidenschenk C, Ouyang W. IL-22, not simply a Th17 cytokine.Immunol Rev. 2013 Mar;252(1):116-32. Liang SC, et al. Interleukin (IL)-22 and IL-17 arecoexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006;203:2271–2279. Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity 2004;21:241–254. Wolk K, et al. IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis. Eur J Immunol 2006;36:1309–1323. Liang SC, et al. IL-22 Induces an Acute-Phase Response. J Immunol 2010;185:5531–5538. Aujla S, et al. IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat Med 2008;14:275–281. Ki S-H, et al. Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3. Hepatology 2010;52:1291–1300.  Kong X, et al. Interleukin-22 induces hepatic stellate cell senescence and restricts liver fibrosis in mice. Hepatology 2012;56:1150–1159. Dudakov JA, et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science 2012;336:91–95. Savan R, et al. A novel role for IL-22R1 as a driver of inflammation. Blood 2011;117:575–584. SonnenbergGF, Nair MG, KirnTJ, Zaph C, Fouser LA, Artis D. Pathological versus protective functions of IL-22 in airway inflammation are regulated by IL-17A. J Exp Med 2010;207:1293– 1305.

*Corresponding author: Dr. Sreenivas Nagarakanti, M.D.S, (PhD), Professor, Department of Periodontics, Narayana Dental College & Hospital, ChinthareddyPalem, Nellore AndhraPradesh, INDIA, Pin: 524003 Phone: +91 9985664566 Email: sreenivasnagarakanti@gmail.com Date of Submission : 08.02.2017 Date of Acceptance : 19.02.2017 Date of Publication : 21.02.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Review Article DOI: 10.21276/AABS.2017.1330

Gastrointestinal Stromal Tumor (GIST): An Update on Its Uncommon and Complex Presentations Dhiraj B Nikumbh1* and Roopali D Nikumbh2 1

Dept of Pathology, ACPM Medical College, Dhule, India Dept of Anatomy, ACPM Medical College, Dhule, India

ABSTRACT Gastrointestinal stromal tumor (GIST) is recently diagnosed as a tumor entity. In the past, these tumors were classified as leiomyomas, leiomyosarcomas and leiomyoblastomas. GISTs are the rare soft tissue sarcomas of the gastrointestinal tract (GIT), accounting for 0.1-3% of all GIT tumors. The most common site of GIST is the stomach (60-70%) followed by small intestine (20-30%) and less common than 5% in colon, esophagus, omentum and mesentry. The epidemiology of GIST is not known completely. Most often these cases were detected on endoscopy, on imaging of the abdomen, at surgery for the other condition or at autopsy. The varied clinical presentations depend on the size of the tumor. Small GIST (less than 2cms) are asymptomatic and incidentally detected at endoscopy or at laparotomy. The large tumors give rise to vague abdominal discomfort or pain (20-50%), acute or chronic GI bleeding, intestinal obstruction (20%) or altered bowel habits and about 35% are detected incidentally. As per literature, the most common (50%) presentation is bleeding. Very large or malignant GIST presented as the exophytic palpable mass. Regarding unusual and complex presentations of GIST, we encountered two cases of GISTs in the twelve years of histopathology practice. Keywords: Gastrointestinal Stromal Tumor, GIT, UNUSUAL, C-KIT

Introduction

Gastrointestinal stromal tumor (GIST) is recently diagnosed as a tumor entity. In the past, these tumors were classified as leiomyomas, leiomyosarcomas and leiomyoblastomas.1 GISTs are the rare soft tissue sarcomas of the gastrointestinal tract (GIT), accounting for 0.1-3% of all GIT tumors.2 The most common site of GIST is the stomach (60-70%) followed by small intestine (20-30%) and less common than 5% in colon, esophagus, omentum and mesentry.3The epidemiology of GIST is not known completely. Most often these cases were detected on endoscopy, on imaging of the abdomen, at surgery for the other condition or at autopsy.1 In view of its complex and unusual presentations over the period of twelve years ,we encountered two cases in sigmoid colon and jejunum respectively.

Discussion

The varied clinical presentations depend on the size of the tumor. Small GIST (less than 2cms) are asymptomatic and incidentally detected at endoscopy or at laparotomy.1 The large tumors give rise to vague abdominal discomfort or pain (20-50%), acute or chronic GI bleeding, intestinal obstruction (20%) or altered bowel habits and about 35% are detected incidentally. As per literature, the most common (50%) presentation is bleeding. Very large or

malignant GIST presented as the exophytic palpable mass.1 Regarding unusual and complex presentations of GIST, we encountered two cases of GISTs in the twelve years of histopathology practice. The first case was presented with perforation and peritonitis in sigmoid colon in a 70 year old male4 .We reported this case in view of its rare site that is sigmoid colon and its unusual presentation as perforation, because colonic GIST rarely perforates. To the best of our knowledge, this was the second documented case of malignant GIST after Hwango Y et al5.The patient presented to surgery with pain in abdomen, on basis of clinical and radiological examination, emergency laparotomy revealed perforation of sigmoid colon and the mass protrudes through the defect. Spindle cell morphology and C-Kit(CD-117) positivity on immunohistochemistry (IHC) confirms the malignant GIST.4 Second case was very interesting in view of its mysterious presentation. A 78 year old male presented with per rectal bleeding and hypotension. The investigations revealed no specific pathology. In view of no relieved in symptoms, emergency life saving laparotomy was performed. The intraoperative findings showed jejunal mass protruding through wall with blackish areas of hemorrhage with multiple nodules on peritoneal suggestive of metastasis. Histopathologically revealed spindle cell morphology with moderate mitotic figures favored malignant GIST it was confirmed by CD-117.6

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R-11 Light microscopically majority of the GIST have spindle cell morphology (80%) with fascicular or storiform pattern of growth.7 Rest (20%) are epitheloid cells with abnormal eosinophilic to clear cytoplasm.7 Surgical resection with negative surgical margin is the treatment of choice in small intestinal GIST and Imatinib mesylate is the standard regimen for inoperable and malignant/metastatic masses.1

AABS; 4(1): 2017 with duodenal neuroendocrine carcinoma (NET) + neurofibromatosis (NF)-Case report and review of the literature,” Zeitschrift f¨ur Gastroenterologie.2005;43(3): 281–288. 3.

Miettinen M, Lasota J.Gastrointestinal Stromal tumors -definition, clinical, histological, immunohistochemical and molecular genetic features &differential diagnosis. Virchows Arch. 2001; 438:1-12.

Jagtap SV, Nikumbh DB, Kshirsagar AY, Bohra A, Khatib W.Malignant Gastrointestinal Stromal tumor of the Sigmoid Colon with Perforation and Peritonitis - An Unusual Presentation. International Jr of Health Sciences and Research (IJHSR). 2012;2(3):104-109.

Hwangbo Y , Jang JY , Kim YW , Park SD, Shim J et al. Spontaneous rupture of a sigmoid colon gastrointestinal stromal tumor manifesting as pneumoperitoneum with localized peritonitis: report of a case. Surg Today. 2011; 41(8): 1085-90.5.

The clinicians needs to aware of these varied ,unusual and mysterious presentations of GIST due to avoid misdiagnosis in view of its aggressive, fatal and malignant nature.

Mujawar P, Nikumbh DB, Suryawanshi KH, Pagare PS, Thakur R. Malignant gastrointestinal stromal tumor (GIST) of the jejunum: the mysterious complex presentation. Int J Med Sci Public Health 2015;4:1168-1171

Reference

7. Fletcher CDM, Berman JJ,Corless C, Gorstein F, Lasota J, Longley BT et al . Diagnosis of Gastrointestinal Stromal tumors: a consensus approach. Hum Pathol.2002 ; 33 : 459-465.

Recently, the new group of GIST was added is “SDH resistant GIST” by WHO in 2013, that is succinate dehydrogenase GIST. Cytogenetically mutation of KIT or PDGFRA occurs in micro GIST (1-10mm).8 GISTs are positive for CD-117 /c-KIT by IHC in 95% cases. New markers DOG1 and PKC-theta are expressed in KIT negative cases.SDH deficient GIST always occur in stomach and pediatric age group with indolent behavior even in presence of metastasis.8

Conclusion

Joensuu H. Gastrointestinal stromal tumors. Annals of Oncology.2006;17(10):280-6.

Kramer K, Siech M, Str¨ater J, Aschoff AJ, and HenneBruns D. “GI hemorrhage with fulminant shock induced by jejunal gastrointestinal stromal tumor (GIST) coincident

Desai SR. Gastrointestinal stromal tumor.Archives of Cytology and Histopathology Research, 2016; 1(3):78-9.

*Corresponding author: Dr Dhiraj B Nikumbh MD, Professor, Dept of Pathology, JMF’S ACPM Medical College, Dhule, Maharashtra, India. Phone: +91 9028522413 Email: drdhirajnikumbh@gmail.com Date of Submission : 08.02.2017 Date of Acceptance : 16.02.2017 Date of Publication : 21.02.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Review Article DOI: 10.21276/AABS.2017.1341

Indigenous Flaps in ENT: Our Experience Manish Munjal1, Archana Arora1*, Gopika Talwar1, Amanjot Kaur1 and Tejinder2 Deptt of ENTHNS, Dayanand Medical college and Hospital, Ludhiana, Punjab, India 2 Dhillon ENT Research Centre, Khanna, Ludhiana, Punjab, India

ABSTRACT The nose is undoubtedly an important facial feature. The rhinologic surgeon with interest in external nasal soft tissue surgery, oncologic or otherwise, may often require reconstruction of a soft tissue defect, from the medial canthus to the nasal ala laterally, to the nasal tip medially. The coverage needs tissues which resemble the adjoining skin texture and have a thickness which would fill the defect appropriately without appearing as a separate patch and upset the patient psychologically. Therefore we utilized local rotation and advancement flaps for the reconstruction of facial defects. Experience with these flaps is being shared. Keywords: Nasolabial Flap, Forehead Transposition Flap, Rintala Flap

Introduction

The history of nasal reconstruction parallels that of plastic surgery. It was begun by Sushruta in India during 600-700 BC [1]. The first procedure that was used for the nasal reconstruction was the midline forehead flap. The present era reconstructive surgeon now has a number of options in his armamentarium like local tissue from the nose, cheek, and forehead and also grafts from the nasal septum, ear, rib, hip, and calvaria [2]. As ENTHNS surgeons, we evaluate lesions which require reconstruction after excision and using simple techniques we can do without the assistance of plastic surgery colleagues. Some simple flaps shall be discussed here. Being a defining feature of the face, the nose is arguably the most prominent aspect. It is a composite structure composed of skin, mucosal lining, cartilage, muscle, subcutaneous tissue, septum, and bone. Nasal reconstruction is thus a very challenging job for most plastic and reconstructive surgeons. Commonly employed local flaps for defects along the medial canthus, nasal dorsum and nasal ala include the Nasolabial flap, Forehead transposition flap and Rintala flap which use the principles of VY advancement and Burows triangle. Our experience with these flaps is being described.

Nasolabial flap

A robust superiorly based myocutaneous flap pedicled on the facial artery, the nasolabial flap is an excellent choice in reconstruction of the nasal alar subunit, nasal tip, dorsum, soft triangle, and partial alar defects or single-stage reconstruction of oral cavity defects. Certain qualities make this flap ideal for nasal reconstruction. The color,

skin tone and texture of the cheek tissue are similar to that of the nose. In addition, the proximity of the nasolabial fold to the nose facilitates easy transposition. [3,4] We used this flap for the reconstruction of defect after excision of an ulcerated malignant lesion of the nasal ala involving the alar cartilage. [Figure 1,2] A sublabial flap may also be used if lip reconstruction is needed along. [Figure 6]

Forehead Transposition Flap

Based on the medial brow area, this vascular flap is pedicled on supratrochlear artery. Advantages of this flap include a good color match, inconspicuous donor and recipient site scars and normal nasal contours. [5] We used this flap for pigmented lesion near the medial canthus which was completely excised and defect reconstructed. [Figure 3] Trapezoid VY advancement flap or the Reigers flap may also be suitable for such a lesion. [Figure 6]

Rintala Flap

It is a superiorly based midline flap dissected on supraperiosteal plane. It has been seen to be excellent for reconstruction over the glabella and the entire nasal dorsum upto the nasal tip, even upto the upper part of columella. It offers a good tissue coverage and flap viability, color match, and nasal contour. [6,7] This flap was used for the reconstruction of the nasal dorsum after removal of black pigmented ulcer on the midline dorsum. [Figure 4,5]

Conclusion

Being ENT surgeons we commonly deal with such lesions on the face like basal cell carcinoma, malignant melanoma, etc which are amenable to complete excision with wide

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Fig. 1: (a) ulcerated lesion on right nasal ala, (b) excising the lesion, (c) raising the nasolabial flap, (d) covering the primary defect.

Fig. 2: (a) principle of VY advancement, (b) complete closure of defect, (c) patient at three month follow up.

Fig. 3: (a) outline of forehead flap, (b) pigmented lesion, (c,d) final outcome at three weeks.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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Fig 4: (a) outline of rintala flap with burrow triangle, (b) pigmented ulcer on lower third of nasal dorsum, (c) rintala flap marked along with burrow triangle, (d) flap dissected and raised.

Fig 5: (a) lesion excised, (b) final closure, (c) patient at six month follow up.

Fig. 6: schematic diagram showing the outline of (a) sublabial flap for lower lip reconstruction, (b) reiger flap for nasal dorsal lesions.

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tissue margins. All that is required is a good reconstruction of the defect after excision. These simple flaps can be easily mastered and utilized as and when required. With this we can limit the Plastic Surgeons being involved in the case and thus avoid additional cost to the patient.

Principles and Techniques of Nasal Reconstruction; Updated: Jul 24, 2015 Author: Joseph Fata http://emedicine. medscape.com/article/1820512-overview:

Acknowledgments

Thornton J, Griffin J, Constantine F. Nasal Reconstruction: An Overview and Nuances.; Semin Plast Surg. 2008 Nov; 22(4): 257–68.

William M. Weathers, MD, Erik M. Wolfswinkel. Expanded Uses for the Nasolabial Flap; Semin Plast Surg. 2013 May; 27(2): 104–109.

Stephen S Park. Forehead Flaps Treatment & Management. Updated: Apr 06, 2015 http://emedicine.medscape.com/ article/880171-treatment#d15

Chiu LD, Hybarger CP, Todes-Taylor N. The Rintala flap revisited. Plast Reconstr Surg. 1994 Nov;94(6):801-7

Kiyoshi O, Emi O. The Rintala flap: a versatile procedure for nasal reconstruction. American journal of otolaryngology; 2014;35:549-688

We are thankful for the assistance provided by the Anasthesia team and operating room staff

Conflict of interest:

all authors declare no conflict of interest

References 1.

Rogers BO. The historical evolution of plastic and reconstructive surgery. Wood-Smith D, Porowski O, eds. Nursing Care of the Plastic Surgery Patient. 1967

*Corresponding author: Dr Archana Arora, 159-C rishi nagar, Ludhiana-141001. Punjab, India Phone: +91 9888780218 Email: Drarchana.ent@gmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 10.02.2017 Date of Acceptance : 23.02.2017 Date of Publication : 28.02.2017

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

Review Article DOI: 10.21276/AABS.2017.1344

Cryptococcal meningitis in HIV (AIDS) patients: A Mini Review Manodeep Sen*, Tanushri Chatterji, Vivek Singh, Pushpa Yadav, Anupam Das and Vineeta Mittal Department of Microbiology, Dr. Ram Manohar Lohia Institute of Medical Sciences (RMLIMS), Lucknow, India

ABSTRACT Meningitis is a major health problem causing inflammation of the protective membranes covering the brain and spinal cord, collectively known as the meninges. Meningitis is generally caused by Meningococcus, Streptococcus pyogenes bacterial species causing bacterial meningitis: BM), Mycobacterium tuberculosis (Tubercular meningitis: TBM), Cryptococcus neoformans (fungal meningitis: CM). In recent times the CM and TBM are two most common types of chronic infectious meningitis. In adults, HIV-associated Cryptococcal meningitis has been reported in many areas of the world having high HIV seroprevalence i.e. in Sub-Saharan Africa. Cryptococcal meningitis is the fourth most commonly recognized cause of life-threatening infection among AIDS patients.Despite availability of antiretroviral therapy, the rise in rate of infections due to HIV/AIDS, suppress the cell mediated immune system and subjects are predisposed for opportunistic infections. Additionally, In AIDS patients, it is characterized by lack of meningeal signs and diminished inflammatory response. Diagnosis of CM is generally based on the collection of CSF sample and/or blood samples. Initially the CSF samples are processed for ‘gold standard’ culture methods, microscopy and India ink preparation. Serologically, Latex agglutination test (LAT) is the most commonly used for detection of Cryptococcal capsular antigen. Enzyme immunoassay (EIA) is another serological tool for detection of capsular polysaccharide antigens of C. neoformans in CSF. Keywords: Cryptococcal Meningitis, HIV-Associated

Introduction

Meningitis is a major health problem causing inflammation of the protective membranes covering the brain and spinal cord, collectively known as the meninges[1]. Meningitis is generally caused by Meningococcus, Streptococcus pyogenes bacterial species causing bacterial meningitis: BM), Mycobacterium tuberculosis (Tubercular meningitis: TBM), Cryptococcus neoformans (fungal meningitis: CM). In recent times the CM and TBM are two most common types of chronic infectious meningitis[2]. In adults, HIV-associated cryptococcal meningitis has been reported in many areas of the world having high HIV seroprevalence i.e. in Sub-Saharan Africa[3, 4]. Though once known to be rare, cryptococcosis has occurred at a high frequency in India in the past two decades[5, 6].Furthermore, prevalence of HIV-associated CM is also found to be common in many parts of India including North-eastern and as well as in Southern India [7, 8].Cryptococcal meningitis is the fourth most commonly recognized cause of life-threatening infection among AIDS patients[9].Despite availability of antiretroviral therapy, the rise in rate of infections due to HIV/AIDS, suppress the cell mediated immune system and subjects are predisposed for opportunistic infections. Additionally, In AIDS patients, it is characterized by lack of meningeal signs and diminished inflammatory response[10]. Clinical picture compatible with a diagnosis of Cryptococcal meningitis includes evidence of fever/

headache/meningismus/altered mental status or any other neurological manifestation with either a cerebrospinal fluid (CSF) abnormal biochemistry and/or pleocytosisand with a cryptococcal antigen titer of ≥ 1:8 and/or Positive India Ink preparation for capsulated yeast cells and/or a Positive CSF culture yielding Cryptococcus neoformans[11]. Diagnosis of CM is generally based on the collection of CSF sample and/or blood samples. Initially the CSF samples are processed for ‘gold standard’ culture methods, microscopy and India ink preparation. Serologically, Latex agglutination test (LAT) is the most commonly used for detection of Cryptococcal capsular antigen[12]. Enzyme immunoassay (EIA) is another serological tool for detection of capsular polysaccharide antigens of C. neoformans in CSF. This is a rapid test that provides visual and numeric result in less than an hour without pre-treatment of the specimen[13, 14].

Conclusion

Since there is limited literature available on Cryptococcal meningitis and its causative agent (Cryptococcus neoformans) therefore, further studies focusing on the significance based on diagnosis, pathogenesis related to the infection would be highly appreciated.

Acknowledgements

The authors acknowledge full support of Department of Microbiology, Dr. Ram Manohar Lohia Institute of Medical Sciences (RMLIMS), Vibhuti Khand, Gomti Nagar. Lucknow.

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Reference 1.

Sáez-Llorens X, McCracken GH. Bacterial meningitis in children.Lancet2003; 361:2139-2148.

Qu J, Zhou T, Zhong C, Deng R, Lü X. Comparison of clinical features and prognostic factors in HIV-negative adults with cryptococcal meningitis and tuberculous meningitis: a retrospective study.BMC infectious disease2017; 17:51.

Williamson PR, Jarvis JN, Panackal AA, et al. Cryptococcal meningitis: epidemiology, immunology, diagnosis and therapy.Nature reviews Neurology2017; 13:13-24. Britz E, Perovic O, von Mollendorf C, et al. The Epidemiology of Meningitis among Adults in a South African Province with a High HIV Prevalence, 2009-2012. PloS one2016; 11:e0163036.

Banerjee U, Gupta K, Chatterjee B, Sethi S. Cryptococcosis at AIIMS.The National medical journal of India1994; 7:51-52.

Banerjee U, Datta K, Majumdar T, Gupta K. Cryptococcosis in India: the awakening of a giant?Medical mycology2001; 39:51-67.

Sharma SR, Hussain M, Habung H. Neurological manifestations of HIV-AIDS at a tertiary care institute in North Eastern India.Neurology India2017; 65:64-68.

AABS; 4(1): 2017 8.

Indira P, Kumar PM, Shalini S, Vaman K. Opportunistic Infections among People Living with HIV (PLHIV) with Diabetes Mellitus (DM) Attending a Tertiary Care Hospital in Coastal City of South India.PloS one2015; 10:e0136280.

Kwon-Drung KJ, Bennette JE, editors, Medical mycology. London: Lea and Febiger, 1992.

10. Chuck SL, Sande MA. Infections with Cryptococcus neoformans in the acquired immunodeficiency syndrome. The New England journal of medicine1989; 32:794-799. 11. Coen M, O’Sullivan M, Bubb WA, Kuchel PW, Sorrell T. Proton nuclear magnetic resonance-based metabonomics for rapid diagnosis of meningitis and ventriculitis.Clinical infectious disease2005; 41:1582-1590. 12. Feldmesser M, Harris C, Reichberg S, Khan S, Casadevall A. Serum Cryptococcal Antigen in Patients with AIDS. Clinical infectious disease1996; 23:827-830. 13. Casadevall A, Mukherjee J, Scharff MD. Monoclonal antibody based ELISAs for cryptococcal polysaccharide. Journal of immunological methods1992; 154:27-35. 14. Sekhon AS, Garg AK, Kaufman L, Kobayashi GS, Hamir Z JM, Moledina N.Evaluation of a commercial enzyme immunoassay for the detection of cryptococcal antigen. Mycoses1993; 36:31-34.

*Corresponding author: Dr. Manodeep Sen, Professor (junior grade), Department of Microbiology, Dr. Ram Manohar Lohia Institute of Medical Sciences (RMLIMS), Vibhuti Khand, Gomti Nagar, Lucknow- 226010 (INDIA), Phone: +91 9839446858 Email: sen_manodeep6@yahoo.com Date of Submission : 10.02.2017 Date of Acceptance : 24.02.2017 Date of Publication : 28.02.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Review Article DOI: 10.21276/AABS.2017.1345

Utility of Interactive Teaching Tools in Classroom Teaching: A Review of literature Dhananjay Shrikant Kotasthane* and Vaishali Dhananjay Kotasthane, Department of Pathology, Mahatma Gandhi Medical College and Research Institute, Pillayarkuppam, Puducherry, India

ABSTRACT Change in medical education is needed to prepare doctors to meet the challenges of changing trends in the health care delivery system.Most of the medical schools have traditional, teacher-centered training.Most of the current undergraduate training is didactic and pedagogical, with the teacher as a source of information, which encourages students for surface learning.The most popular and widespread teaching method is lecturing.In a lecture, learners are passive.It haslowestretention value of all teaching techniques. After reviewing literature, it was noted from various studies that incorporation of various interactive techniques, viz,brainstorming,think pair and share,Multiple choice questions,role play, word puzzles, quiz, group tasks,Problem based learning,case based learning help to achieve learning objectives in a student centric manner..Also, from review of literature it is found that interactivity makes the students more attentive and enthusiastic. Incorporation of interactivity in lectures has been found to improve teaching-learning process by educators who utilized interactivity in classroom teaching. Interactive teaching in medical education is need of hour to be introduce to achieve goals of learning outcomes of undergraduate teaching Keywords: Medical Education, Medical Education Technology, Interactive Teaching Tools.

Introduction

Change in medical education is currently a worldwide phenomenon. It is needed to prepare doctors to fulfill the expectations of society, to cope with the exponential growth of medical and scientific knowledge, to inculcate physiciansâ&#x20AC;&#x2122; ability for lifelong learning, to ensure mastery in information technology and to adjust medical education to changing trends in the health care delivery system. These trends have been introduced in the different health institutes in most of the developed countries.(1) In India, greater efforts are to be taken to orient teachers about these trends to bring desired changes in medical education to produce need-based human resources for health in the region. However, such a shift from traditional approach to a need-based approach requires a fundamental change of the roles and commitments of educators.(1)Most of the medical schools have traditional, teacher-centered and hospital-based training with a few exceptions only.(1,2,3,4) The broad goal of this study was to assess the effect of utilizationof interactive tools in lectures. Self-directed learning involves the learner as an active participant and encourages the development of deep learning. Most of the current undergraduate training is didactic and passive with teacher as a centre for learning and as a source of information. In contrast to this, Learnercentered learning is an active process, where the student learns through his own study.(5) Moreover, the approach

motivates students to adapt to the new knowledge, challenges, and problems he willencounter in future in his professional life. The key features of self-directed learning concord with the principles of adult learningand also with the findings of research in cognitive psychology(6,7,8) Strategies that have been developed as self-directed learning include problem-based learning, interactive learning, discovery learning, task-based learning, experimental and reflective learning, portfolio-based learning; smallgroup, self instructional and project-based learning, peerevaluation and learning contracts.(6) In traditional didactic teaching, the teacher is responsible for learning, teaching is an instructive process, learners are passive, the teacher is an instructor and a lecturer, the learner has only written, taped or broadcast material and the learner receives information.(9) In contrast to this, in collaborative learning,the learner is responsible for learning,teaching/learning is a constructive process,learners are active,the teacher is a facilitator and a counsellor (the teacher acts as a tutor),the learner has a possibility to reach a wide range of information via new educational technology and the learner is a creative person who solves problems and uses informationcollaborative work. (9) Consequently, the typical teaching methods and resources/ activities which take place in the two scenarios also differ.(9)

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In traditional didactic teaching process - resources/ activities include Lectures, Seminars, and Films, audio and video tapes, printed material, Radio broadcasts and TV broadcasts. While incollaborative learning - resources/ activities include Project learning and problem solving, formulating a question/thesis/problem, answering a question or solving a problem,preparing a report/proposal/ plan, designing a prototype/ conducting a project.(9) Wagner defined interactivity as “reciprocal events that require at least two objects and two actions.Interactions occur when these objects and events mutually influence one another”.(10)Interactive teaching is a two-way process of active participant engagement with each other, the facilitator, and the content.(9)When compared to traditional learning, collaborative learning promotes more critical thinking skills such as analysing, evaluating, synthesising and applying information. It also promotes more social skills and a self directive way of learning. Self-direction means that the learner has to take more responsibility of his/her own learning. Collaborative learning methods are becoming more and more common and popular, but are often difficult, time consuming or expensive to put into practice. (9)In Studies that focus on the interactivityand its derivatives, Daniel and Marquis (11), Moore(12), Wagner (13), Markwood and Johnstone(14), Barnard (15), Parker (16) and Blurtoon(17) have found interactive teaching tools to be far superior to traditional didactic methods. Moore offers three types of interactivity. (12, 18, 19) In Moore’s typology we have learner-content, learner-instructor, and learner-learner interactivity. Learner-content interactivity is illustrated by a student reading a book or a printed study guide. Instructorlearner interaction is the core of the teaching process. The

success of the course design will depend largely on whether the conversation between teacher and learner is such that the learner can increase self-direction and construct new knowledge or not. Learner-learner interaction involves students working together to discuss, debate and attempt to solve problems that arise in their study of the course materials. (17). Markwood and Johnstone provide fourth type of interactivity.The fourth type of interaction is one of interaction through electronic exchange, with students electronically or digitally sharing the results of newly formed knowledge over a period of time (14).Anderson and Garrison described the three common types of interaction viz, student-student; student-teacher; student-content as illustrated in Figure 1.(20) Interactive teaching values student’s prior ideas and aims at empowering students to be independent learners. For teachers it offers an opportunity to learn along with the students and to use their interactive skills to listen carefully and challenge misconceptions where possible. To find the time to diagnose what students understand, either individually or as a group, and then to challenge them to enrich their thinking, requires a class management style that allows teachers space to interact.(9) Mathew L Costa et al have found in their study that interactive teaching methods are superior to conventional didactic teaching,(21)There was a consistent suggestion in the literature that shifting the balance of interaction in classrooms towards the dialogic end of the scale would bring improvements to the learning process and consequently to attainment outcomes.(22)

Fig. 1: Types of interaction as described by Anderson and Garrison.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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Review Article

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Interactive teaching methodology is becoming one of the important teaching modalities as against didantic lectures, thanks to recent brainstorming in the field of medical education technology(MET) in developing countries like India. Developed countries are way ahead in this MET and have already successfully implemented their student centric, skilled based curriculum. Accordingly, many medical institutes/universities in India where medical education unit is highly functional, have already started to implement innovations in medical curriculum. In this, interactive teaching is becoming a stepping stone/step forward in student-centric learning process. After reviewing articles from different corners of India for interactive teaching , it was interesting to see that wherever it was introduced ,students’ feedback was very positive and demanded for continuation of this format. Study from D Y Patil deemed university showed perspectives of II MBBS students in Pathology regarding interactive teaching methods such as group discussions, brainstorming session, question answer sessions, multiple choice questions(MCQs),confusion technique and summaries.(23) Their study showed good internal consistency with Crohnbach SQ alpha coefficient of 0.9.They found most popular interactive mode to be MCQs(76%) followed by brainstorming (64%) and confusion technique(53%).End result of this learning outcomes student noted were improved communication skills, retention of topic, improved attention span.Another study from Northern India(Haryana) introduced interactive teaching in the form of case based scenario,think pair and share,quiz and role play.(24)The feedback from students showed encouraging results regarding their perceptions of interactivities with 75.2% of the students strongly agreed that sessions were enjoyable than traditional didactic lectures. Also, it stimulated thinking(78%) regarding the topic. They were also more enthusiastic, motivated to take part in discussion. Similarly, one more study from Amritsar also took feedback in the form of questionnaire on interactive teaching techniques such as small group activity, brainstorming ,think pair and share sessions, demonstrations with the help of audio-visuals , role play, problem-solving, directed listening, case-based examples ,pre and post testing.(25) The result of questionnaire showed that 85% students favored computer aided teaching as most reliable technique,90% found audiovisual aids develops the ability to understand the topic better, 70% responded that Group teaching to be good learning experience, 90% found Role plays and simulation more interactive. Thus, from the findings of our study and the literature reviewed, incorporation of interactivity in classroom teaching definitely improves teaching- learning process. It promotes self directed learning, prolongs retention of the

subject in one’s memory and creates more interest in the subject. Shift from the traditional teacher centered, pedagogic teaching approach to new student centered adult learning approach is the need of the present hour. Introduction of innovative tools like interactivity in class room teaching can help to achieve this goal. In the present study, we have focused on interactivity in lectures as a method of collaborative learning. Possibly the most popular and widespread teaching method is lecturing, in which the teacher gives information and the students listen or take notes. Lecturing is a useful way of imparting a great deal of information quickly, but it is passive for students. Keeping the students’ attention is a major dilemma educators have to face. The best use of lecturing is in combination with other methods; this helps students retain their interest and attention, allows for more student participation, and emphasizes different learning styles. Lecturing delivers “concepts”, delivers a lot of information in a short amount of time andconveys information that is difficult to present in another way. Overuse of lecturing should be avoided because in a lecture your learners are passive, doesn’t guarantee understanding, no feedback from learners, easily bores the audience unless well prepared and has lowest retention value of all teaching techniques. People generally listen for only 15-20 minutes without a break, people learn more when given an opportunity to process what they are learning. People retain more if they review or use the information immediately after learning it. Interactive Teaching involves facilitator and learners, encourage and expect learners to participate, use questions to stimulate discussion, emphasizing the value of answers, give participants hands-on experience, use teaching aids to gain and retain attention.Participants like to be actively involved .Participants want to share knowledge and ideas. Teacher doesn’t have to be an expert and answer all questions, because learners can address questions as well. Judicious incorporation of interactivity in lectures leads to learner –content, learner-learner and learner-student interaction, which makes teaching learning process easier, interesting and fruitful.

References 1.

Majumder A, D’Souza U, Rahman S. Trends in medical education: Challenges and directions for need-based reforms of medical training in South-East Asia. Indian J Med Sci 2004;58:369-80

Majumder MA. Today’s students tomorrow’s physician: Emerging challenges for undergraduate medical education. Bangladesh med J 2003;32:84-7.

Majumder MA. Medical Education in Bangladesh: Past Successes, Future Challenges. Bangladesh Med J 2003; 32:37-9.

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Majumder MA. Issues and priorities of Medical Education Research in Asia. Ann Acad Med Singapore 2004;33:257-63

16. Parker, Angie. Interaction in Distance Education: The critical conversation, Education Technology Review.1999; 13.

Barrows HS, Tamblyn R. Problem-based Learning: An Approach to Medical Education. New York: Springer, 1980.

Spencer JA, Jordan RK. Learner-centered approach in medical education. BMJ 1999; 318:1280-3.

17. Bluurton, C. New Directions of ICT –use in education, UNESCO’s World Communication and Information Report 1999.

Knowles M. The adult learner: a neglected species.4th ed. Houston, TX: Gulf Publishing, 1990.

Regehr G. Norman GR. Issues in cognitive psychology: implications for professional education. Acad Med 1996; 71:988-1001.

Michael Hegarty, Anne Phelan, Lisa Kilbride.Blueprint for Interactive Classrooms.In Classrooms for Distance Teaching and Learning - A Blueprint.Belgium:Leuven University Press,1998. (Site updated: 30 April 2002) available from:http://bic.avnet.kuleuven.be/products/ handbook/handbookchapters/08chapter2.html

10. Wagner, J. Learning from a distance, The International Journal of Multimedia, 1994;19 (2): 12-20. 11. Daniel, J., C. Marquis. Independence and interaction: Getting the mix right, Teaching at a Distance, 1993;15:445-460. 12. Moore, M. Editorial: distance education theory, The American Journal of Distance Education.1991; 5(3): 1-6. 13. Wagner, E.D In support of a functional definition of interaction. American Journal of Distance Education,1994; 8(2): 6 – 26. 14. Mark wood, R. S. John stone. New Pathways to a degree: Technology opens the college, Western Cooperative for Educational Telecommunications, Western Interstate Commission for Higher Education, Boulder, CO.1994 15. Barnard, R. Interactive Learning: A Key to successful distance delivery, The American Journal of Multimedia, 1995;12: 45 – 47.

18. Moore, M. Three types of interaction, The American Journal of Distance Education, 1992;3 (2):1-6. 19. Moore, M. Theory of transactional distance Education.In Desmond Keegan (Ed) Theoretical Principles of Distance Education, London & New York: Routledge. 1993 p:22-38 20. Anderson, T., and Garrison, D. R. Learning in a networked world: New roles and responsibilities. In C. Gibson (Ed.), Distance Learners in Higher Education. Madison, WI: Atwood Publishing.1998.p.97-112 21. Matthew L Costa, Lee Van Rensburg, Neil Rushton.Does teaching style matter? A randomised trial of group discussion versus lectures in orthopaedic undergraduate teaching. Medical Education2007;41(2):214–217 22. Gary Beauchamp,Steve Kennewell. Interactivity in classroom and itsimpact on learning.Computers and education 2010:54(3): 759-766. 23. B u c h A C , C h a n d a n w a l e S S , B a m n i k a r S A . Interactiveteaching:Understanding perspectives of II MBBS students in Pathology.Med. J DY PatilUniv2014;7:693-695 24. Gupta A, BhattiK,WaliaR,AgnihotriP,KaushalS. Implementation of Interactive Teaching Learning Methods in Large Group in Endocrine Pharmacology.Ind J Pharmac and Pharmaco ,October-December 2015;2(4);197-202 25. Kaur D, Singh J, Seema,MahajanA,KaurG.Role of interactive teaching in Medical Education .Inter J Basic and App Med Scie,Sept- Dec 2011;1(1):54-60

*Corresponding author: Dr Dhananjay Shrikant Kotasthane, Professor and HOD Department of Pathology, Mahatma Gandhi Medical College and Research Institute, Pillayarkuppam, Puducherry – 607402, India. Email: dskotasthane@gmail.com Date of Submission : 10.02.2017 Date of Acceptance : 24.02.2017 Date of Publication : 28.02.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Review Article DOI: 10.21276/AABS.2017.1315

Giant Cell Lesion and Central Giant Cell Granuloma of Jaw: A Brief Review Nilesh Kumar1*, Aaditee V. Vande2, Shivsagar Tewary2 and Sameer Anil Zope3 1

Department of Oral & Maxillofacial Surgery, School of Dental Sciences, KIMSDU, Karad, Maharashtra, India. 2 Department of Prosthodontics, School of Dental Sciences, KIMSDU, Karad, Maharashtra, India. 3 Department of Periodontics, School of Dental Sciences, KIMSDU, Karad, Maharashtra, India.

ABSTRACT This review discusses giant cell lesion with main focus on central giant cell granuloma of jaw. Review of the lesion with regard to its nomenclature, pathogenesis and its clinical, radiological & histopathological features is presented. Surgical therapy along with recently reported conservative management protocol is discussed in detail. Keywords: Craniofacial, Oral Pathology, Intraosseous Lesion

Introduction

Giant cell lesion comprises an interesting and controversial group of lesion, characterized by presence of giant cells in their histological section. Giant cells are cells with more than one nucleus. These cells are found in many physiological as well as pathological conditions. It is multinucleated and larger in size in comparison to other body cell. Two main mechanisms have been proposed for the formation of multinucleated giant cell; multinuclear division without cytokines and fusion of multiple cells. The precursor of giant cell remains unknown. However it is believed to be derived from osteoclast and macrophage. They are found in variety of lesions, which range from reactive pathology to neoplasm and from cyst to hormone related disorders. All these lesions are characterized by presence of giant cells histologically (table 1). Though giant cells are found in variety of diseases and conditions, it is not a characteristic or pathognomonic feature of all such lesions. Lesions in which Giant cells are a characteristic histological feature are grouped under giant cell lesion.

Classification of Giant Cell Lesions of Oral Cavity

Significant controversy exists regarding classification of giant cell lesion of maxillofacial region (table 2). Based on its location it can be intraosseous (e.g. central giant cell granuloma) or extraosseous (e.g. peripheral giant cell granuloma). The amount on giant cells is variable in various groups of diseases. Based on propensity in which giant cells are present it can be classified as; true giant cell lesion (lesion with predominant giant cells) which include central giant cell granuloma, peripheral giant cell granuloma, giant cell tumor and giant cell lesion of hyperparathyroidism. Giant cell lesion with moderate number of giant cells

includes cherubism, giant cell granuloma of Pagetâ&#x20AC;&#x2122;s disease and aneurysmal bone cyst. Fibrous dysplasia and pyogenic granuloma have minimal number of giant cells. Clinically it may be benign or malignat. Benign lesion are either non-aggressive (e.g. central giant cell granuloma) or aggressive (aggressive variant of Central giant cell granuloma). Malignant giant cell lesion include metastatic giant cell tumor. Table 1: Variety of lesions having Giant cells: 1. Infections Bacterial (T.B., Leprosy, Syphilis) Viral (Measles, HSV infections, Herpetic Gingivostomatitis) Fungal (Candidiasis, Blastomycosis, Histoplasmosis) Parasitic (Leismaniasis) 2. Fibro-osseous lesions Cherubism Pagetâ&#x20AC;&#x2122;s disease Fibrous dysplasia 3. Reactive lesion Central giant cell granuloma Peripheral giant cell granuloma Periapical granuloma 4. Hormonal lesion Brown tumor of Hyperparathyroidism 5. Cystic lesion Traumatic bone cyst 6. Neoplastic lesions Benign neoplasm (Giant Cell Tumor) Malignant neoplasm (Osteosarcoma, Chondrosarcoma)

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R-23 Table 2: A working classification of giant cell lesion of the oral cavity 1. Lesions where giants cells are pathognomonic i. Central giant cell granuloma ii. Peripheral giant cell granuloma iii. Giant cell tumor iv. Giant cell lesion of Hyperparathyroidism 2. Lesions where giant cells are characteristic i. Cherubuism ii. Giant cell granuloma of pagets disease iii. Aneurysmal Bone Cyst 3. Lesion associated with presence of giant cells i. Orofacial granulomatosis ii. Foreign body reaction iii.Fibrous Dysplasia

Central Giant Cell Granuloma (CGCG)

W.H.O. defines CGCG as an intraosseous lesion consisting of fibrous tissue that contains multiple foci of hemorrhage, aggregation of multinucleated giant cells and occasionally trabeculae of woven bone. Nomenclature: CGCG of jaws in past were grouped under giant cell tumor. Re-evaluation of the lesion suggested that is reparative and is formed in response to injury. Jaffe named this group of lesion as central giant cell reparative granuloma [1]. He believed the lesion was a reparative response to injury rather than neoplasm. General dissatisfaction with the term central giant cell reparative granuloma has been expressed by many authors. Waldron and Shafer pointed out that; the lesions are not a reparative process and comment that ‘it does not seem logical that a destructive, locally invasive and potentially continuously enlarging lesion could represent a reparative’ reaction. Most oral & maxillofacial pathologists have dropped the term ‘reparative’. Today these lesions are designated as ‘central giant cell granuloma’ or by a more non-committal term ‘giant cell lesion’ Etiopathogenesis: The cause of CGCG continues to be a mystery. These lesions do not fit into the concept of either reactive or neoplastic disease, and as a group may exhibit feature of both. Jaffe was the first to separate CGCG from the giant cell tumor. He believed CGCG to be a reactive / reparative lesion arising subsequent to injury. However current experience indicates that giant cell lesion of jaws exhibits a range of activity from that of a reparative granuloma to that of an aggressive neoplasm. How to distinguish between them remains unanswered. Perhaps there is a reactive and a neoplastic form of CGCG (as group exhibit features of both). Alternatively one could speculate that the subset of tumors that behave as a neoplasm develop Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 from a reactive (reparative) lesion though an epigenetic event occurring in the spindle mesenchyme cells of bone. CGCG are composed of a large number of monocyte derived multinucleated giant cell osteoclasts in a stroma of mononuclear spindle shaped mesenchymal cells. While giant cell is a prominent feature of these lesions, it is the mononuclear spindle cells that are the proliferating cells (neoplastic cells) in CGCG. It is believed that spindle cells recruits monocytes and induces them to differentiate into osteoclastic giant cells through the release of cytokines (figure 1). Osteoblast mediated bone resorption and proliferation of spindle cells leads to the growth of CGCG. Clinical Features: CGCG is a rare tumor of jaw and accounts to about 7% of benign jaw tumors. It is exclusively seen in the jaws (not found in extragnathic skeleton). It predominantly occurs in children and young adults (common in 1st to 3rd decade of life). It is more common in females than in males (in ratio of 3-2: 1). Either jaw may be involved, but mandible is more commonly involved than maxilla (60-70% of the reported cases arises in mandible). It is commonly seen in anterior portion of jaw [2]. Most giant cell granulomas of the jaw are asymptomatic and first come to notice during routine radiographic examination or as a result of painless expansion of bone. A minority of cases however may be associated with pain, parasthesia or perforation of the cortical bone plates, occasionally resulting in ulceration of mucosal surface by the underling lesion (figure 2). Displacement of tooth and increased tooth mobility may also be seen [3]. Based on the clinical and radiographical features, several groups of investigators have suggested that CGCG may be divided into two categories; non-aggressive lesion and aggressive lesion. Non-aggressive lesion makes up most cases. It exhibits no or few symptoms (found on routine radiography). It demonstrates slow growth and usually does not show cortical perforation or root resorption of the teeth involved in the lesion. Aggressive lesion is characterized by pain, rapid growth, cortical perforation, root resorption, parasthesia (nerve involvement and perineural spread is rare though). It has marked tendency to recur after removal (as compared to non-aggressive type). Radiographical Features: Radiographically CGCG appear as a radiolucent lesion, which may be unilocular or multilocular [4]. The radiolucency is usually well delineated, but the margins are generally non-corticated. The lesion may vary from small sized incidental finding on radiograph to large destructive lesion (Figure 3). The radiographic findings are not specifically diagnostic. e-ISSN: 2349-6991; p-ISSN: 2455-0396

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Fig. 1: Flow-chart explaining the pathogenesis of CGCG

Fig. 2: Clinical presentation of the lesion as extra-oral swelling (a), intra-oral view showing buccal cortical bone expansion.

Fig. 3: Radiological features of patient with CGCG in figure 2 showing a defined radiolucent lesion of mandible with resorption of involved tooth roots (a), sectional computed tomography images showing expansion and thinning of the cortical bones (b) and three dimensional formatted image of the lesion showing ballooning and resorption of mandible (c,d).

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R-25 Small unilocular lesions may be confused with periapical granuloma or cysts. Multilocular giant cell lesions can’t be distinguished radiographically from ameloblastoma or other multilocular lesions. The multilocular or soap bubble radiolucent lesion appears similar to ameloblastoma, odontogenic myxoma, ameloblastic fibroma, etc. The septa between the loculations are usually fibrous in nature but occasionally may be residual, atrophied and remodeled trabeculae. A thin sclerotic rim is often seen at the periphery, which is typically scalloped. Expansion and thinning of the facial and to a lesser extent the lingual, cortex is common for the larger lesions and may be marked because the central giant cell lesion may achieve a large size over period of time (hyperparathyroidism should be suspected with large lesions). Enlargement is characteristically slow but may be alarmingly rapid in the early stages, especially if associated with infection, trauma (extraction) or hyperparathyroidism. Differential Diagnosis: CGCG presents as swelling which appear as unilocular or multilocular radiolucency of jaw. The various differential diagnoses to be considered include [5,6]: •

•

If the suspected region is in maxillary molar segment of the jaw, a multilobed maxillary sinus must be considered as the differential diagnosis, especially if the pattern is bilateral and the region is asymptomatic. If the lesion is situated in the posterior part of the mandible, in a patient over 30 years of age, it is more likely to be an ameloblastoma, especially if there is an accompanying paresthesia of lips. If the multilocular lesion is situated anteriorily in the jaws of the patient under 30 years of age, it is much more likely to be a giant cell granuloma than ameloblastoma If the patient complains of polydypsia and polyurea or has a history of renal disease along with abnormal serum calcium or phosphorous and alkaline phosphatase levels, the lesion is more likely to be a giant cell lesion of hyperparathyroidism. If the lesion occurs in a child and is multiple, and if there is a family history of such lesion the diagnosis is most certainly cherubism.

Histological Feature: The central giant cell lesion is composed of mesenchymal stroma with moderate cellularity. The lesional cells appear as spindle-shaped or ovoid with moderate amounts of cytoplasm and a moderately large open nucleus. Collagen fibers are seen in variable amounts in the stroma along with multinucleated giant cells having randomly located nuclei (up-to 60 per cell, usually less Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 than 10 per cell) scattered throughout. Focal areas of erythrocyte extravasation scattered throughout is usually seen. It is not unusual to see more blood outside vessels than within vessels. The lesion is described as a blood-soaked sponge with oozing blood. The focal hemorrhage gives the cut surface on gross examination a mottled tan and brown appearance. Hemosiderin deposits may be seen as a sign of old hemorrhage. The stroma has dense collagen in older lesions. Lesions in very young persons (less than 7 years of age) appear biologically active, with numerous mitotic figures. The stroma may also be myxomatous in some lesions or regions of lesions, and at least 10% of cases show reactive new bone formation, located especially toward the periphery of the lesion. Occasional areas of necrosis and healing mimic aneurysmal bone cyst, and some authors have considered these two lesions as simple variations of the same phenomenon [7]. The histological differential diagnosis for central giant cell lesion includes the brown tumor of hyperparathyroidism, aneurysmal bone cyst and cherubism. The lesion is, for all practical purposes, indistinguishable from the brown tumor, but it can be separated from the aneurysmal bone cyst because it lacks the large blood-filled spaces without endothelial lining of the latter lesion. Also, it does not have the connective tissue septae with embedded multinucleated giant cells which are so characteristic of the latter entity. The stroma of cherubism is much loose and less cellular than that of the giant cell lesion. Number of giant cells is considerably less, with often only a few surrounding occasional blood vessels. Extravasation of erythrocytes is not a feature of cherubism. Additionally, the clinical and radiographic presentation of cherubism is usually quite different, with multiple quadrants of involvement. The pathologist must, as usual, evaluate the stroma for signs of dysplasia which might indicate osteosarcoma or fibrosarcoma of bone. Treatment and Prognosis: The traditional treatment of CGCG is surgical removal. However the extent of tissue removed ranges from simple curettage to enbloc resection. Curettage has been supplemented with peripheral ostectomy in many of the reported cases [8]. Treatment of CGCG with cryosurgery and radiation therapy has also been reported in literature. CGCG have also been treated by nonsurgical methods such as systemic dose of calcitonin and intralesional injection of corticosteroids. Surgical Treatment: The surgical treatment of CGCG varies from simple curettage to enbloc resection depending on extent of tissue involvement. Small lesions may be curetted out completely, while large lesions should be biopsied first for histological confirmation of the diagnosis. e-ISSN: 2349-6991; p-ISSN: 2455-0396

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Several sites should be selected for representative sampling of the lesion. As with all central lesions of the jaw, the pathologist should be furnished with clinical and radiographical details. If biopsy report confirms CGCG it can be treated by surgical excision. Local curettage is the conventional treatment of CGCG. However enbloc resection is treatment of choice for locally aggressive CGC (figure 4). The primary treatment of CGCG with no evidence of cortical perforation and when the mucosa appears to be clinically intact is through curettage via an intraoral approach under GA. The surgical steps in management of CGCG are enumerated below: • •

•

• • •

•

The incisions are made at least 1cm away from the radiographic extent of the lesion on either side. A classical buccal mucoperiosteal flap is commonly raised. The thin buccal bone overlying the lesion is removed (a reciprocating saw or micromotor driven bur is used to cut the cortical bone around the lesion). A through curettage is done to remove the entire lesion until clinically healthy appearing bone is encountered. Compartmental or separated lesions are rendered unilocular by excision of intervening bony septa (radiographs act as a guide to ensure that compartments of the lesion well insulated by bone are not overlooked). The lesion usually bleeds excessively. However on its complete removal the bleeding gets easily controlled. The bone cavity may be saucerised by reducing the height of its osseous walls, to facilitate visual and mechanical access. Peripheral ostectomy is performed by trimming the cancellous bone from the walls of bony defect (0.5 bone removed) using surgical burs. In some lesions hemorrhage may be troublesome but can be controlled by small, hot, wet dressings or iodine impregnated gauze pack. Bleeding usually stops after removal of the lesion. The teeth in the area of lesion should be preserved (such teeth should be endodontically treated before surgery). However if adequate bone support to the roots are lacking or recess are created by roots from which lesion can’t be removed, the teeth should be extracted. After through debridment of the bone cavity and attaining hemostasis, the flap is repositioned and sutured. In case of large bony cavity, packing with ribbon gauze soaked in either whitehead varnish or iodoform paste is done (that is retained insitu with loose sutures for 4 to 5 days after surgery, to protect the wound from food and other debris and allow healing by secondary intention).

• •

If possible, pressure dressing should be applied to prevent hematoma formation. The patient is instructed to use frequent hot saline rinse starting the day after surgery to keep the wound free of food debris. All treated patients should be reviewed clinically and radiographically until healing is completed [9].

Medical Therapy: Besides the traditional surgical treatment of CGCG, an alternative medical line of treatment has been widely studied and used recently. The agents used for the treatment of CGCG are; calcitonin, corticosteroid and antiangiogenic drug (interferon). Non-surgical medical modality of treatment of CGCG is usually recommended for: •

Aggressive, large rapidly growing lesions; surgical removal of which will lead to mutilation of face and severe compromise in function, loss of tooth & tooth germs (especially in children and young adults). • In recurrent lesions, especially where the lesion recurs after multiple surgical excision. • When lesion is large and/or is in close proximity to important vital structuresn (medical therapy can be used independently or as an adjunct to surgery to preserve the vital structure and prevent its damage). Calcitonin Therapy: Giant cells in CGCG are osteoclasts. Indirect evidence of this was provided by the behavior of the giant cells in the cortical bone, showing bone excavation typical of osteoclasts. It has been shown that giant cells express calcitonin receptors. Calcitonin binds on these receptors and inhibits the osteoclastic activity of giant cells (figure 5). Calcitonin therapy for management of CGCG is base on these above findings. CGCG develop from mononuclear precursor cells, and as such are part of granulocyte/macrophage lineage or are of primarily fibroblastic origin. In this case the effect of calcitonin might be explained by the fact that calcitonin receptors are found on mononuclear cells as well. Calcitonin induces involution and inhibition of DNA synthesis in these cells. Two forms of calcitonin are available for therapeutic use; salmon calcitonin and human calcitonin. Human calcitonin is preferred over salmon calcitonin as it has lower antigenic properties than animal calcitonin. Routes of administration include subcutaneous injection and nasal spray. After subcutaneous injection peak in plasma is reached in an hour, with half life of 70-90 minutes. Dosage for subcutaneous route is 100 IU per day, whereas nasal Spray measuring 200 IU is administered per day for 12-24 months [10]. Therapy is continued until there is no further resolution of the lesion radiographically (resolution usually takes 12-24 months).

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Fig. 4: Lesion involving the right parasymphis region of mandible (a), intraoperative view showing exposure of the pathology (b), enbloc resection of the lesion (c,d).

Fig. 5: Proposed mechanism of action of calcitonin.

Drawbacks of calcitonin therapy include the long duration of treatment. Recurrence after therapy is stopped have been reported if few cases. Side effects although usually minor and infrequent include; flushes, headache, diarrhea and nausea. Calcitonin as a therapy for CGCG of the jaws is a promising alternative to surgical curettage, particularly for large lesions [11]. Further investigations into dosage and duration of treatment are necessary before an optimal treatment protocol can be suggested.

decrease in secreted levels of these enzymes (figure 6). In addition it has been experimentally shown that steroids induce apoptosis of osteoclasts. On basis of the above evidence, it is possible to hypothesize that the result obtained by using intralesional steroids in treatment of CGCG of the jaw bones may be due to; inhibition of extracellular production of lysosomal protease and steroidal apoptotic action on osteoclasts like cells [12]. Both the mechanism causes cessation of bone resorption in CGCG.

Corticosteroid Therapy: Giant cell osteoclasts in CGCG accomplish bone resorption by secreting lysosomal proteases (e.g. cathepsin, gluccoronidsae). These proteases mediate osteoclastic bone resorption by creating an acidic extracellular medium. Steroids produce a dose dependent

Treatment protocol of corticosteroid therapy includes:

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Pretreatment biopsy is done to establish the diagnosis. Laboratory investigations of parathyroid hormone (PTH), calcium, and phosphorus are done to rule out hyperparathyroidism. An Intralesional injection is prepared e-ISSN: 2349-6991; p-ISSN: 2455-0396

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of 50% mixture by volume of traimcinolone acetate 10 mg/ mL with 2% lidocaine. The solution is administered with a disposable syringe with 22G needle. The average dosage of the solution is 6 mL for adults (equivalent to 30 mg of triamcinolone) and 5 mL for the pediatric patients (equivalent to 25 mg of triamcinolone) [13]. The solution is injected by clinically estimating the site where the cortical bone is more expanded and therefore, it is thinnest at that point. The bone is not trephinated. Once inside the lesion, the needle is redirected to inject small amounts into different areas. The treatment is terminated when there is a significant amount of resistance caused by the bone being formed and calcified, thus avoiding the need for trephination. Steroid therapy should not be used in patients with diabetes mellitus, peptic ulcer, infections and in immunecompromised patients [14]. Atiangiogenic Therapy (Using Interferon): Interferon (INF) is a cytokine with antiviral and anti-angiogenic properties. INF is either produced by recombinant DNA technology or it is purified from cultured human cells. Interferon alpha was the first angiogenesis inhibitor used. It is used in treatment of life threatening haemangiomas and other vascular tumors of various organs. The use of INF-alpha in CGCG is based on the hypothesis that CGCGâ&#x20AC;&#x2122;s has proliferative vascular component. Though CGCGâ&#x20AC;&#x2122;s are not true vascular lesion, it has a vascular

component to it. In various published cases reports INF has been successfully used in combination with surgery for treatment of aggressive CGCG [15]. INF-alpha used as a monotherapy for aggressive CGCG was capable of terminating the rapid growth of the lesion. But only partial remission of the lesion is achieved in all reported cases, which stopped after few months [16,17]. The reason that total remission canâ&#x20AC;&#x2122;t be achieved is probably due to the fact that the proliferating tumor cells are of fibroblastic origin (mononuclear spindle cells) and not endothelial cells. INF has no direct inhibiting effect upon the proliferating tumor cells. INF therefore seems capable of terminating the rapid growth and even causes regression, probably by inhibiting neo-angiogenesis in the lesion [18]. Dosage of interferon therapy in CGCG is 3,000,000 units/m2 administered once per day by subcutaneous injection. Possible side effects of therapy include fever, flu like symptoms, lethargy, postnasal drip, skin rashes and hair loss [19].

Conclusion

Central giant cell granulomas are rare and a variable group of disorder. Its presentation varies from benign intraosseous pathology discovered on routine radiographs to more aggressive lesion presenting with large swelling, involvement of large area of jaw, tooth displacement along with involvement of surrounding vital structures. Knowledge of its pathogenesis and presenting features helps to effectively diagnose the condition. Conventional surgical intervention as well as more recently reported medical therapies has been successfully used for management of this pathology.

Fig. 6: Proposed mechanism of action of corticosteroid.

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References 1.

Edwards PC, Fox J, Fantasia JE, Goldberg J, Kelsch RD. Bilateral central giant cell granulomas of the mandible in an 8-year-old girl with Noonan syndrome. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:334-40

De Lange J, Van den Akker HP. Clinical and radiological features of central giant-cell lesions of the jaw. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:464-70

Kruse-Lรถsler B, Diallo R, Gaertner C, Mischke KL, Joos U, Kleinheinz J. Central giant cell granuloma of the jaws: A clinical, radiologic, and histopathologic study of 26 cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:346-54

Ciorba A, Altissimi G, Giansanti M. Giant cell granuloma of the maxilla: case report. Acta otorhinolaryngol Ital 24, 2629, 2004

De Lange J, Van den Akker HP. Radiologic features of central giant cell granuloma of the jaw. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;81:720-6

Sidhu MS, Parkash H, Sidhu SS. Central giant cell granuloma of jaws-review of 19 cases. Br J Oral Maxillofac Surg. 1995 Feb;33(1):43-6.

Stavropoulos F, Katz J.Central giant cell granulomas: A systematic review of the radiographic characteristics with the addition of 20 new cases. Dentomaxillofac Radiol. 2002 Jul;31(4):213-7.

Bataineh AB, Al-Khateeb T, Rawashdeh MA. The Surgical Treatment of Central Giant Cell Granuloma of the Mandible. J Oral Maxillofac Surg. 2002 Jul;60(7):756-61. Rawashdeh MA, Bataineh AB, Al-Khateeb T. Long-term clinical and radiological outcomes of Surgical management of central giant cell granuloma of the maxilla. Int J Oral Maxillofac Surg. 2006 Jan;35(1):60-6.

10. Pogrel MA. Calcitonin Therapy for Central Giant Cell Granuloma. J Oral Maxillofac Surg 61:649-653, 2003.

AABS; 4(1): 2017 11. de Lange J, Rosenberg AJ, van den Akker HP, Koole R, Wirds JJ, van den Berg H. Treatment of central giant cell granuloma of the jaw with calcitonin. Int. J Oral Maxillofac. Surg. 1999; 28:372-376. 12. Kurtz M, Mesa M, Alberto P. Treatment of a central giant cell lesion of the mandible with intralesional glucocorticosteroids. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001; 91:636-7. 13. Carlos R, Sedano HO. Intralesional corticosteroids as an alternative treatment for central giant cell granuloma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002; 93:161-6. 14. Adornato M., Paticoff K.A. Intralesional corticosteroid injection for treatment of central giant-cell granuloma. J Am Dent Assoc. 2001 Feb; 132(2):186-90. 15. de Lange J, van den Akker HP, van den Berg H, Richel DJ, Gortzak RA. Limited regression of central giant cell granuloma by interferon alpha after failed calcitonin therapy: a report of 2 case. Int J Oral Maxillofac Surg. 2006 Sep;35(9):865-9. 16. Kaban LB, Troulis MJ, Ebb D, August M, Hornicek FJ, Dodson TB. Antiangiogenic Therapy with Interferon Alpha for Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg. 2002 Oct;60(10):1103-11; 17. Kaban LB, Mulliken JB, Ezekowitz RA, Ebb D, Smith PS, Folkman J. Antiangiogenic Therapy of a Recurrent Giant Cell Tumor of the Mandible with Interferon Alfa-2a. Pediatrics. 1999 Jun;103(6 Pt 1):1145-9 18. Kaban LB, Troulis MJ, Ebb D, August M, Hornicek FJ, Dodson TB. Antiangiogenic therapy with interferon alpha for giant cell lesions of the jaws. J Oral Maxillofac Surg. 2002 Oct;60(10):1103-11 19. Goldman KE1, Marshall MK, Alessandrini E, Bernstein ML. Complications of alpha-interferon therapy for aggressive central giant cell lesion of the maxilla. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005 Sep;100(3):285-91.

*Corresponding author: Dr. Kumar Nilesh, Dept. of Oral & Maxillofacial Surgery, School of Dental Sciences, Krishna Hospital, Karad, Satara , Maharashtra 415 110 INDIA Phone: +91 9158542384 Email: drkumarnilesh@yahoo.com Date of Submission : 05.02.2017 Date of Acceptance : 02.03.2017 Financial or other Competing Interests: None. Date of Publication : 11.03.2017

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Review Article DOI: 10.21276/AABS.2017.1402

Clusterin: It’s Implication in Health and Diseases Sheikh Ishaq, Harnam Kaur2* and Sonam Bhatia2 1

Department of Biochemistry, Government Medical College, Srinagar, Jammu and Kashmir. India 2 Department of Biochemistry, ESIC Medical College, Faridabad, Haryana, India

ABSTRACT Clusterin (CLU) is a glycoprotein with a nearly ubiquitous tissue distribution that has been reported to be implicated in several physiological processes as well as in many pathological conditions including ageing, diabetes, atherosclerosis, degenerative diseases and tumorigenesis including tumors of prostate, colon, and breast. Two distinct CLU mRNA isoforms,the conventional secreted form of CLU (sCLU) is thought to be a component of high density lipoprotein-cholesterol. sCLU functions as a chaperone for misfolded proteins and it is thought to promote survival by reducing oxidative stress. Nuclear CLU, formed by alternative splicing, is responsible for promoting apoptosis via a Bax-dependent pathway. This review will summarizes our understanding of the importance of CLU in various physiological functions and speculate on its role in disease. Keywords: Clusterin, Cell Interactions, Complement, Injury, Lipoprotein

Introduction

Apolipoprotein-J (ApoJ), also known as Clusterin (CLU), is 80 k Daglycoprotein with a nearly ubiquitous tissue distribution. It has been implicated in such diverse processes as sperm maturation, regulation of complement activation, programmed cell death, tissue remodeling and lipid transport. Expression of ApoJ is upregulated in acute myocardial infarction, atherosclerosis, myocarditis, oxidative stress, inflammation and after injury in general. [1]Blaschuk et al. in 1983 identified a high-molecular weight protein in ram rete testis fluid and named CLU for its ability to cluster sertoli cells [2]. Additional analyses indicated that this unknown protein was capable of eliciting the “Clustering” of Sertoli cells, mouse testis TM-4 cells, and erythrocytes resulting in the name CLU. In 1984, Griswold and colleagues purified a dimeric acidic glycoprotein from the Sertoli cells of rat testes. [3] In humans, it was firstly purified from serum and the cloned gene was named CLI (complement cytolysis inhibitor) [4] or ApoJ [5] due to similarities with other known apolipoproteins.CLU consists of two polypeptide chains connected by four to five disulfide bonds [6]. The protein is one of the most prominent extracellular chaperones. The chaperone activity of CLU has been intensively studied [7, 8]. With regard to its chaperone activity, CLU is designated as a protein that allows clearing of cellular debris and misfolded proteins, as well as the clearance of Aβ via the blood-brain barrier [9]. The resolute action of chaperone activity, scavenging- and clearance-function, may be the basis for the tissue defensive role of this protein [10]

The Human CLU gene

The CLU gene is a single 9-exon expressed at dissimilar levels in virtually all tissues in mammals. CLU gene is

located on chromosome 8p21 proximal to the lipoprotein lipase gene locus [11].CLU gene is organized into nine exons, ranging in size from 47 bp (exon I) to 412 bp (exon V) spanning a region of 16580 bp [12]. In humans, at least two distinct CLU mRNA isoforms that differ in their unique exon I exist as a result of alternative transcriptional initiation start sites, (Isoform 1, NM_001831.2 and Isoform 2, NM_203339.1).These two transcripts are probably originated from two transcriptional initiation start sites and only produced in humans and chimpanzees. Both isoforms contain 9 exons (and 8 introns) and a terminal 5’-untranslated region [13].The mRNA isoform 1 is a major form of CLU mRNA, whereas other forms comprising mRNA isoform 2 account for <1% of total CLU mRNA [14]. This protein is then directed to the lumen of the endoplasmic reticulum by a leader signaling sequence and further undergoes an extensive N-linked glycosylation process, where it becomes a 75-80kDa mature precursor after transportation from the endoplasmic reticulum (ER) to the Golgi apparatus [15].Six N-linked Glycosylation sites of human CLU were identified by Kapron, three on the alpha chain (α 64N, α 81N, α 123N) and three on the beta chain (β 64N, β 127N, β 147N) [16]. The mature 75-80kDa protein undergoes intracellular cleavage between amino acid residue Arg205 - Ser206 to form alpha (α) and beta (β) subunits, which are linked together by five disulfide bonds upon extracellular secretion (sCLU) [17]. Under reduced conditions, the subunits of CLU can be detected with the approximate size of 35-40kDa [18]. But, when cells are subjected to stress, post-translational modifications, such as glycosylation and internal proteolytic cleavage could be blocked [15]. Failure of protein cleavage leads to the

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R-31 retrograde transport of uncleaved and non-glycosylated (55-60kDa) form of CLU from the Golgi back to the ER. Thus, the secretion of CLU is reduced but not eliminated and intracellular nonglycosylated precursor CLU (pCLU) increased. Another form of CLU is found in the nucleus and is known to be induced after stress, such as ionizing radiation (IR) [15,19]. Many theories for the synthesis of nuclear CLU (nCLU) have been proposed. One theory postulates that CLU is generated from an alternative splicing event, which results in an N-terminal truncated mRNA lacking exon 2, where the ER reader peptide sequence and the first AUG codon reside [19]. This alternative 47 transcript is translated into a precursor protein (pnCLU), which is not cleaved into α or β subunits. Upon cell damage, pnCLU undergoes post-translational modification to form a mature ~55-kDa protein, exposing both of its nuclear localization signals (NLSs), and migrates to the nucleus to induce apoptosis [19].

The functions of CLU

The biological roles of CLU has proved challenging due to two main properties. Firstly its ability to interact with a variety of molecules, including itself, amyloid proteins, lipids, immunoglobulins and components of the membrane attack complex. Secondly, its upregulation during many biological and pathological states. These include developmental remodeling, epithelial cell differentiation, in response to injury and other stresses, as well as in apoptotic disease states.

Clusterin: chaperone activity

CLU, a ubiquitous and highly conserved secreted protein, is an efficient extracellular chaperones (20) CLU potentially inhibits stress-induced protein aggregation by ATP-independent binding to non-native proteins to form soluble, high molecular weight complexes. CLU binds to hydrophobic ligands of misfolded proteins, it was suggested that its interaction with these ligands is as a result of its chaperone properties (21, 22). There is a highly conserved 14-bp element, the heat shock element (HSE), in the CLU proximal promoter in vertebrates which is capable of binding the transcription factor heat shock factor 1 (HSF1) and that then induces CLU expression following heat shock in transient expression assays in cell culture. The conserved HSE motif alone was shown to be capable of driving reporter gene expression in response to heat shock (23).

AABS; 4(1): 2017 showed CLU as a part of the membrane attack complex and has a complement inhibitory role (25,26). Membrane attack complex causes cell lysis by transmembrane channel formation. It is formed on the surface of pathogens as a result of activation of complement system cascade (C1C9). Based on CLU association with the MAC in some experiments and human diseases, and its ability to inhibit complement mediated cytolysis in vitro, an important role for CLU as a complement regulator.

Clusterin function in lipid transport

As an apolipoprotein, CLU is found in a subset of dense HDL particles containing apoA-I and paraoxonase[27]In plasma, CLU forms HDL particles with apoA-I and apoE and may play an important role in reverse cholesterol transport from peripheral tissues to the liver,[28] CLU can promote cholesterol and phospholipid export from macrophage-foam cells which constitute the hallmark cell type of atherosclerotic lesions.[29]The protective effect of sCLU and its peptides is possibly due to the property of CLU in removing toxic and harmful substances to prevent an immune reaction and the subsequent atherosclerotic changes of the vessel wall (30).

Clusterin - cell interactions

CLU is capable of inducing cell aggregation of a variety of cell types hence the name. CLU is considered an adhesion molecule that promotes cell-cell and cell-substratum interactions. CLU may promote these contacts during development when critical cell interactions are taking place, and also during tissue damage hence maintains tissue integrity [31]

Clusterin in neurodegenerative disorders

CLU protein was initially identified in glomerular immune complexes in a pattern similar to other terminal complement components [24]. Several other reports subsequently

Genome-wide association studies provide evidence of genetic variation in the CLU gene, CLU in susceptibility of Alzheimer’s disease (AD). Its strongest association was found for the common single nucleotide polymorphism (SNP) rs11136000 located in a non-coding, intronic CLU region[32]. CLU expression is highest in the brain and is markedly up regulated under situations of stress and inflammation .This gives rise to glycosylated heterodimeric protein that is constitutively secreted and referred to as soluble clusterin (sCLU), when found in association with lipoproteins which by far we already know are shorter forms of the precursor CLU have been detected intracellularly and named cytosolic, truncated or nuclear CLU [33-35].The function of intracellular CLU (iCLU) is not completely understood. Of relevance to AD, it has been recently shown that iCLU levels increase quickly in cultured primary neurons exposed to amyloid-b peptides (Ab), and that this iCLU elevation is required for the neurotoxic downstream signaling effects of Ab[36].

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sCLU influences Alzheimer’s disease (AD) pathogenesis; its levels are found to be raised in AD-affected brain regions[37]. As shown by Western blot and ELISA increased levels of CLUCLare seen in hippocampus, frontal cortex, in cerebrospinal fluid and plasma of AD patients. While there are other studies that demonstrate levels of CLU did not differ significantly between control and AD cases in the frontal cortex, temporal cortex, or thalamus in postmortem human brain[38,39]. On other hand certain other studies are suggestive of elevated plasma CLU levels and associationwith disease prevalence and severity of AD and with increased amyloid deposition and brain atrophy. Also experimental findings suggest that CLU increases both in amyloid-β (Aβ) aggregation and clearance, raising the question of whether elevated CLU levels are beneficial or harmful[40]. The explanation for these differences is unclear;the challenge of understanding the role of CLU in AD goes beyond quantification of protein levels partially that can be explained by alternative splicing variants, single nucleotide polymorphisms, and post-translational modifications[41], which are difficult to ascertain with immunological methods and likely associated with development of AD is needed to facilitate clarification of the role of CLU in AD.

observed to promote angiogenesis and chemo resistance. Other pathways CLU participates in to downplay apoptosis in tumor cells include the PI3K/AKT/mTOR pathway and NF-κB pathway.As evident by its varied key roles in cancer development, CLU can serve as a therapeutic target for fighting tumor growth and chemoresistance[49].

Conclusion

CLU has been implicated as a significant risk factor for the development of numerous diseases, however, an extensive gap exists in the literature in understanding functions of CLU. As CLU isoforms appear to mediate various physiological processes, the tendency to focus on the effects of CLU as a singular protein could lead to conflicting reports in the literature that are currently unresolved. Therefore, before researchers can fully ascertain the therapeutic potential of CLU from a clinical perspective, it is imperative that key deficiencies are addressed at the genetic level.

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Fritz IB, Burdzy K, Setchell B, Blaschuk O. Ram rete testis fluid contains a protein (CLU) which influences cell-cell interactions in vitro. Biology of Reproduction 1983; 28(5): 1173-88.

Sylvester SR, Skinner MK, Griswold MD. A Sulfated glycoprotein synthesized by sertoli cells and by epididymal cells is a component of the sperm membrane. Biology of Reproduction 1984; 31(5): 1087-101.

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Role of clusterin in cancers

CLU, a multifunctional protein have been found to be involved in a number of biological processes like complement cascade, lipid transport, membrane recycling, cell adhesion and programmed cell death[42]. In humans, two proteins: secretory CLU protein (sCLU, secretory CLU) (75-80 kDa) and nuclear CLU protein (nCLU, nuclear clusterin) (55 kDa) encoded by gene isoform 1 ORF [isoform 1, NM_001831, mRNA encodes the functional protein and isoforms 2 , NR_038335, non-coding RNA; and isoform 3, NR_045494, non-coding RNA[43]are found on chromosome 8p21-p12[44,45].It has been found that sCLU enhances tumorigenesis by facilitating binding between Klu70 and apoptotic protein Bax consequently, preventing BAX from localizing to the outer mitochondrial membrane and to stimulate cell death because of its proapoptotic activity[46]. Another mechanism involved in sCLU prosurvival activity is the upregulation of the phosphatidylinositol 3- kinase (PI3K)/protein kinase B (AKT) pathway and insulin-like growth factor (IGF)1 activating PI3K/AKT pathway through upregulation of sCLU[47], sCLU has other actions related to tumor behavior. For example, in clear cell renal carcinoma, sCLU regulates tumor cell migration, invasion and metastasis by modulating extracellular signal-regulated kinase (ERK)1/2 signaling and matrix metallopeptidase (MMP)-9 expression[48].In epithelial ovarian cancer, CLU has been

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AABS; 4(1): 2017 9 complex of complement and in the immune deposits in glomerulonephritis.” Journal of Clinical Investigation 1988;81(6): 1858-1864. 25. Jenne, D. E. and J. Tschopp Molecular-structure and functional-characterization of a human-complement cytolysis inhibitor found in blood and seminal plasma identity to sulfated glycoprotein-2, a constituent of rat testis fluid. Proc Nat Acad of Sciences of the United States of America 1989;86(18): 7123-7127. 26. Kirszbaum L, J A Sharpe, et al. Molecular-cloning and characterization of the novel, human complement-associated protein, sp-40,40 - a link between the complement and reproductive systems.” Embo Journal 1989;8(3): 711-718. 27. Baralla A, Sotgiu E, Deiana M, Pasella S, Pinna S, Mannu A, et al. Plasma clusterin and lipid profile: A link with aging and cardiovascular diseases in a population with a consistent number of centenarians. PLoS One. 2015;10:e0128029. 28. Bettuzzi S. Conclusions and perspectives. Adv Cancer Res. 2009;105:133–50. 29. Gelissen IC, Hochgrebe T, Wilson MR, Easterbrook-Smith SB, Jessup W, Dean RT, et al. Apolipoprotein J (clusterin) induces cholesterol export from macrophage-foam cells: A potential anti-atherogenic function? Biochem J. 1998;331(Pt 1):231–7. 30. Klock, G., M. Baiersdorfer, et al. (2009). Cell Protective Functions of Secretory Clusterin (sCLU). Advances in Cancer Research, 2009;104:S. 104: 115. 31. Rosenberg, M. E. and J. Silkensen. Clusterin - physiological and pathophysiologic considerations.” International Journal of Biochemistry & Cell Biology 1995; 27(7): 633-645. 32. Lambert JC, Heath S, Even G, Campion D, Sleegers K, Hiltunen M, et al.Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer’s disease. Nat Genet. 2009;41:1094–9. 33. Nizard P, Tetley S, Le Dre´an Y, Watrin T, Le Goff P, et al. Stressinduced retrotranslocation of clusterin/ApoJ into the cytosol. Traffic 2007;8: 554–565. 34. Bettuzzi S, Rizzi F. Nuclear CLU (nCLU) and the fate of the cell. Adv Cancer Res 2009;104: 59–88. 35. Prochnow H, Gollan R, Rohne P, Hassemer M, Koch-Brandt C, et al. (2013) Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-kB signaling pathway. PLoS One 2013;8: e75303. 36. Killick R, Ribe EM, Al-Shawi R, Malik B, Hooper C, et al. Clusterin regulates b-amyloid toxicity via Dickkopf1-driven induction of the wnt-PCPJNK pathway. Mol Psychiatry 2014; 19: 88–98. 37. Rahul S. Desikan, Wesley K. Thompson, Dominic Holland,The role of clusterin in amyloid-β associated neurodegeneration JAMA Neurol 2014; 71(2): 180–187

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39. Baig S, Palmer LE, Owen MJ, Williams J, Kehoe PG, Love S: Clusterin mRNA and protein in Alzheimer’s disease. J Alzheimers Dis 2012; 28:337–344. 40. Yu JT, Tan L. The role of clusterin in Alzheimer’s disease: pathways, pathogenesis, and therapy.Mol Neurobiol. 2012; 45(2):314–26. 41. Zhou W, Ross MM, Tessitore A, Ornstein D, Vanmeter A, Liotta LA, Petricoin EF 3rd: An initial characterization of the serum phosphoproteome. J Proteome Res 2009; 8:5523–5531 42. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin 2015; 65: 87-108 [PMID: 25651787 DOI: 10.3322/ caac.21262] 43. Wong P, Borst DE, Farber D, et al. Increased TRPM-2/ clusterin mRNA levels during the time of retinal degeneration in mouse models of retinitis pigmentosa. Biochem Cell Biol 1994; 72: 439-446 [PMID:7605616] 44. Zhang Q, Zhou W, Kundu S, et al. The leader sequence triggers and enhances several functions of clusterin and is instrumental in the progression of human prostate cancer in vivo and in vitro. BJU Int 2006; 98: 452-460

45. Leskov KS, Araki S, Lavik JP, et al. CRM1 proteinmediated regulation of nuclear clusterin (nCLU), an ionizing radiationstimulated, Bax-dependent pro-death factor. J Biol Chem 2011; 286: 40083-40090 46. Sintich SM, Steinberg J, Kozlowski JM, Lee C, Pruden S, Sayeed S, Sensibar JA. Cytotoxic sensitivity to tumor necrosis factor-alpha in PC3 and LNCaP prostatic cancer cells is regulated by extracellular levels of SGP-2 (clusterin). Prostate 1999; 39: 87-93 [PMID: 10221563] 47. Gleave M, Miyake H. Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer. World J Urol 2005; 23: 38-46 48. Trougakos IP, So A, Jansen B, Gleave ME, Gonos ES. Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic andoxidative stress. Cancer Res 2004; 64: 1834-1842 [PMID: 14996747] 49. Nakouzi Al N , Wang CK , Beraldi E , et al Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis. EMBO Molecular Medicine 2016; 8(7):761-768.

*Corresponding author: Dr Harnam Kaur, Professor & Head Department of Biochemistry, ESIC Medical College, Faridabad, Haryana, India121001 Phone: +91 9910196404 Email: drharnam@gmail.com Date of Submission : 10.03.2017 Date of Acceptance : 16.03.2017 Date of Publication : 21.03.2017 Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/aabs.2017.1248

Association between the Bone Turnover Markers and The Gynecologic Factors Among Moroccan Women Basma Nejjar1,2*, Abdellatif Bour2, Anis Sfendla3, Adil Najdi4, Olivier Bruyère5 and Etienne Cavalier1 Department of Clinical Chemistry, Unilab Lg-CIRM, Faculty of Medicine, University of Liège, CHU Sart-Tilman, 4000 Liege, Belgium 2 Department of Biology, Team of Food and Nutritional Transition (ETAN), Laboratory of Biological assays, Faculty of Sciences, University Ibn Tofail, 14000 Kenitra, Morocco 3 Department of Biology, Faculty of Sciences, Abdelmalek Essaadi University, 93000 Tetouan, Morocco 4 School of Medicine of Tangier, Morocco and Laboratory of Epidemiology, Clinical Research and Community Health, School of Medicine of Fez, 3000 Fez Morocco 5 Department of Public Health, Epidemiology and Health Economics, Faculty of Medicine, University of Liege, CHU Sart-Tilman, 4000 Liege, Belgium 1

ABSTRACT Background: The action of bone cells on the bone mineral density can be highlighted from their enzymatic activities, by the bone alkaline phosphates (BAP) and the Cross Laps-telopeptide terminal of the collagen type 1 (β-CTX). They are reliable to explore the activity of bone turnover. We aim to put in perspective the association between gynecological factors and the concentration of bone turnover markers (BTMs) among Moroccan women. Methods: This is a cross-sectional study of 125 women from February to April 2015. A randomized sample was made. The biochemical parameters of bone metabolism concerned serum calcium, 25 hydroxyvitamin D (25OHD), parathyroid hormone (PTH), phosphorus , BAP and β-CTX. All data related to gynecologic, sociodemographic and anamnesis parameters was collected by standardized questionnaire. Result: Among 125 women, 64(51.42) were menopausal, the mean BAP was 22.1±7.6 ng/L, the median of the interval between successive births “IBSB” and of β-CTX were 36.0[24.0-58.0] months, 368.5 [237.0-504.8]ng/L. A positive correlation was observed between the β-CTX and IBSB (r=0.2,p=0.05). The BAP concentration was higher in menopausal women (p<0.001). The linear regression showed that 12% of BAP concentration was associated to the menopause (R2=0.12, p=0.001) but no associated factors was founded with concentration of β-CTX. Conclusion: Our study shows that the menopause is associated factor with concentration of BAP and IBSB is correlated with the concentration of β-CTX. Keywords: Bone Turnover, Markers, Gynecologic, Women, Morocco.

Introduction

The bone remodeling is a complex physiological process during which the osteoclasts, cells of bone resorption, and the osteoblasts, cells of bone formation, act in sequential and coupled frequencies to destruct and renew the bone matrix. The objective of this physiological process is to lead an adequate bone mineral density (BMD) depending on the age and the sex (1). This bone remodeling process is under the action of several factors such as biological mediators, cited the vitamin D and the calcium, also under the action of sexual hormones and under the action of some external parameters related to the lifestyle of the person (1, 2). The action of these cells on the bone can be highlighted from their enzymatic activities by the biochemical markers of bone turnover: Bone Alkaline Phosphatase (BAP) and

C-telopeptide Terminal Collagen type 1 (β-CTX). The BAP is an enzyme of the bone formation specific to the osteoblasts whereas the β-CTX is an enzyme of bone resorption specific to the osteoclasts (2). The use of these enzymes are reliable to their non invasive exploration of bone turnover (3), they allow in association with the bone densitometry to estimate the loss of bone and to monitor the response of therapeutic effect of several bone diseases (4). The trend to establish a link between markers and BMD was also subject to research among Moroccan researchers (5, 6). Yet, to our knowledge, no study so far in Morocco has been undertaken to study the associated factors with the concentration of biochemical of bone turnover markers (BTMs). Related to this background, we conducted this present study in Meknes region of Morocco, with the aim to put into

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perspective the association between gynecological factors and the concentration of BMTs among women in the general population.

are serum calcium, phosphorus, 25 hydroxyvitamin D (25OHD), parathyroid hormone (PTH), β-CTX and BAP.

Materials and Methods

The biological analysis of calcium serum levels (reference range: 2.1- 2.6 mm) and phosphorus (reference range: 0.74 to 1.51 mm) were carried out by Cobas 8000. The biological analysis of 25OHD (reference range of >20 ng/ ml), the BAP (reference range: 6.1-22.2 µg/L), the PTHi (reference range: 4 to 26ng/L) and of the β-CTX (reference range <700ng/L) were carried out by CLIA.

This research included Caucasian women, aged 18 years old and older, not presenting any diseases affecting the bone metabolism, such as thyroid and parathyroid disease, kidney and liver disease, chronic intestine inflammatory and tumors.

The quantification method of 25OHD recognizes 25 (OH) D2 and D3. The threshold of hypovitaminosis D was fixed at 20 ng/ml as recommended by the American Institute of Medicine (10).

Subjects: The present cross-sectional study was conducted from February to April 2015 among 125 women of Meknes. The participants were recruited from a variety of sources, including workers in private and public companies, patients consulting at ambulatory medicine, medical staff of a laboratory and from “word of mouth “.

Our institutional committee approved this study. The ethical procedures of study were in accordance with the Declaration of Helsinki. The 125 women recruited gave their oral consent to participate in this investigation and were formed in advance about its objective and conditions of study. Any subject didn’t want to be included or didn’t want to continue answering could leave the study without any effect on her treatment process. The investigation was conducted in the medical laboratory, each participant came to the laboratory to have a fasting blood test and to complete questionnaire. The standardized questionnaire was used to collect the data and was divided in three sections related to sociodemographic, anamnesis, anthropometric parameters, gynecological parameters and biological parameters. Sociodemographic, Anamnesis, Anthropometric Parameters: The first component of the questionnaire concerned sociodemographic variables e.g. date of birth, socio-professional status, marital status and level of instruction. The anamnesis variables “ personal and family history of fractures, the feeling of diffuse pain, defines as unpleasant sensory at the level of the bones and muscles (7)” and the anthropometric parameters “weight and the waist-hip ratio “WHR” calculated by dividing the waist by hip in meter”. Gynecologic Parameters : The second component related to the gynecological characteristics including the age at menarche, parity, interval between successive births “IBSB” defined as the time separated two successive births (8), and the menopause defines as the ending of menstrual periods in a natural or surgical way due to the stop production of ovarian hormones(9). Biological Parameters: The third component referred to the biochemical parameters of bone metabolism which

Statistical Analysis: The normality of the variables has been tested by Shapiro-Wilk. The Gaussian variables were reported as means with standard deviation (SD), the no Gaussian ones as median with interquartile ranges [IQR]. The categorical variables were expressed as absolute (n) and relative frequencies (%). The Spearman coefficient was used to determinate the correlation. Student’s t test and Mann-Whitney U test were used to compare the average of BAP and the median of β-CTX in qualitative variables. The multiple regression models, we introduced the variables which were statistically significant at the univariate analysis, was used to determinate the associated factors with the concentration of BTMs.

Result

In this study of 125 women, 64(51.42) of them were menopausal, the mean± (SD) age and BAP were 50.3(15.3) years, 22.1±7.6 ng/L, the median with interquartile ranges [IQR] of the IBSB and β-CTX were 36.0[24.0-58.0] months, 368.5 [237.0-504.8]ng/L. The serum concentrations of calcium (2.3±0.08 mm) and phosphorus(1.0±0.1 mm) were within normal range whereas for the 25-OHDconcentration (8.8±3.7 ng/ml) was in the low range. The characteristics of our sample in the univariate analysis are shown in Table 1. For the threshold less than 20ng/ml, the prevalence of hypovitaminosis D was 98.4% (n=123). Among the deficient vitamin D women, 87 (70.7%) of them had a hyperparathyroidism (PTH=35.4±13.9 ng/L), 14 (11.4%) a high bone resorption (β-CTX= 376.6 [239.6-513.0] ng/L), and 56 (45.5 %) a high bone formation (BAP =22.2±7.7µg/L). Correlation BTMs with Biological and Gynecological Parameters: A significant positive correlations between BAP and the age(r=0.3, p<0.001) and the PTH (r=0.1,

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A-3 p=0.04) were observed there was a positive correlation between the β-CTX and IBSB (r=0.2,p=0.05). In addition, we noted significant negatives correlations between the PTH and the 25OHD (r=0.2,p=0.02), serum calcium (r=-0.2,p=0.002) and phosphorus (r=-0.1,p=0.02) but no correlations were found between 25OHD and BAP or β-CTX. Comparison of BTMs with Amnesic and Gynecological Parameters :The BAP concentration was higher in women according to the menopause (p<0.001) and the diffuse pain (p<0.001). However, no difference in the BAP concentration was found according to the personal

AABS; 4(1): 2017 and familial history of fractures (p=0.6 and p=0.7) (Table 2). The β-CTX median concentrations didn’t show any differences according to the qualitative variables mentioned above (Table3). Linear Regression Model for Btms: The significant variables of the study in the univariate analysis were introduced in descending linear regression model for the BAP and β -CTX. The results showed that 12% of BAP concentration has been associated to the menopause (R2=0.12; beta =-0.29, p=0.001), whereas the β-CTX concentration appeared to not be associated with any variable of this model ( p=0.7).

Table 1: Database of the sample Age

Total N

Characteristics

125

50.32±15.4

Socio-demographic characteristics Marital status

125

Married

57 (45.6)

Not married Socioprofessional status

68(54.4) 125

With the employment

57(45.6)

Without employment

68(54.4)

Level of Education

125

Illiterate

38(30.4)

Primary school

27(21.6)

Secondary education

34(27.2)

Superior

26(20.8)

Anamnesis characteristics Widespread pain

125

Yes

83(66.4)

Non

42(33.6)

Personal history fractures

125

Yes

34(27.2)

Non

91(72.8)

Familial History fractures

125

Yes

29(23.2)

Non

96(76.8)

Anthropometric characteristics Weight (kg)

123

72.9±14.2

WHR (m)

123

0.96±0.13

Age at menarche

106

12.2±1.6

Parity

3.0[2.0-5.0]*

Gynecologic characteristics

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Total N

Menopause

Characteristics

125

Yes

64(51.42)

61(48.8)

Space btween births

36.0[24.0-58.0]*

Calcium (mm)

125

2.3±0.08

Phosphorus (mm)

125

1.0±0.1

25OHD (ng/ml)

125

8.8±3.7

PTHi (ng/L)

125

35.3±13.8

β-CTX (ng/L)

125

368.5[237.0-504.8]*

BAP (µg/L)

125

22.1±7.6

Biological parameters

* Mediane [IQR]

Table 2: Comparison of concentrations of BAP with amnesic and gynecologic parameters using Student t test BAP

P Value

Menopause Yes

(64)24.5±6.9

Non

(61)19.7±7.6

<0.001

Widespread pain Yes

(83)23.2±8.0

(42)20.0±6.4

0.016

Personal history of fractures Yes

(34)21.9±7.6

(91)22.2±7.7

0.8 (NS)

Familial history of fractures Yes

(29) 23.0±7.9

(96)21.9±7.6

0.5 (NS)

Table 3: Comparison of β-CTX with amnesic and gynecologic parameters using Mann-Whitney test Β-CTX

P Value

Menopause

U 1860.0

Yes

(64)382.6[266.5-521.9]

Non

(61)354.6[219.3-493.1]

0.6(NS)

Widespread pain Yes

(83)367.8[248.9-496.6]

(42)408.7[216.8-534.3]

0.9 (NS)

Personal history of fractures

1732.0 1515.0

Yes

(34) 364.3[212.5-488.5]

(91) 376.6[239.6-513.0]

0.8 (NS)

Familial history of fractures Yes

(29) 360.1 [213.4-617.2]

(96)385.4 [241.9-474.3]

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0.9 (NS)

1388.0

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Discussion

The objective of this study was to determine the associated gynecological factors with the concentration of BTMs among Moroccan woman. The results showed that the menopause was an associated factor with the BAP and the IBSB was correlated with the β-CTX but wasn’t considered as an associated factor with it concentration. The literature reported that the BAP is an sensitive marker of bone remodeling during menopause (11,12)including secondary hyperparathyroidism, aluminum-related lowturnover bone disease, osteomalacia, adynamic osteopathy, osteoporosis, and skeletal beta2-microglobulin amyloid deposits. In spite of the enormous progress made during the last few years in the search of noninvasive methods to assess bone metabolism, the distinction between high- and low-turnover bone diseases in these patients still frequently requires invasive and/or costly procedures such as bone biopsy after double tetracycline labeling, scintigraphicscan studies, computed tomography, and densitometry. This review is focused on the diagnostic value of several new serum markers of bone metabolism, including bonespecific alkaline phosphatase (bAP. Some researchers find similar results to ours, their studies have highlighted the correlation between the BAP and the sexual hormones during menopause; they showed that the concentration of BAP is related to the concentration of the follicle stimulating hormone (FSH) among menopausal women (13-15) . A Chinese investigation reported that the concentration of FSH explained 1.9-11.8% of BAP concentration among postmenopausal women (13). A Moroccan study, among menopausal women found an correlation between ovarian hormones Estradiol and the Osteocalcin but the authors didn’t consider the BAP in their study (5). In the contrast to others studies (16) we didn’t find the same ascertainment. The variation in outcomes can be attributable to the technical’s methods and the genetic variety of population’s studies which must be taken into account during BTMs interpretation (17). The positive correlation between the BAP and PTH allows to suggest that the increase of BAP is associated to hyperparathyroidism leading the hyperactivity of bone and predict the risk of osteomalacia and secondary osteoporosis among women (12,18). Our results showed that the IBSB was correlated with the β-CTX concentration. In fact, there is a physiological change in calcium metabolism during pregnancy which allows a mineral transfer from the mother to the fetal Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 skeleton to ensure its growth and its mineralization following a hormonal adjustment (19,20). So after the birth, the maternal bone needs time to be restore but the successive pregnancies affect the bone mineralization due to increase a process of bone resorption (21 ) To our background, no Moroccan study has been performed among mothers population addressing the influence of IBSB on BTMs. We can suggest that the IBSB affect the bone by taking into account the hypothesis of the strong parity of the Moroccan population ,with average of fertility equal to 7.2 children per woman in 1962 which are considered as menopausal women in our sample (22), and the psychosocial dimensions of the Moroccan society which impose the status of procreation for the women (23). The results reported high prevalence of hypovitaminosis D in our population such as the reported by the study of El Allali et al. among Moroccan women where the prevalence of hypovitaminosis D was 91% (6). The 25OHD deficiency can be related to many causes such as the individual factors regarding to the lifestyle of women , to the geography factors taking into account the position of the city, to the technical parameters and finally to the political factors due to the lack of vitamin D supplementation for foodstuffs. Our investigation didn’t demonstrate any link between the 25OHD and BTMs along with our study some authors didn’t find any relationship (24)and thus to increased bone loss. We investigated 72 elderly subjects (16 men and 56 women but others demonstrated that a deficiency in 25OHD leads to an increase in the serum concentration of BTMs (25). Those differences could be due to the season and geographical place of the study, the age and ethnic of the subjects and interlaboratory variation for the measurement of BTMs and 25OHD. Our study presents strengths and limitations. The main limitations lie to the procedure to select the subjects, we cannot exclude that some subjects did not reported remote traumas, and the lack of the bone densitometry measurement. By contrast, the strengths of our research consisted to control the analytical part of our study using standard procedures undertaken by an experienced medical staff to manage and conduct the study also in standardization of the biological analysis among all subjects.

Conclusion

This study allows to determinate the gynecological factors associated with the concentration of biochemical e-ISSN: 2349-6991; p-ISSN: 2455-0396

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BTMs parameter which is the menopause. We also demonstrated that the IBSB was a correlated factor with the β-CTX. Longitudinal perspectives studies could be initiated to establish a link between these factors and the BTMs.

Acknowledgements:

The authors would to thank Dr Mohammed Khalid NEJJAR and his medical staff for their help, devotion and general support.

Reference: 1.

J.P. Salles, I. Gennero. Développement précoce de l’os et marqueurs biologiques. In: Le Bouc Yves ; Tauber, Maithé. Aspects biologiques, moléculaires et cliniques de l’axe GH/ IGF-I .Paris : Springer,2012.110-118.

Garnero P, Hausherr E, Chapuy MC, Marcelli C, Grandjean H, Muller C, et al. Markers of bone resorption predict hip fracture in elderly women: the EPIDOS Prospective Study. J Bone Miner Res. oct 1996;11(10):1531-1538.

Honkanen RJ, Honkanen K, Kröger H, Alhava E, Tuppurainen M, Saarikoski S. Risk factors for perimenopausal distal forearm fracture. Osteoporos Int. 2000;11(3):265-270.

Siggy Rausch, Marco Hirsch, Raphael La Schiazza, JeanLuc Dourson, Bernard Weber, Ernest Wilwert(Conseil scientifique Domaine de la santé).Texte long. OSTEOPOROSE , GT Laboratoire; 21 Juillet 2010. El Maataoui A, El Maghraoui A, Biaz A, Elmachtani SI, Dami A, Bouhsain S, et al. Relationships between vertebral fractures, sex hormones and vitamin D in Moroccan postmenopausal women: a cross sectional study. BMC Womens Health. 2015;15(41):1-8. Allali F, El Aichaoui S, Khazani H, Benyahia B, Saoud B, El Kabbaj S, et al. High Prevalence of Hypovitaminosis D in Morocco: Relationship to Lifestyle, Physical Performance, Bone Markers, and Bone Mineral Density. Seminars in Arthritis and Rheumatism. juin 2009;38(6):444-451. Les enseignants du groupe Pitié-Salpêtrière. Faculté de médecine Pierre & Marie Curie- Université Pierre et Marie Curie. Minimum vital Niveau A 2001-2002. Chapitre 5 : Douleur. Faculté de Médecine Pitié-Salpêtrière. Fév. 2002 :43-52. France.

Beauchemin C, Hamel C, Simon P, Héran F. Trajectoires et origines: enquête sur la diversité des populations en France : Grandes enquêtes. Catalogue 2015, Institut national d’études démographiques ; 2015. Rapport n° : 23837411469432445, Ined editions;. France. Département Prévention, pôle Santé publique et soins (PSPS)-INCa. Traitements hormonaux de la ménopause et risques de cancers : Etat des lieux et des connaissances : Fiches repères. Institut National du Cancer; Févr. 2015. France.

10. Ross AC. The 2011 report on dietary reference intakes for calcium and vitamin D. Public Health Nutr. mai 2011;14(5):938-939.

11. Ureña P, De Vernejoul MC. Circulating biochemical markers of bone remodeling in uremic patients. Kidney Int. juin 1999;55(6):2141-2156. 12. Garnero P, Sornay-Rendu E, Chapuy MC, Delmas PD. Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res. mars 1996;11(3):337-349. 13. Chen C, Liang M-K, Zhang H, Peng Y-Q, Wu X-P, Wu X-Y, et al. Relationships between age-related biochemical markers of bone turnover and OPG, TGF-β1 and TGF-β2 in native Chinese women. Endocr Res. 2014;39(3):105-114. 14. Wu X-Y, Wu X-P, Xie H, Zhang H, Peng Y-Q, Yuan L-Q, et al. Age-related changes in biochemical markers of bone turnover and gonadotropin levels and their relationship among Chinese adult women. Osteoporos Int. févr 2010;21(2):275-285. 15. Lambrinoudaki I, Christodoulakos G, Aravantinos L, Antoniou A, Rizos D, Chondros C, et al. Endogenous sex steroids and bone mineral density in healthy Greek postmenopausal women. J Bone Miner Metab. 2006;24(1):65-71. 16. van Geel TACM, Geusens PP, Winkens B, Sels J-PJE, Dinant G-J. Measures of bioavailable serum testosterone and estradiol and their relationships with muscle mass, muscle strength and bone mineral density in postmenopausal women: a cross-sectional study. Eur J Endocrinol. avr 2009;160(4):681-687. 17. Cavalier E, Bergmann P, Bruyère O, Delanaye P, Durnez A, Devogelaer J-P, et al. The role of biochemical of bone turnover markers in osteoporosis and metabolic bone disease: a consensus paper of the Belgian Bone Club. Osteoporosis International. juill 2016;27(7):2181-2195. 18. El Maghraoui A, Ouzzif Z, Mounach A, Rezqi A, Achemlal L, Bezza A, et al. Hypovitaminosis D and prevalent asymptomatic vertebral fractures in Moroccan postmenopausal women. BMC Womens Health. avr 2012;12(11):1-8. 19. Holick MF. The vitamin D deficiency pandemic and consequences for nonskeletal health: mechanisms of action. Mol Aspects Med. déc 2008;29(6):361‑368. 20. Winkvist A, Rasmussen KM, Habicht JP. A new definition of maternal depletion syndrome. Am J Public Health. mai 1992;82(5):691-694. 21. Shahtaheri SM, Aaron JE, Johnson DR, Purdie DW. Changes in trabecular bone architecture in women during pregnancy. Br J Obstet Gynaecol. mai 1999;106(5):432-438. 22. Haut Commissariat au Plan. Répartition géographique de la population d’après les données du recensement général de la population et de l’habitat de 2014. Rapport Final. Direction de la statistique : 2014. Maroc.

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A-7 23. Mohamed Saïd SAADI : L’expérience marocaine d’ intégration de la femme au développement. Institut Supérieur de Commerce et d’Administration des Entreprises.2004 : 1-8.Maroc. 24. Siggelkow H, Schulz H, Kaesler S, Benzler K, Atkinson MJ, Hüfner M. 1,25 dihydroxyvitamin-D3 attenuates the confluence-dependent differences in the osteoblast characteristic proteins alkaline phosphatase, procollagen

AABS; 4(1): 2017 I peptide, and osteocalcin. Calcif Tissue Int. mai 1999;64(5):414‑421. 25. Kruger MC, Kruger IM, Wentzel-Viljoen E, Kruger A. Urbanization of black South African women may increase risk of low bone mass due to low vitamin D status, low calcium intake, and high bone turnover. Nutr Res. oct 2011;31(10):748‑758.

*Corresponding author: Basma Nejjar, Department of Biology, Team of Food and Nutritional Transition (ETAN), Laboratory of Biological assays, Faculty of Sciences, University Ibn Tofail, 14000 Kenitra, Morocco. Email: Basma.nejjar02@gmail.com Date of Submission : 06.01.2017 Date of Acceptance : 19.01.2017 Date of Publication : 22.01.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Original Article DOI: 10.21276/AABS.2017.1306

Hepatitis B Versus Hepatitis C in Blood Donors Ashok Yadav*, Ravi Jain Department of Pathology, MGM Medical College, Indore. India

ABSTRACT Introduction: Transmission of infectious diseases through donated blood is of concern to blood safety as transfusion forms an integral part of medical and surgical therapy. Blood transfusion carries the risk of transfusion-transmissible infections, including HIV, hepatitis, syphilis, malaria and infrequently toxoplasmosis. With every unit of blood, there is 1% chance of transfusion-associated problems including transfusion-transmitted diseases.Among all infections HIV and hepatitis are the most dreadful. Aims & Objectives: To find out the seroprevalence of hepatitis B virus and hepatitisC virus in blood donors , to determine the incidence of transfusion related disease in blood donors, to find the incidence of spectrum of diseases in blood bank donation, to find the age distribution of the cases studied and to find the sex distribution of the cases studied. Material & Methods: The present study is being undertaken in the Department of Pathology MGM Medical College Indore. This is a retrospective study that will be conducted, during the period 2008 –2013. Tests are routinely done on every blood unit to exclude HIV, HBV, HCV, syphilis and malaria. Donors were selected by the standard criteria for donor fitness. The screening for HIV was done by ELISA using kits. HBS Ag was detected by ELISA. Anti-HCV test was done by ELISA. Results: In the present study, 94721 blood donors are observed in the year 2008-13 in the M. Y. Blood Bank.Majority of donors are voluntary donors 76.25 % as compared to replacement/relative donors 23.74 %.Out of total 94721 blood donations, majority of donors are male donors 94.9 % (89900) as compared to female donors 5.08 % (4821).The seroprevalence of HBV&HCV is 1.83% & 0.07% respectively. Seroprevalence is higher in the age group 26-35 year for HBV (0.93 %). HCV (0.025 %) is prevalent higher in age group 18-25 year. Over all Seroprevalence of transfusion transmitted disease in all donations in the year 2008-13 is 1.91 %. Seroprevalence of transfusion transmitted disease is higher in voluntary donors 1.44 % as compared to replacement/relative 0.532 % donors. Conclusion: voluntary blood donation should be encouraged for prevention of transfusion-transmissible diseases. The time and cost involved in screening donated blood can be reduced by an effective donor education and selection program that promotes self-exclusion by donors at risk of transfusion-transmissible infections. Introducing nucleic acid testing (NAT) for HBsAg and HCV is recommended to detect the infection during window period. Keywords: Hepatitis B, Hepatitis C, Transfusion transmitted diseases, Voluntary donors, Replacement donors

Introduction

Transmission of infectious diseases through donated blood is of concern to blood safety as transfusion forms an integral part of medical and surgical therapy. Blood transfusion carries the risk of transfusion-transmissible infections, including HIV, hepatitis, syphilis, malaria and infrequently toxoplasmosis, Brucellosis and some viral infections like CMV, EBV and herpes. With every unit of blood, there is 1% chance of transfusionassociated problems including transfusion-transmitted diseases.Among all infections HIV and hepatitis are the most dreadful. The first case of transfusion-associated AIDS was described in an infant given transfusion for erythroblastosis foetalis. There after, many cases were reported all over the world in which transfusion of blood and its products was the only risk factor.The improved screening and testing of blood donors has significantly reduced transfusion-transmitted diseases in most developed

countries. This has not been so in developing nations. Poor health education and lack of awareness result in the reservoir of infections in the population.

Material and Methods

The present study is being undertaken in the Department of Pathology MGM Medical College Indore. This is a retrospective study that will be conducted, during the period 2008 –2013. Tests are routinely done on every blood unit to exclude HIV, HBV, HCV, syphilis and malaria. Donors were selected by the standard criteria for donor fitness. The screening for HIV was done by ELISA using kits. HBS Ag was detected by ELISA. Anti-HCV test was done by ELISA. ABO and Rhesus (Rh) blood groups were determined using blood grouping antisera: anti-A, anti-B, anti-AB, and anti-D. Selection of cases for the study included the donors of MYH Blood Bank.

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

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Aim 1. To find out the seroprevalence of hepatitis B virus and hepatitisC virus in blood donors. 2. To determine the incidence of transfusion related disease in blood donors. 3. To find the incidence of spectrum of diseases in blood bank donation. 4. To find the age distribution of the cases studied. 5. To find the sex distribution of the cases studied.

Results

Graph 1: Number of blood units collected during the year 2008-13. Out of total 94721 blood donations, majority of donors are voluntary donors 78.83 % as compared to replacement/relative donors 21.17 %

Graph 2 : Number of male and female donors during the year 2008-13.Out of total 94721blood donations, majority of donors are male donors 94.9 % as compared to female donors 5.08%

Graph 3 : Seropositive donors for HBV and HCV in 200813 Seroprevalence of HBV and HCV are 1.83 % and 0.07 % respectively.

Graph 4: Age wise distribution of HBV in the year 200813. Seroprevalence is higher in the age group 26-35 year

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

The present study is conducted in the Department of Pathology MGM Medical College Indore and M. Y. Hospital blood bank. This is a retrospective study that was conducted, during the period 2008 â&#x20AC;&#x201C;2013. In the present study, 94721 blood donors are observed in the year 200813 in the M. Y. Blood Bank. The data collected from donor register record book, donors form, master record book, HIV, HBV and HCV positive beg number records. The results and observations studies are presented below:

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Graph 5 : Age wise distribution of seroprevalence of HCV in the year 2008-13. Seroprevalence is higher in age group 18-25 yrs in year 2011,12& 2013.

Graph 6: Overall age distribution of seroprevalence of HBV and HCV in 2008-2013. HBV is more prevalent in age group 26-35 years while HCV is more prevalent in 18-25 years.Â

Graph 7 :Seropositivity of transfusion transmitted disease in 2008-13.Seropositivity is higher in Voluntary donors in all years except year 2008 where seropositivity is higher in replacement donors.

Discussion

There are four main groups of micro-organisms known to cause infections namely viruses, bacteria, protozoa and fungi. Only first three groups of microbes - viruses, bacteria and protozoa - have been reported to be transmitted by blood transfusion. Individuals with fungal infections are usually too sick to be accepted as blood donors. Viruses are most commonly transmitted by transfusion.

Recently, a new form of infectious agent - the prion - has been identified. At this time, there is no evidence to suggest that they could be transmitted by blood transfusion. Viruses are the simplest forms of life. They infect all forms of life, they lack certain components needed to live and their growth hence depend on the host cell that they infect to provide these missing components.Following are some of the viruses which are known to be transmitted through blood:

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1. Human immunodeficiency virus (HIV) 2. Hepatitis B virus 3. Hepatitis C virus

drinking glasses, kissing, hugging, coughing, sneezing, or breastfeeding.4 The transmission of HBV is mainly by parental route which involves direct contact with body fluid. The most common route of infection are:

4. Hepatitis A virus 5. Hepatitis G virus 6. Non - A, Non - B Hepatitis

•

Contact with infected blood, either by exposure of wounds to infected blood or to contaminated needles and syringes used in injecting drugs, tattooing, ear piercing, or acupuncture.

•

Sexual contact

•

Neonatal or perinatal transmission

•

Transfusion of infected blood or blood products.1

7. Epstein Barr Virus 8. Cytomegalo virus (CMV) 9. Human T Lymphocytic virus (HTLV - 1 & HTLV - 2) Hepatitis B is an infectious inflammatory illness of the liver caused bythe hepatitis B virus (HBV) that affects hominoidea, including humans. Originally known as “serum hepatitis”,2 the disease has caused epidemics in parts of Asia and Africa, and it is endemic in China.3 About a third of the world population has been infected at one point in their lives,4 including 350 million who are chronic carriers.5 The virus is transmitted by exposure to infectious blood or body fluids such as semen and vaginal fluids, while viral DNA has been detected in the saliva, tears, and urine of chronic carriers. Perinatal infection is a major route of infection in endemic (mainly developing) countries.6 Other risk factors for developing HBV infection include working in a healthcare setting, transfusions, dialysis, acupuncture, tattooing, extended overseas travel, and residence in an institution.4,7 However, Hepatitis B viruses cannot be spread by holding hands, sharing eating utensils or

Hepatitis C is an infectious disease affecting primarily the liver, caused by the hepatitis C virus (HCV).[8] The infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis, which is generally apparent after many years. In some cases, those with cirrhosis will go on to develop liver failure, liver cancer or life-threatening esophageal and gastric varices.9 HCV is spread primarily by blood-to-blood contact associated with intravenous drug use, poorly sterilized medical equipment and transfusions. Hepatitis C only infects humans and chimpanzees.10 Hepatitis C is the leading cause of liver transplantation, though the virus usually recurs after transplantation.11 No vaccine against hepatitis C is available.

Figure 1: Transmission

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Voluntary or Replacement/Relative Donor -In our study, out of total 94721 blood donations, majority of donors are voluntary donors 76.25 % as compared to replacement/ relative donors 23.74 %. Similarly majority of donors are voluntary in another study out of 19135 blood donors, 11165 (58%) were voluntary and 7970 (42%) were replacement/ relative donors by Nagarekha Kulkarni12Associate Professor, Department of Pathology, Vijayanagara Institute of Medical Sciences, Bellary - 583104, Karnataka, India. Male or Female Donor: In our study, out of total 94721 blood donations, majority of donors are male donors 94.9 % (89900) as compared to female donors 5.08 % (4821). Similarly another study is comparable for majority of donors are male 96.22 % by Dimple Aroraand Bharti Aroraet al13in Haryana. In the another study, the percentage of male patients was 73% (860/1178) as compared with 27% (318/1178) for female patients by Manisha Jain and Anita Chakravarti et al14. conducted in New Delhi. Seroprevalence of HBV: In our study, the seroprevalence of HBV is 1.83 % in total blood donations in the year 2008-13. Seroprevalence of HBV is comparable to another study with seroprevalence of HBS Ag was 1.7 % by Dimple Aroraand Bharti Aroraet al13conducted in Haryana.The seroprevalence of hepatitis B surface antigen was 0.87% noted in hospital-based population by SmitaSood and ShirishMalvankar et al15 conducted in Rajasthan.In another study conducted among donors of interior Sindh (Pakistan) by Mujeeb et al18, the seroprevalence of HBV was 6.2% . Seroprevalence of HCV: In our study, the seroprevalence of HCV is 0.07 % in total blood donations in the year 200813.It is comparable to another study, theseroprevalence of HCV 0.072% by Murphy EL, Fang J,Tu Y, et al. conducted in USA. This seroprevalence is much lower than the 0.45.2% seroprevalence reported in an earlier study conducted in various European countries by Hahneet al17. Another study by Viet Le at al19 found HCV prevalence to be 0.17%. Age wise distribution: In our study, Seroprevalence is higher in the age group 26-35 year for HBV (0.93 %). HCV(0.025 %) is prevalent higher in age group 18-25 year .In another study,the highest seroprevalence of antiHCV was found in males above the age of 61 years. The highest seroprevalence for anti-HIV was found in the age group 31-40 years by SmitaSood and ShirishMalvankar et al15conducted in Rajasthan.In another study, 3 positive cases belonged to 21-40 years age group by S Mishra and N Chayani et al16 conducted in Orissa.

Summary and Conclusion

The present study is conducted in the Department of Pathology MGM Medical College Indore and M. Y.

Hospital blood bank. This is a retrospective study that was conducted, during the period 2008 –2013. Tests are routinely done on every blood unit to exclude HIV, HBV and HCV. Donors were selected by the standard criteria for donor fitness. The data collected from donor register record book, donors form, master record book, HIV, HBV and HCV positive beg number records. Out of total 94721 blood donations, majority of donors are voluntary 76.25 % as compared to replacement/relative donors 23.74 %.Seroprevalence of HBV and HCV are 1.83 % and 0.078 % respectively. Seroprevalence of HBV is higher than HCV.Seroprevalence is higher in the age group 2635 year for HBV-0.904 %. For HCV-0.025 %, it is higher in age group 18-25 yearsAge group 26- 35 year are show higher Seroprevalence (0.952 %) for HBV & HCV.. Over all Seroprevalence of transfusion transmitted disease in all donations in the year 2008-13 is 1.91 %. Seroprevalence of transfusion transmitted disease is higher in voluntary donors 1.44 % as compared to replacement/relative 0.532 % donors.HBV and HIV are the most prevalent transfusiontransmissible diseases among blood donors in Indore. Screening and better selection of donors are necessary to improve blood safety in the regional blood transfusion centre of M. Y. Hospital. Therefore, it is concluded that voluntary blood donation should be encouraged for prevention of transfusion-transmissible diseases. The time and cost involved in screening donated blood can be reduced by an effective donor education and selection program that promotes self-exclusion by donors at risk of transfusion-transmissible infections. Introducing nucleic acid testing (NAT) for HBsAg and HCV is recommended to detect the infection during window period.

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Haedicke, J.; Brown, C.; Naghavi, H. (Aug 2009). “The brain-specific factor FEZ1 is a determinant of neuronal susceptibility to HIV-1 infection”. Proceedings of the National Academy of Sciences 106 (33): 14040–14045.

Arauz-Ruiz P, Norder H, Robertson BH, Magnius LO (August 2002). “Genotype H: a new Amerindian genotype

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Coffin CS, Mulrooney-Cousins PM, van Marle G, Roberts JP, Michalak TI, Terrault NA (April 2011). “Hepatitis B virus (HBV) quasispecies in hepatic and extrahepatic viral reservoirs in liver transplant recipients on prophylactic therapy”. Liver Transpl 17 (8): 955–62

Schwartz O, Maréchal V, Le Gall S, Lemonnier F, Heard JM (March 1996). “Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein”. Nat. Med. 2 (3): 338–42.

Alpert E, Isselbacher KJ, Schur PH (July 1971). “The pathogenesis of arthritis associated with viral hepatitis. Complement-component studies”. The New England Journal of Medicine 285(4): 1859.

Pungpapong S, Kim WR, Poterucha JJ (2007). “Natural History of Hepatitis B Virus Infection: an Update for Clinicians”. Mayo Clinic Proceedings 82 (8): 967–975.

10. Schaefer S (January 2007). “Hepatitis B virus taxonomy and hepatitis B virus genotypes”.World Journal of Gastroenterology : WJG 13 (1): 14–21. 11. Pope M, Haase A (2003). “Transmission, acute HIV-1 infection and the quest for strategies to prevent infection”. Nat Med 9 (7): 847–52. 12. Nagarekha Kulkarni “Analysis of the seroprevalence of HIV, HBsAg, HCV and syphilitic infections detected in the pretranfusion blood: A short report” http://www.ijbti.com/ archive/2012-archive/100006IJBTINK2012-kulkarni/index.php

AABS; 4(1): 2017 13. Dimple Arora, Bharti Arora, Anshul Khetarpal “Seroprevalence of HIV, HBV, HCV and syphilis in blood donors in Southern Haryana” Year 2010 Vol: 53(2) Page308-309 14. Manisha Jain, Anita Chakravarti, VikasVerma, PreenaBhalla”Seroprevalence of hepatitis viruses in patients infected with the human immunodeficiency virus” Year : 2009 Vol: 52( 1)Page : 17-19 15. SmitaSood and ShirishMalvankar “Seroprevalence of Hepatitis B Surface Antigen, Antibodies to the Hepatitis C Virus, and Human Immunodeficiency Virus in a HospitalBased Population in Jaipur, Rajasthan”Year : 2010 , Vol : 35 (1)Page : 165-169 16. S Mishra, N Chayani, G Sarangi, B Mallick, SB Pati “Seroprevalence of anti HCV antibody in and around Cuttack, Orissa”year : 2002 Vol : 20 ( 1) Page : 40-41 17. Hahné SJ, Veldhuijzen IK, Wiessing L, Lim T-A, Salminen M, Laar M van de. Infection with hepatitis B and C virus in Europe: a systematic review of prevalence and costeffectiveness of screening. BMC Infectious Diseases. 2013;13:181. doi:10.1186/1471-2334-13-181. 18. Mujeeb SA, Pearce MS. Temporal trends in hepatitis B and C infection in family blood donors from interior Sindh, Pakistan. BMC Infectious Diseases. 2008;8:43. doi:10.1186/1471-2334-8-43. 19. Viet L, Lan NTN, Ty PX, et al. Prevalence of hepatitis B & hepatitis C virus infections in potential blood donors in rural Vietnam. The Indian Journal of Medical Research. 2012;136(1):74-81.

*Corresponding author: Dr Ashok Yadav, Yadav clinic, New panchsheel colony, Musakhedi, Indore, India Phone: +91 9893273236 Email: drashokmyh@gmail.com, ravijainpatho@gmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 04.02.2017 Date of Acceptance : 10.02.2017 Date of Publication : 15.02.2017

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

Original Article DOI: 10.21276/AABS.2017.1306

Analysis of Critical values in NABL (National Accreditation Board for Testing and Calibration Laboratories) accredited Hematology and Clinical Pathology laboratory. Killol Nathubhai Desai and Sanjay Chaudhari Department of Pathology, GMERS Medical College, Junagadh, Gujarat, India Department of pathology, Pramukhswami Medical College, Karamsad, Gujarat, India 1

ABSTRACT Background: Reporting of laboratory critical values has become an issue of national attention as illustrated by recent guidelines described in the National Patient Safety Goals of the Joint Commission on Accreditation of Healthcare Organizations. They may be considered an important laboratory outcome measurement because they reflect clinical effectiveness, patient safety and operational efficiency Aims: To improve effectiveness, patient safety and operational efficiency by improving laboratory outcome measurement. Settings and Design: Cross-sectional study done at Shree Krishna Hospital, Karamsad, from January 2012 to December 2013. Methods and Material: All data were obtained from reports generated by hematology and clinical pathology laboratory that has been recorded into critical call back log. Statistical analysis used: The parameters were evaluated using descriptive statistical analysis with IBM Statistical Package for the Social Sciences v 20.0 and Microsoft Office Excel 2007 software. Results: The hematology and clinical pathology laboratory reported 19,423 critical values. The majority of critical callbacks (78.4%) resulted from testing performed in the hematology laboratory. The analytes most commonly called back were Hemoglobin and Urine Ketone. We have recorded maximum 52.7% call back from inpatient department followed by emergency department 34.2% and outpatient department 13.1%. The mean time between entering value in the critical callback register and conveying the information to the patient location or ordering clinician was 21 minutes for IPD, 30 minutes for OPD and 20 minutes for ED. Conclusions: â&#x20AC;&#x153;Every laboratory should have at its disposal a procedure to notify critical Keywords: Critical call back, Turn around time, LIS (Laboratory information system)

Introduction

Critical value reporting originally was highlighted by Lundberg, who defined a critical value as a result suggesting that the patient is in imminent danger unless appropriate therapy is initiated promptly. [1] In the 30 years since Lundbergâ&#x20AC;&#x2122;s observations, the concept of defining critical values and systems for reporting have been adopted widely by laboratories throughout the world. [2] In the United States, laboratory accrediting agencies such as the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) and the College of American Pathologists have made critical value reporting part of the requirements for accreditation. [3, 4] The recent national focus on patient safety has brought increased attention to the issue of laboratory critical value reporting. The JCAHO has made improving the process of critical value reporting a National Patient Safety Goal for the years 2004 through 2006. [3] The JCAHO requires health care

organizations to track and improve the timeliness of reporting and receipt of critical test results by the responsible licensed caregiver. Moreover, the JCAHO has defined critical test results as not only laboratory tests but also imaging studies, electrocardiograms and other diagnostic studies. Therefore, the process of critical value reporting is of interest across the health care organizations. Critical value reporting parameters may be considered an important laboratory outcome measurement because they reflect clinical effectiveness, patient safety and operational efficiency. For the critical value reporting process to be effective, the organization must understand and address the variables involved in the process. This information is not readily available in the literature. Most reports have analyzed only a few analytes for short periods or have reviewed a small number of critical values in a number of different institutions. [5, 6, 7] In the present study, we analyzed 24 months of critical value data and

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19,423 individual critical results to understand the scope of critical value reporting and identify opportunities for process improvement.

using the IBM SPSS (Statistical Package for the Social Sciences v 20.0) and Microsoft Office Excel 2007 software.

Materials and Methods:

During the period of the study, the hematology and clinical pathology laboratories reported 19,423 critical values. During the same period, these laboratories reported 90,000 test results. Therefore, tests with critical values represented approximately 21.6% of the total test results reported.

This prospective cross-sectional study was conducted at Central Diagnostic Laboratory, Hematology and Clinical pathology section, Shree Krishna Hospital from January 2012 to December 2013. All major medical and surgical specialities are supported by the hospital, along with pediatric and obstetric services and extensive primary care and specialty outpatient practices. From January 2012 to December 2013, the laboratories performed 90,000 reportable tests, of which 34% were for inpatients, 55% for outpatients and 11% for emergency department (ED) patients. Critical Callback Procedures: Table 1 shows the critical callback list for hematology and clinical pathology laboratory that is in use at our institution. Responsible laboratory personnel (Resident Doctors, Lab Technologist) will ensure the validation of the result by repetition of test or by recalibration of parameter if necessary and/ or by checking quality control result. If the result is in the critical value (upper or lower) the lab personnel would communicate with the responsible caregiver [Consultant, Registrar, Medical Officer, Nurse and Operations associate (Clerical staff who perform clinical support function)] to inform the values over phone. At the same time he/she will also inform the result to the concerned laboratory doctor (consultant, senior doctor and incharge). For outpatient critical value would be informed to responsible consultant or his/her medical assistant (medical officer). The laboratory personnel will record details of the critical call back (parameter, critical value, informed person, date, time) in a register. Data Collection and Analysis: All data were obtained from reports generated by hematology and clinical pathology laboratory that has been recorded into critical call back register.

Statistical Analysis

The obtained parameters were evaluated using descriptive statistical analysis. Statistical analyses were performed

Results

The majority of critical callbacks (78.4%) resulted from testing performed in the hematology laboratory (Table 2). The clinical pathology laboratory accounted for 21.6% of critical callbacks. The analytes most commonly called back were hemoglobin (5212 results; 26.8% of critical results) and Urine Ketone (3100 results; 16% of critical results) (Table 3). We have recorded maximum 10,242 (52.7%) call back from inpatient department (IPD) followed by emergency department (ED) 6651 (34.2%) and outpatient department (OPD) 2530 (13.1%) (Table 4). Critical value calls were prominent 11,577 (59.6%) in morning shift and minimum 561 (2.9%) in night shift (Table 5). The â&#x20AC;&#x153;in-laboratoryâ&#x20AC;? turnaround time for each critical value was determined to assess the timeliness of critical value reporting. Turn around time is the time period between receiving of a sample and generation of report. [3] For the 19,423 critical values, the mean time between entering value in the critical callback register and conveying the information to the patient location or ordering clinician was 21 minutes (12-30 minutes) for IPD, 30 minutes (1545 minutes) for OPD and 20 minutes (10-30 minutes) for ED (Table 6). Delay in critical value reporting correlated with testing performed on outpatients and testing ordered on requisitions lacking the name of the ordering clinician or the ordering location. Tests performed in settings where there is continuous technologist presence (e.g. Coagulation study) were called back faster than tests performed in other areas. This information was useful as we began to implement measures to improve critical value reporting in all areas of the laboratory.

Table 1: List of Critical values in hematology and clinical pathology laboratories. SL. NO

Discipline

Test parameter

Critical value

Hematology

Hemoglobin

<5.0 gm/dl

Hematology

Platelet

<50000/uL

Hematology

INR (International normalised ratio)

>5.0

Hematology

Total leucocyte count

>20000/cmm<4000/cmm

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Hematology

Absolute neutrophils count

<1800/cmm

Hematology

Immature: Total neutrophils ratio

>0.2 (Pediatric patients)

Hematology

Malaria parasite

Present

Hematology

Malaria antigen

Positive

Hematology

Blast cells

Present

10.

Clinical Pathology

Urine Ketone

Positive

11.

Clinical Pathology

CSF- cell count

>5 cells

Table 2: Evaluation of Critical calls back in hematology and clinical pathology laboratories. Laboratory

Hematology

Clinical pathology

Total

Critical calls back

15,223 (78.4%)

4200 (21.6%)

19,423

Table 3: Evaluation of Critical values by parameters Test parameters

No. Of Critical calls back

Hemoglobin

5212 (26.8%)

Platelet

2022 (10.4%)

INR (International normalised ratio)

1958 (10.1%)

Total leucocyte count

3331 (17.1%)

Absolute neutrophils count

521 (2.7%)

Immature: Total neutrophils ratio

412 (2.1%)

Malaria parasite

1556 (8%)

Malaria antigen

148 (0.8%)

Blast cells

63 (0.3%)

Urine Ketone

3100 (16.0%)

CSF- cell count

1100 (5.7%)

Total

19,423

Table 4: Evaluation of critical values by clinical care areas Clinical care areas

No. Of Critical calls back

Inpatient department (IPD)

10,242 (52.7%)

Outpatients department (OPD)

2530 (13.1%)

Emergency department (ED)

6651 (34.2%)

Total

19,423

Table 5: Frequency of Critical Calls back in each shift. Timing

Early morning

Afternoon

Evening

Night

Total

No. Of Critical calls back

11,577 (59.6%)

4566 (23.5%)

2719 (14%)

561 (2.9%)

19,423

Table 6: Evaluation of Critical values by TAT (Turn Around Time) Clinical care areas

TAT (Turn Around Time) (Minutes) Minimum

Maximum

Mean

Inpatient department (IPD)

Outpatients department (OPD)

Emergency department (ED)

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Discussion:

In this study, we provide a comprehensive view of the critical value reporting process in a large medical center. We provide details regarding the scope, volume, timing and operational aspects of critical value reporting. Many of these parameters should be applicable to a variety of settings. This analysis provides a context for comparison and process improvement. Increasing workload in the clinical laboratory makes it important to achieve efficient use of laboratory resources to maximize clinical benefits. Expansion of critical callback lists to include testing that does not meet the criterion of the “imminent danger” standard may dilute the urgency of a critical value call and lead to unnecessary interruptions for clinicians. For example, critical value calls for high INR levels will not be of clinical value for patients receiving heparin in cardiac operations. In addition, there are many clinical settings (chemotherapy, malignancy) in which the “critical” test result is expected and reporting of this value may not contribute to improved patient care. Communication by telephone, especially when performed by technologists, is a costly practice in terms of the resources required to complete the phone calls and document the process. For this reason, it is helpful to try to reduce the number of phone calls by careful review of the critical values list. In addition to determining which tests are to be included in the critical values list, another important strategy is to examine the consequences of changing the boundaries for critical value reporting. These boundaries must be defined in consultation with clinicians. Small changes in critical value reporting parameters may result in the addition or loss of thousands of phone calls for the laboratory staff. Outpatient critical values present unique challenges in timely reporting to clinicians. One of the strongest correlates of delayed reporting of critical values was the samples being obtained from an outpatient. Outpatient critical values are challenging to communicate to the responsible clinician because there often are different approaches in various practices for determining patient coverage. Unlike inpatients, there is no fixed patient location that can be phoned.

AABS; 4(1): 2017 Another contributor to delay in outpatient critical value reporting is the heterogeneity of the outpatient population, with specimens arriving from health centers, clinics, urgent care centers, dialysis centers, and physicians’ offices. Each of these areas is likely to have a different call schedule, answering service and cross-coverage procedure, making reliable communication with the responsible licensed caregiver challenging. The nature of outpatient specimen transport and processing often results in outpatient test results being generated in the evening when the outpatient clinic or physician’s office is closed. The laboratory must have a mechanism to determine on-call coverage and work with outpatient practices to improve the communication processes. The potential for technological solutions to improve the process of critical value reporting is evident in numerous reports. [8, 9] The use of information technology to automatically communicate with the responsible provider has been demonstrated to reduce the critical value reporting time in controlled settings. For implementation of automated critical value reporting, interfaces from the LIS to technologies that facilitate bidirectional communication (such as e-mail or 2 - way pagers) need to be developed. An important component in such a system is the ability of the automatic reporting system to reliably determine the identity of the responsible provider. At larger medical centers, this task can be challenging because there may be different coverage lists, tests ordered by consultants unknown to the primary caregiver and patient transfers to different locations. An electronic reporting system potentially could create dangerous delays in communication if not properly implemented. The system needs to have an “acknowledgment” function such that the laboratory can ensure that the responsible caregiver received the result. [10] Electronic systems also require an escalation procedure so that lack of acknowledgment of the critical result prompts an alternative approach for communication.

Another factor we identified as causing delay for outpatients was illegible or missing patient information. We have noted that recent improvements in the critical value communication times have coincided with increased awareness of critical value monitoring. We presently are working with our outpatient practices to improve communication between the laboratories and the outpatient care centers.

Development of LIS middleware with alert reporting software should permit highly nuanced approaches to critical value reporting in the near future. Rules-based logic can be applied to laboratory values to build alerts that take into account not only the result value, but also other related results, a change in the current test result from previous results (delta checks), patient demographics, ordering provider and other parameters to customize the alerting to the patient’s condition and the needs of the clinical team for notification. For example, many oncology physicians do not want to be notified regarding patients with neutropenia. The ability to provide a physician specific critical values list could eliminate a large number of unnecessary critical value calls. These systems, when interfaced

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with automated alerting systems, will have the potential to improve patient safety and provide more context-sensitive critical value reporting. At present, practical implementation of this scenario would be constrained by regulations (particularly the JCAHO National Patient Safety Goals) that require all critical results to be communicated and do not allow for more nuanced approaches.

http://www.jcaho.org/accredited+organizations/patient+safety/ npsg.htm. Accessed June 22, 2014. 4.

Center for Medicare and Medicaid Services. Department of Health and Human Services. Clinical Laboratory Improvement Amendments of 1988: 68 Federal Register (2003) (codified at 42 CFR 493.1291(g)).1047

Lum G. Assessment of a critical limit protocol for point-of care glucose testing. Am J Clin Pathol 1996;106:390-395.

“Every laboratory should have at its disposal a procedure to notify critical results. A consensus should be reached with clinicians to establish a specific list of critical limits according to the type of patient and the timeliness of laboratory test”. We are proud to introduce the system of critical call back system in our laboratory which is NABL accredited.

Howanitz PJ, Steindel SJ, Heard NV. Laboratory critical values policies and procedures: a College of American Pathologists Q-Probes study in 623 institutions. Arch Pathol Lab Med 2002;126:663-669.

Kuperman GJ, Boyle D, Jha A, Rittenberg E, Ma’Luf N, Tanasijevic MJ et al. How promptly are inpatients treated for critical laboratory results? J Am Med Inform Assoc 1998;5:112-119.

References:

Lundberg GD. When to panic over abnormal values. Med Lab Obs 1972;4:47-54.

Bates DW, Pappius E, Kuperman GJ, Sittig D, Burstin H, Fairchild D et al. Using information systems to measure and improve quality. Int J Med Inform 1999;53:115-124.

Lundberg GD. Critical (panic) value notification: an established laboratory practice policy (parameter). JAMA 1990;263:709.

Tate KE, Gardner RM, Weaver LK. A computerized laboratory alerting system. MD Comput 1990;7:296-301.

Joint Commission on the Accreditation of Healthcare Organizations: National Patient Safety Goals. Available at:

Conclusion

10. Dighe AS, Rao A, Coakley AB, Lewandrowski KB. Analysis of laboratory critical value reporting at a large academic medical center. AM J Clin Pathol 2006;125:758-764.

*Corresponding author: Dr. Killol Nathubhai Desai, A-301, Staff Quarters, GMERS Medical College, Paddock road, Junagadh, Gujarat, India. Pin: 362001, Phone: +91 09428050253 Email: drkilloldesai@gmail.com Date of Submission : 06.02.2017 Date of Acceptance : 10.02.2017 Financial or other Competing Interests: None. Date of Publication : 15.02.2017

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Original Article DOI: 10.21276/AABS.2017.1327

Exposure to Video Games Shortens Simple Visual Reaction Time: A Study in Indian School Children Paramita Bhattachariyya1, Subhasis Das1* and Ashwin R.2 1 Dept. of Physiology, Pondicherry Institute of Medical Sciences, Kalapet, Puducherry, India Dept. of General Surgery, Madurai Medical College, Alwarpuram, Madurai, Tamil Nadu, India

ABSTRACT Background: Electronic gaming media has become an integral part of the lives of school children. Previous research suggested that action video game playing improves cognitive performance, leading to better visual and attentional skills. On the contrary, other studies have concluded that such gaming activities are more likely to have multiple ill effects on the psychological and physical health of young adults. Visual reaction time has already served to be a good indicator of aptitude in sports involving quick reflexes. However, there is insufficient knowledge about the effects of gaming media exposure on cognitive patterns, as assessed by visual reaction time, especially in Indians. Hence this study was undertaken to estimate the effect of the usage of electronic gaming media on the simple visual reaction time of Indian school children. Methods: Healthy male school-going children in the age group 9-12 years, who were frequently exposed to electronic gaming media for not less than 7-9 hours per week along with children who did not play video games but had normal physical activity (n=38 in each group) were included in this study. Visual reaction time, using a red light, was estimated in the participants. Result: Visual reaction time was found to be significantly decreased in children who played computer games regularly, as compared to controls. A significant negative correlation was also seen, in the study group, between visual reaction time and duration of hours of playing games. Conclusion: Thus, this study concludes that video game playing improves performance in tasks involving hand-eye coordination and quick reflexes. Keywords: Action Video Games, Visual Reaction Time, Hand-Eye Coordination, Attentional Skills,

Introduction

In the 1970s and â&#x20AC;&#x2DC;80s video game parlors and arcades first came into existence and soon became a favorite pastime for adolescents and adults. With the turn of the century, the popularity of these games has spread among children also. This was made possible by the advent of computer games, gaming consoles and gaming apps on tablets and mobile phones, which have brought these apparently risk-free, often action-packed entertainment right into our homes. Devices using virtual reality have taken the gaming experience into an entirely different level. With the availability of online gaming environments video game players (VGPs) have even begun to compete over the internet and the social aspect of such gaming has also got enhanced. In the industrialized countries, the incidence of exposure to these gaming media, among children aged 9 to 16 years, ranges from 98% to 100%. [1] The average media consumption of the children in this age group is >4 hours/day.[2] Most evaluations have focused on the negative influence of excessive media consumption on child health. There

has been a lot of speculation by many health professionals across the globe claiming that these gaming media are the major cause for the development of childhood obesity, impulsivity and aggressive behavior. Studies in Germany and Romania have demonstrated that prolonged exposure to computer games have negative influences on sleep, learning and memory, leading to many psychiatric symptoms especially emotional and behavioral symptoms, somatic complaints and family interaction problems.[3,4] In a study correlating cognitive skills with neuro-psychiatric behavior, Chinese children exhibited more impulsivity and various psychiatric disorders including anxiety, depression and poor interpersonal relations.[5] Excessive internet usage has been shown to be associated with Attention Deficit Hyperactivity Disorder (ADHD).[6] Online game addiction in school children has shown positive correlation with aggressiveness and narcissistic personality traits which may predispose to hostile behavior, frequent arguments with teachers and poor school performance.[7,8] A study conducted on Spanish school children reported that chronic exposure to video games gives rise to distress in social life, occupational activities and school performance.[9]

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Behavioral health aside, on the physical aspect also, changes have been reported in VGPs, obesity and visual impairments being the most common of these. Despite the gamut of studies highlighting the negative aspects of video games on behavior, much research has gone into examining the cognitive augmentation that VGPs exhibit. Visuospatial experience and the prolonged engagement in a demanding visual task, obtained through playing action video games, enhances several visual and cognitive processes.[10] Playing action-video-games can alter fundamental characteristics of the visual system, such as the spatial resolution of visual processing across the visual field. [11] Action games not only widen the range of cognitive abilities, including attention, memory, and executive control skills, but also significantly improve eye-hand motor co-ordination.[12,13] There may be an enhancement in attentional skills in VGPs [14] and such avid players of these fast paced video games demonstrate better target detection capabilities when given attention-demanding tasks.[15] A significant reduction in reaction times has also been reported.[16] Reviewing the literature, it was found that despite the enormous progress in research done across the world to assess the impact of these electronic gaming media on the health status of school children, opinion is divided between the negative and the positive aspects. Also, there is insufficient knowledge about the effects of excessive media exposure on simple reaction times in Indian children, a deficit that this study has sought to rectify.

Materials and Methods

This observational study was initiated after obtaining due approval from the Institutional Research Committee (Review Board) and Ethics Committee. The study was conducted in two groups, with 38 healthy male children, in the age group 9-12 years, randomly selected from schools in and around Pondicherry, as per selection criteria. Children in the “study group” played computer games for not less than 7-9 hours/week, while those in the “control group” did not play computer games but had normal physical activity. Children with history of visual or auditory impairment, epilepsy, ADHD, mental retardation or any other psychiatric disorder were excluded. Children with history suggestive of eye, ear, and throat infections, at the time of the study, were also excluded. Variables such as the time of the day for the tests, ambient temperature, humidity and posture of the participant in relation to the apparatus were constant for all the participants. Other constants ensured were freedom from

exams or tests, adequate sleep on the night prior to the testing and a normal breakfast the same morning. After obtaining permission from the school authorities, informed written consent from the parents of each recruited child, and assent from each of the children, all the participants underwent a thorough physical examination, including tests for vision with torch, Snellen’s chart, Jaeger’s chart and Ishihara’s charts and tests of hearing using tuning forks, prior to starting the procedure. With help of the parents of the recruited children and their teachers, a questionnaire, with relevant demographic and anthropometric data and data related to video game playing, was filled up. Audio-visual Reaction time apparatus (Anand Agencies, Pune) was used for the measurement of reaction time. Each participant had to undergo 5 sets of 10 recordings of simple visual reaction time using a red-light test stimulus, with a 1 minute break in between sets. The mean of the 50 recordings from each subject was used for analysis.

Result

For the purpose of statistical analysis, GraphPad Prism 4 software package was used. Statistical significance was considered at a p value < 0.05. The mean values of anthropometric variables such as age, height, weight and BMI were statistically similar between the two groups, i.e., the study group (n=38) and the control (n=38). (Table-1) The mean simple visual reaction times of the participants in the study group and control group were 157.4 ± 21.9 ms and 218.8 ± 36.14 ms respectively. Comparison of the mean simple visual reaction times in the two groups by unpaired 2 tailed Student “t” test showed a statistically significant difference (p < 0.0001). (Fig.-1) Among the participants in the study group, the mean duration of video game playing was 6.59 ± 3.59 hours per week. A significant negative correlation (Pearson) between visual reaction time and duration of hours of playing games was observed in the study group participants (r = -0.0381, p < 0.05). (Fig.-2) On analysis of the correlation between reaction time and BMI, no statistically significant correlation was observed in either group.

Discussion

This observational study aimed at assessing the effect of video game playing on simple visual reaction time, showed that there was a statistically significant decrease in visual reaction time in children playing computer games (p < 0.0001) as compared to controls. The results of this study are consistent with the earlier finding that action

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Table 1: Comparison of anthropometric parameters between Study and Control groups. Study group (Mean ±SD)

Control (Mean ±SD)

12.26 ± 0.95

12.42 ± 0.86

> 0.05

Height (cm)

149 ± 8.33

146.24 ± 8.36

> 0.05

Weight (kg)

40.53 ± 9.29

37.97 ± 6.28

> 0.05

BMI (kg/m2)

17.68 ± 3.59

17.24 ± 1.92

> 0.05

Age (yrs)

Fig. 1: Comparison of simple visual reaction time Between the Study and Controls groups.

Fig.-2: Correlation between reaction time and duration of playing video games among Study group participants.

video games modify visual selective attention, leading to improvement in basic cognitive skills.[12] A decrease in reaction time indicates an improved sensorimotor performance and could be due to an enhanced

information processing ability of the central nervous system.[17] However, the underlying mechanisms for this improvement remains unclear. It has been suggested that alterations in visual skills such as those brought about by

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playing action video games are of importance in promoting visual plasticity.[18] That action video games modulate early sensory processing resulting in greater sensitivity to exogenous sensory inputs,[10] could also be an explanation for improvement in cognitive skills. Reaction time is known to be affected by various factors like age, practice, fatigue and distractions.[19] In this study, a significant negative correlation was found between visual reaction time and duration of hours of playing video games. This might indicate that repeated video game playing results in training of the central nervous system in improving the ability to ignore and/or inhibit extraneous distracting stimuli. It could also be a result of traininginduced enhancement of performance which abound in the field of perceptual learning.[20] Conventional exercise training protocols as well as pranayamic practice are known to improve attention, reduce distractibility, reduce mental fatigability and induce alertness and arousal, especially to extraneous stimuli.[17,21] Our study did not demonstrate any significant correlation between reaction time and body mass index in both study and control groups. However, adolescents spending more than 6-7 hours weekly on video games showed a trend towards higher body mass index than those who did not play at all. This finding could be attributed to a reduction in overall physical activity owing to more time spent in front of a screen. As video-game playing has become a ubiquitous activity in today’s society, it is worth considering its potential consequences on perceptual and motor skills, and that was the focus of this study. However, this study has a few limitations, especially in the age group and gender that was selected as well as the fact that a longitudinal study could not be undertaken. Moreover, one might argue that the reduced reaction time was what led to these adolescents excelling in, and therefore spending more time on computer games, and not the other way round. Such a premise can only be verified by a crossover cohort study.

Conclusion

This study concludes that playing action video games, by virtue of their very nature, enhances visual processing and thinking strategy in school children. It not only ensures better sensorimotor performance but also significantly improves cognitive skills of these individuals. Therefore, they exhibit faster reaction time than those who do not play video games. This faster reflex leads to better concentration, alertness, muscular co-ordination and improves performance in speed and accuracy tasks.

Such games, if used for training could lead to a better preparation for professions where faster reflexes and better hand-eye coordination are an asset. However, caution must be exercised, since excessive use of such games could have multiple deleterious effects as documented in earlier studies.

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15. Mishra J, Zinni M, Bavelier D, Hillyard SA. Neural basis of superior performance of action videogame players in an attention-demanding task. J Neurosci. 2011;31(3):992-8. 16. Dye MWG, Green CS, Bavelier D. Increasing speed of processing with action video games. Curr Dir Psychol Sci. 2009;18(6):321-6. 17. Borker AS, Pednekar JR. Effect of Pranayam on visual and auditory reaction time. Indian J Physiol Pharmacol. 2003;47(2):229â&#x20AC;&#x201C;30.

20. Green CS, Bavelier D. Action video game modifies visual selective attention. Nature. 2003 May 29;423(6939):534-7. 21. Mouelhi Guizani S, Bouzaouach I, Tenenbaum G, Ben Kheder A, Feki Y, Bouaziz M. Simple and choice reaction times under varying levels of physical load in high skilled fencers. J Sports Med Phys Fitness. 2006 Jun;46(2):344-51.

*Corresponding author: Subhasis Das, Dept. of Physiology, Pondicherry Institute of Medical Sciences Ganapathychettikulam, Kalapet, Pondicherry â&#x20AC;&#x201C; 605014, INDIA Phone: +91 9003547368 Email: subhasiscmc@gmail.com Date of Submission : 07.02.2017 Date of Acceptance : 12.02.2017 Financial or other Competing Interests: None. Date of Publication : 15.02.2017

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Original Article DOI: 10.21276/AABS.2017.1309

Client Satisfaction in Thalassemia Control Unit, North Bengal Medical College and Hospital, Darjeeling District, West Bengal Nilanjana Ghosh1 and Indranil Chakrabarti2* Department of Community Medicine, North Bengal Medical College, West Bengal, India 2 Department of Pathology North Bengal Medical College, West Bengal, India

ABSTRACT Background: Health of a nation lies in hands of its people. India harbours a huge load of thalassemia , the fatal yet preventable condition, in various forms. Haemoglobin E has prevalence of 3-10% in West Bengal and is believed to be harboured mostly by Rajbanshis, who form majority of the local population in this terrain. Hence effective utilization of services provided is of utmost importance. Satisfaction of clients determines optimum service utilization, an indicator of effective health outcome and desirable health indicators. Accountability, accessibility, availability and sensitivity to felt needs of community by health staffs engaged in service delivery ensure satisfaction among beneficiaries. Thalassemia control unit (TCU) is a state government endeavour which aids in case and carrier detection through screening programs and there further management. The objectives of the study were to assess client satisfaction among beneficiaries regarding various aspects of services provided to them at TCU and resolve issues with managerial skills. Methods: Study was conducted in TCU of North Bengal Medical College for six months. Study subjects were selected by systematic random sampling technique pertaining to study criteria. 120 beneficiaries were studied. Predesigned, pretested schedule adapted from CSQ8 Questionnaire was applied and exit interview was done. Satisfaction of clients in different components was assessed and means scores compared. Health staffs were also interviewed. Results: Among selected beneficiaries 40 were known to health staffs at clinic. Majority were adults, Hindus, females and illiterates. Satisfaction level regarding different components varied among respondents. Significant difference was noted among two groups of respondents regarding behaviour of staffs at the clinic. Managerial issues were addressed after prioritization and categorization. Conclusion: Satisfaction levels varied for different components among respondents. Significant difference was noted regarding behaviour of staff to known beneficiaries .Various issues existed which were neglected but could be addressed with managerial skills. Appropriate health education and sensitive interventions may prove beneficial. However, a larger study with more representative sample is warranted. Keywords: Thalassemia, Client Satisfaction, Management

Introduction

Client centred, need based, demand driven services are strongly interwoven between service providers and beneficiaries. They need to work in unison and develop mutual trust and accountability to ensure appropriate service delivery, adequate service utilization, effective health outcomes and desirable health indicators1. Thalassemias the dreaded yet preventable disease with fatal complications are the most common inherited hemoglobinopathies in India and pose a huge economic burden to the country. Hemoglobinopathies, namely hemoglobin (Hb) variants, thalassemia syndromes, hereditary persistence of foetal haemoglobin (HPHF) and their combinations show a wide variation of prevalence in different regions and different populations. Around 1.5% of the worldâ&#x20AC;&#x2122;s population carries the beta thalassemia gene. Hemoglobin E has a prevalence of 3-10% in West Bengal and is believed to be harbored mostly by Rajbanshis, who form the majority of the local population2.

TCU (Thalassemia control unit ) has been set up in various tertiary care hospitals by GoI and state governmentâ&#x20AC;&#x2122;s initiative to aid in carrier and case detection through screening, awareness generation and counselling sessions. They provide curative , promotive ,preventive and rehabilitative care in form of premarital counselling, screening programs and arranging for chelation therapies3. This endeavour not only helps treat the cases and decrease the disease burden but also provide a host of services including field activities and monitoring and supervising other small hospitals engaged in similar service. Clients in health sector are either patients who come solely for curative purpose or beneficiaries who avail preventive and promotive and curative services. It is well known that for effective health outcome and desirable health indicators optimum service utilization by the beneficiaries is an essential prerequisite. Hence ensuring their satisfaction for whom the services are designated and designed is of paramount importance. Standards being main drivers of

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A-25 quality, client satisfaction among beneficiaries regarding various aspects of services provided are of utmost concern and a burning issue4. Health staffs engaged in the service delivery and the beneficiaries utilizing them are very vulnerable unless driven, owned and firmly embedded by each other. Appropriate utilization of these essential client centred, need based, demand driven services largely depends on awareness, involvement, motivation and attitude of beneficiaries towards them. Moreover, illiteracy, ignorance, lack of awareness, age old customs, fixed firm false beliefs, deep rooted faith in traditional healers and lack of credibility of public health facilities and other related areas of their dissatisfaction are some other burning issues which need to be addressed5. Better health care decisions can be made by the beneficiaries only if they find the designated health care service planned for them accessible, affordable and available. Rural and hilly terrains are perceived to be neglected. Moreover dearth of literature on the issue in the area pertains. Thus in the aforesaid context the present study was conducted with the following objectives. To determine satisfaction among beneficiaries regarding various aspects of services provided to them. Categorization and prioritization of the relevant issues according to their varying degree of severity and addressing them by appropriate strategy designing and planned interventions.

Materials and Methods

AABS; 4(1): 2017 tape recorder , IEC materials were tools used and study techniques applied were exit - interview method for beneficiaries , in-depth interview with health staffs engaged in delivering the services, relevant record review, focused â&#x20AC;&#x201C;group-discussions (FGDs) to elicit and address issues and demonstration of IEC materials. Data Collection and Analysis: Ethical clearance was obtained from the institutional ethics committee, NBMCH and permission from requisite higher authorities and the concerned were duly obtained. Purpose and benefits of the study was explained to health staff and respondents. Anonymity and confidentiality of the study was ensured. Informed verbal voluntary consent from the respondents and other health staffs engaged in service delivery was obtained. Data was collected using appropriate tools and techniques. Questionnaires were administered not disrupting their routine work. FGDs conducted after their routine work also highlighted certain issues which were addressed simultaneously. Data was cleaned, collated cross-checked, compiled and entered in Microsoft Excel. Principles of descriptive and inferential statistics were applied for data analysis. Appropriate statistical software was used. Data was represented in forms of tables and charts in the result section. Assessment of Satisfaction: Satisfaction of the clients regarding various aspects of services provided to them at sub-centre level was assessed as follows:

Predesigned, pretested semi-structured schedule for beneficiaries and health staffs , relevant record review ,

Satisfaction was assessed using a modified questionnaire adapted from Client Satisfaction Questionnaire 8(CSQ8), which is commonly used to assess satisfaction of clients in health sector in and outside the country 3. The questionnaire after modification was translated, back translated then validated and pretested before final application. The questionnaire had 8 components each with one single item. The components were range of services provided, quality of services provided, crisis management services available, duration of waiting time spent before availing the service, availability of health staff at the clinic, behaviour of health staff, perceived involvement of the health staff in fields and recommending others. Each reported a single score measuring a single dimension of overall satisfaction. Thus for each component minimum response was 1and maximum response 5. Each respondent gave a score/ response for the single item in each component. Satisfaction for each component was assessed using a 5 point Likertâ&#x20AC;&#x2122;s scales where 1 - very dissatisfied,2- dissatisfied, 3- neither satisfied nor dissatisfied, 4- satisfied, 5- very satisfied 4.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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Descriptive, cross-sectional, hospital based study for six months (April â&#x20AC;&#x201C;August 2016) was conducted in TCU, NBMCH, a 500 bedded hospital situated in foot-hills of Darjeeling district. All beneficiaries attending the clinic on outdoor days comprised the sampling frame. Taking prevalence as 50% , 10% as allowable error and 20% non responders, final sample size was calculated to be 120. Applying systematic random sampling technique, the required study subjects were selected after calculating sampling interval. Inclusion criteria included those having outdoor ticket of the hospital and respondents not willing to participate in the study, seriously ill and having documented mental illness were excluded. It was noted that 40 beneficiaries (33.33%) were known to the service providers and 80 (66.66%) were unknown to them. Health staffs engaged in service delivery were also interviewed.

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Accordingly the level of satisfaction was categorized and compared. Collected data was analyzed and presented using the principles of descriptive statistics and studentâ&#x20AC;&#x2122;s t test was applied to compare the mean score of satisfaction between the two groups, respondents known to the health staffs working at the clinic and those not known to them, who had come on their own.

Result

120 beneficiaries were studied. It was noted that among them 40 respondents were known to the health staffs working at the TCU. There were no non-responder. Basic Descriptors: Majority of beneficiaries were Hindus. 72.5% and 46.7 % of beneficiaries were females and belonged to general caste respectively. 28.4% were illiterates though most of the beneficiaries resided in urban areas. Among respondents, 53.4% were adults and for the 17.2% minors, their respective caregiver opined on their behalf. (Table1, Fig.1) 80 beneficiaries (66.6%) were not known to the staff and had come on their own for availing the service. Respondents opined that they frequently visited the tertiary care hospital for availing various services. It was found that as high as 50% were aware about the purpose of functioning of TCU. Other findings: Client satisfaction among the beneficiaries was assessed on a 5 point Likertâ&#x20AC;&#x2122;s scale5. Levels of satisfaction of respondents were assessed for each of the 8 components individually, in relation to the various aspects of services provided to them. 53.4%, 47.5%, 49.2% and 53.4% of total respondents were satisfied with range of services / facilities provided to them, waiting time spent before availing the service, availability of the health staffs at the clinic and behaviour of health staffs at the clinic respectively. 50.8% were neither dissatisfied nor satisfied with quality and extent of regular services provided. Interestingly regarding the crisis management services available like arranging for blood transfusion, or reaching out a patient in times of acute need, respondents who came on their own were dissatisfied but those who knew the health staffs were neither satisfied nor dissatisfied with the service. However regarding recommending others to the clinic and regarding the involvement of the health staffs with the community during field visits, majority of respondents were neither satisfied nor dissatisfied. The mean score of satisfaction in 7 components among the two groups of respondents, those known and those not known to the health staffs of the TCU were found to be not significantly different. However regarding behaviour and humaneness of the health staffs, the mean score of

satisfaction was observed to be significantly different among the two groups. (Table 3) In depth interview with health staffs engaged in the clinic was conducted for ascertaining the various aspects of services provided and identifying the bottlenecks faced by them, if any. It was noted that though majority of respondents who were unknown to health staffs were dissatisfied with crisis management services. Most of the respondents were neither satisfied nor dissatisfied with quality of services available. However, they said they would recommend others as they had no other option. The health staffs engaged cited logistic constraints, poor amount of technical knowhow and improper functioning of the machines as their limitations. They also opined that beneficiaries unknown to them had no accountability and viewed them with suspicion and wanted more centres to be run, more deployment of other staffs during field visits, starting of chelation therapy so that accountability could generate among respondents. A designated senior staff who could monitor and provide supportive supervision would help further. Moreover duty performance would improve if appropriate infrastructural support and division of labour is ensured. However few issues could be resolved locally with apt managerial skills, proper technical knowhow, careful understanding of the topic and culture sensitive interventions. The problems were prioritized and categorized into four sections according to their varying degrees of severity. Thus remedies applying appropriate managerial skill and technical knowhow were attempted. (Table 4)

It was noted with intense awareness generation the attendance of the patients visiting the outdoor of the thalassemia clinic had increased. Respondents asked for pamphlets for their neighbours. Many adolescents and teenagers turned up and asked for relevant literature on the topic which was an important finding indicating the positive effect of awareness generation. Discussion

Community based studies are representative of actual situation prevailing in the area, as the tests and interviews are carried out in their natural settings. Rajbanshis emerged as of the most predominant ethnic group in this area. They are survivors of an aboriginal race.7 Significant association between Rajbanshis, living in the northern

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AABS; 4(1): 2017

Table 1a: Distribution of Study Subjects According to Age Total ( n=120 )

Age (yrs)

No. 64 35 21 120

Adults ( >18 yrs) 15 yrs.-18 yrs. Minors (<15 yrs) Total

Percentage 53.4 29.1 17.5 100

Table 1b: Distribution of respondents according to highest literacy status Total Respondents ( n=120)

Highest literacy status Illiterate Just literate Passed primary school Completed X Completed XII Graduate/above Total

No.

Percentage

34 29 25 14 12 6 120

28.4 24.2 20.8 11.7 10.0 05.0 100

Table 2: Assessing satisfaction among respondents Table 2 a: Distribution according to satisfaction levels regarding range of services provided Levels of satisfaction

A (n=80)

B (n=40)

Total (n= 120)

No.

Percentage

No.

Percentage

No.

Percentage

2.5

3.3

13.7

14.2

26.2

22.5

53.7

52.5

53.4

3.7

4.1

Table 2 b: Distribution according to satisfaction levels regarding waiting time spent Satisfaction levels

A (n=80)

B (n=40)

Total (n= 120)

No.

Percentage

No.

Percentage

No.

Percentage

16.2

7.5

13.3

27.5

22.5

47.5

11.2

12.5

11.7

Table 2 c: Satisfaction levels regarding availability of health staffs at clinic Satisfaction levels

A (n=80) No.

B (n=40)

Total (n= 120)

Percentage

No.

Percentage

No.

Percentage

2.5

4.2

22.5

18.4

27.5

12.5

22.5

41.2

49.2

3.7

5.8

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Table 2d: Satisfaction level regarding behaviour and humaneness of health staffs Satisfaction levels

A (n=80)

B (n=40)

Total (n= 120)

No.

Percentage

No.

Percentage

No.

Percentage

3.3

13.3

27.5

22.5

25.8

51.2

57.5

53.4

3.7

4.2

Table 2e: Satisfaction level according to the crisis management services available A (n=80)

Satisfaction level

No.

B (n=40)

Percentage

Total (n= 120)

No.

Percentage

No.

Percentage

7.5

8.4

38.7

37.5

13.7

12.5

3.7

4.2

Table 2 f: Satisfaction levels regarding quality and extent of regular services provided Satisfaction levels

A (n=80) No.

B (n=40)

Percentage

Total (n= 120)

No.

Percentage

No.

Percentage

3.7

7.5

27.5

26.7

51.2

50.8

12.5

14.2

3.3

Table 2 g: Satisfaction levels regarding involvement of health staffs in field Satisfaction levels

A (n=80)

B (n=40)

Total (n= 120)

No.

Percentage

No.

Percentage

No.

Percentage

3.7

5.8

27.5

28.4

38.7

35.8

22.5

24.2

7.5

5.8

Table 2 h: Satisfaction level regarding recommending others to the clinic Satisfaction levels

A (n=80)

B (n=40)

Total (n= 120)

No.

Percentage

No.

Percentage

No.

Percentage

3.7

7.5

27.5

26.7

51.2

50.8

12.5

14.2

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AABS; 4(1): 2017

Table 3: Mean satisfaction score in relation to several components of satisfaction Components of Satisfaction Mean score A (n=80) MeanscoreB(n=40) Range of services Quality of services Crisis management services Waiting time Availability of health staffs at clinic Behaviour of health staff Perceived involvement of health staff in fields Recommending others to the clinic

Statistical tests

3.43±0.87 2.84±0.80 2.68±0.94 3.44±1.05 3.39±0.88 3.16±0.99 3.00±0.94

3.38±0.98 2.83±0.93 2.65.±0.98 3.55±0.99 3.48±0.93 3.39±0.88 2.88±1.11

t = 0.28, p = 0.77 t = 0.76, p = 0.93 t = 0.13, p = 0.89 t = -0.56, p = 0.57 t = -0.50, p = 0.61 t = -2.91, p = 0.00 t = 0.64, p = 0.52

3.29 ±0.87

3.43 ±0.87

t = 0.32, p = 0.71

The values depict Mean ± Standard Deviation Table 4: Table categorizing and prioritizing the various relevant issues noted A B C D Low impact on performance Medium to high impact - low Requiring attention but Problems difficult to resolve severity easy to resolve Delay in arriving the centre, Not utilizing the service due to Logistic constraints – cited Ignorance of respondents so missed that day cultural festivals by health staff regarding the purpose of clinic Personal vengeance with More field involvement of health Improper inventory control Cultural perceptions of health staffs staff desired mechanism, condemnation respondents False beliefs procedures taboos of respondents , Came on a holiday Health staff on leave Illiteracy of caregiver No chelation therapy available Came for re-screening even Delay in counselling d/t mild Failure of co-ordination in Fear of loss of daily wage of if know not a carrier physical illness. the clinic respondent -

Forgot clinic location

Absence of monitoring and supportive supervision.

May be ignored

Low priority problem

Dissatisfaction of respondents Behaviour of health staff Take action to resolve

part of W.Bengal and HbE hemoglobinopathies exists. Prevalence of HbE trait was found to be highest amongst all hemoglobinopathies. Increased occurrence of HbE in this area is similar to found in Assam and Tripura hinting at lineage simulation between the Rajbanshis and these states. Hill tribes, mainly adivasis, also showed a high HbE occurrence, thus indicating intra racial marriages with Rajbanshis and acculturation. Adivasis, who emigrated into Northern part of West Bengal for tea cultivation from Chotanagpur plateau, their results also match with the residents of central east coast of India.2,5 A detailed comparative insight becomes imperative to understand the dynamics of adequate service utilization for assuring effective health outcomes. The study was a hospital based descriptive, cross-sectional study with a small sample size and may not be comparable to other studies in true sense but an attempt to interpret findings of present study in light of other studies will help create an overall view and a holistic approach. Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Lack of specific advice provided by the health care facility Dissatisfaction of respondents regarding crisis management services Prepare alternative solution to resolve

Majority of the respondents were illiterates. Similar to present a study conducted on utilization and coverage quality of antenatal care services in Bankura,W.Bengal found 52.4% respondents to be illiterates.6 Illiteracy poses a major hindrance for utilization of services. Client satisfaction is a burning issue and warrants immediate attention and interventions. Satisfaction of beneficiaries was thus assessed and compared with other studies. Various studies have been conducted on client satisfaction in health care settings, both inside and outside India. In a study on community health workers, Berman concluded that involvement in field activities embed the health workers are part of community and make them more acceptable enhancing overall service coverage. Study also noted that accessibility of workers combined with equity in services increases the trust and morale of the beneficiaries, especially those who are not known to the e-ISSN: 2349-6991; p-ISSN: 2455-0396

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health staffs. Vaccination camps were evaluated in urban areas of Mexico and in communities dispersed along a river in Amazon, Ecuador. It was seen that round the clock availability and good behaviour of the health staffs resulted in improved coverage.7

should be attempted with vigour and convergence of various health services and alerting physicians in the entire hospital for conveying the appropriate advice and referring the needy to TCU is required.

In another study it was seen that 95% respondents opined that health workers had friendly attitude and provided a range of good quality services. However inconvenient timings and long waiting time was a major obstacle in their service utilization. A study carried out in Delhi found that average total satisfaction score was 84.79% of maximum possible scores. It was found that human factors were more related with concepts of satisfaction than the physical factors8.

Acknowledgements

A study by U. Kapil on utilization of health care facilities by at risk children, found that in 37% cases prolonged waiting time , in 12.03 % cases unpleasant behaviour of hospital staff, in 16% cases non availability of drugs, in 35% cases inconvenience of timings and in 14% cases unsatisfactory treatment were reasons for not availing health services.8

Conclusion:

Present study concludes that satisfaction levels among the respondents vary. Alarmingly, significant difference exists between respondents known and unknown to health staff regarding their behaviour . Many local issues persist in the health care setting which can be resolved by monitoring, understanding and applying managerial skills. However some issues warrant larger and higher level intervention. On categorizing the problems according to their severity and priority various issues could be identified promptly. Timely interventions, supportive supervision go a long way in successful implementation of any program.

Recommendations

It is unfortunate for a tertiary care hospital with specialist services to suffer from lack of convergence of service. Appropriate referrals to TCU and opening of chelation therapies are need of the hour. Intensive awareness generation and appropriate health education imparted on relevant and related issues pave way for better service utilization. Efforts to improve community participation

However it is imperative that a larger community based study with liberal fund allocation and other logistic support is strongly recommended.

We are grateful to the staff of Thalassemia Control Unit for their support

Reference 1.

Goswami BK, Pramanik R, Chakraborty S, Pal PP, Banerjee S, Bandopadhyay A.Spectrum of Hemoglobin Variants in the Population of Northern Region of West Bengal: An Ethnogenetic Proposition, J Family Med Prim Care. 2014 Jul-Sep; 3(3): 219–223

Verma IC, Saxena R, Thomas E, Jain PK. Regional distribution of β -thalassemia mutations in India. Hum Genet. 1997;100:109-113.

Larsen DL, Attkisson CC, Hargreaves WA, Nguyen TD. Assessment of client/patient satisfaction: Development of a general scale, Evaluation and Program Planning. 1979; 197-207. Available from http://www.CSQscales.com /.[last accessed on 2016 July 26]

Tabish S A. Health Planning. In: Tabish SA,editor. Hospital and Health Services administration: Principles and Practice,3rd ed. New Delhi: Oxford University Press; 2005. p. 43-51.

Discussion Paper on Scales for Measuring Customer Satisfaction. Available from: www.marketdirectionsmr. com/wp-files/wp.../02/SurveyScales.pdf [last accessed on 2016 June 7]

Sinhababu A, Mahapatra BS, Das D, Mundle M, Soren AB, Panja TK. A study on utilization and quality of coverage of antenatal care services at the subcentre level. Indian J Public Health. 2006;50(1):49-52.

Aikat A, Biswas R. In search of a new accountability: the voice of patients. The Health 2011; 2(2):48-50.

Kapil U. Utilisation of health care facilities by “At risk” children. Indian J Community Med 1989; 14(2):83-4.

*Corresponding author: Dr Indranil Chakrabarti, Associate Professor, Department of Pathology, North Bengal Medical College, West Bengal, India Phone: +91 9433187448 Email: drinch@rediffmail.com Date of Submission : 04.02.2017 Date of Acceptance : 17.02.2017 Financial or other Competing Interests: None. Date of Publication : 21.02.2017

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Original Article DOI: 10.21276/AABS.2017.1313

A Study on Prevalence and Causative Factors of Megaloblastic Anaemia in Hadoti Region Lakshmi agarwal1*, Jayant Ramawat1, Manmohan Agrawala2 and Naresh Rai1 1 Govt Medical College, Kota, Rajasthan, India Cosultant Oncosurgeon, Pushpadi Cancer Care Centre, Kota, Rajasthan, India

ABSTRACT Background: Anaemia is the most common problems encountered by clinicians in Hadoti Region. The affected population includes male, female as well as children. Over the last two decades, it has been found that incidence of megaloblastic anaemia is increasing. Folic Acid and Vitamin B12 deficiency are the most common cause of megaloblastic anemia. Of these two micronutrients, Vitamin B12 deficiency is more common now, due to vegetarian life style of people. At present, Anemia control or prophylaxis program give only Iron and Folic acid. This study has been chosen to focus on this issue. The cases for increasing incidences of Folate / Vit. B12 deficiency needs to be elucidated. Objectives: To focus on the incidence of megaloblastic anaemia in Hadoti Region. & Probable Causative factors will be analysed Methods: All patients presenting to our hospital over a period of 2 months with a haemoglobin <10 g/dl and peripheral smear findings consistent with megaloblastic anaemia will be included in the study. Diet, drug intake, previous blood transfusion, presenting symptoms and other relevant history will be taken into consideration. Complete blood counts, peripheral film examination, reticulocyte count and cobalamin and folate assays will be recorded. Patients with chronic disease like renal disease, cancer, tuberculosis, liver disease etc will be excluded from the study. All data will be collected and evaluated statistically. Result: In the present study, total 500 patients who were admitted in medicine, paediatric and gynaecology ward were evaluated. All these patients met the inclusion criteria. Depending on the MCV value, serum assay and peripheral smear finding, they were categorised into 3 groups- Macrocytic, normocytic and microcytic anaemia. The normal MCV value but with megaloblastic blood film or low serum markers were considered into macrocytic anaemia. Total 100 patients were diagnosed as macrocytic anaemia. The sex distribution were-70(male), 30(female). Fifty five per cent of patients with cobalamin deficiency and 08% of patients with folate deficiency were found. All the patients were vegetarian and from middle class and low socio economic group. Conclusion: Cobalamin deficiency was responsible for megaloblastic anaemia in the majority of our patients. The supplementation program for Anemia control and prophylaxis should vary according to the regional requirements. Vitamin B12 should be included in the nutritional programme along with iron and folic acid. Awareness camp and Education program about megaloblastic anemia can be implemented for the prevention. Keywords: Megaloblastic Anemia, Increasing, Vitamin B12, Health Programme

Introduction

Anaemia is the most common problems encountered by clinicians in Hadoti Region. The affected population includes male, female as well as children. According to World Health Organization (WHO), the global prevalence of anaemia is 24.8%, which means about 1.62 billion people worldwide1.Overall health status of a person is judged on level of haemoglobin of a person.The high prevalence of anaemia is a serious health hazards for the economic development and productivity of the country Over the last two decades, it has been found that incidence of megaloblastic anaemia is increasing. Folic Acid and Vitamin B12 deficiency are the most common cause of megaloblastic anaemia. Of these two micronutrients, Vitamin B12 deficiency is more common now, due to

vegetarian life style of people.Vegetarianism has become increasingly popular among people including adolescents in current years perhaps because this diet is believed to offer health benefits. Cobalamin is synthesized by bacteria and is found in soil and in contaminated water. Foods of animal origin(e.g.meat,eggs,and milk) are the primary dietary sources. The amount of cobalamin in the average Western diet (5 to 15 mg/day) is more than sufďŹ cient to meet normal requirements. The body can store large amounts of cobalamin. Because of this, it can take 2 to 5 years for adeďŹ ciency to develop even in the presence of severe malabsorption2. As the totally vegetarians do not consume any animal products, they are at high risk of developing vitamin B12

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deficiency. It is generally agreed that in some communities the only source of vitamin B12 is from contamination of food by microorganisms. When vegetarians move to countries where there are high standards of hygiene and the rules of sanitation are stringent, there is good evidence that risk of vitamin B12 deficiency increases in adults and particularly in children born to and breastfed by women who are strict vegans3. In India with diverse ethnic populations, different dietary and social customs, the incidence of megaloblastic anaemia and its associated problems have not been adequately documented. Unfortunately, from public health view point, deficiency of B12 / folate has been regarded to contribute little to nutritional anemia4. Various workers have brought this to the forefront but in vain. At present, anaemia control or prophylaxis program give only Iron and Folic acid. This study has been chosen to focus on this issue

Material and Methods

It is a prospective and retrospective study. All patients presenting to our hospital over a period of 2months from 1staugust 2016 to 30th September 2016 with a haemoglobin <10 g/dl and peripheral smear findings consistent with anaemia were included in the study. Diet, drug intake, previous blood transfusion, presenting symptoms and other relevant history were taken into consideration. Complete blood counts, peripheral film examination, reticulocyte count and cobalamin and folate assays were recorded. Patients with chronic disease like renal disease, cancer, tuberculosis; liver disease etc was excluded from the study.

Results

In the present study, total 500 patients who were admitted in medicine, paediatric and gynaecology ward were evaluated. All these patients met the inclusion criteria. Depending on the MCV value, serum assay and peripheral smear finding, they were categorised into 3 groups-Macrocytic, normocytic and microcytic anaemia (table-1). The normal MCV value but with megaloblastic blood film or low serum markers were considered into macrocytic anaemia. Total 100 patients were diagnosed as macrocytic anaemia. The sex distribution were-70(male), 30(female). Fifty five per cent of patients with cobalamin deficiency and 08% of patients with folate deficiency were found(table-2). All the patients were vegetarian and from middle class and low socio economic group. Presentation: The predominant symptoms were • • • •

Neurological symptoms (50%), Fatigue (38%) Anorexia, Gastritis,

• Low grade fever, • Palpitation. Laboratory findings: TheMCV ranged from 70fL to 128fL. Peripheral smear revealed Macro-ovalocytes, Tear drop cells, Basophilic stippling, Polychromasia, Hyper segmented neutrophils or Pancytopenia. 30 patients had received blood transfusions for anaemia Table 1: distribution of different types of anaemia. Microcytic

350

Normocytic

Macrocytic

100

Total

500

Table 2:- Distribution of megaloblastic anaemia according to the causative factors Pure cobalamin deficiency

55%

Pure folate deficiency

10%

Combined deficiency

Unknown(reports not available)

27%

Discussion

Megaloblastic anaemiais a heterogeneous group of disorders that have common blood abnormalities and symptoms. It is a macrocytic anaemia that is usually accompanied by leukopenia and thrombocytopenia and specific bone marrow morphology affecting erythroid, myeloid and platelet precursors.5. Megaloblastic anaemia most commonly results from folate or cobalamin (vitamin B12) deficiency. Paul Ehrlich in 1880 first used the term megaloblast to describe the abnormal cells in the bone marrow of a patient with pernicious anaemia. Thomas Addison first described pernicious anaemia, the best known of the megaloblasticanaemia, in 1855. For a number of years the disease was known as Addisoniananaemia6. Anton Biermer in 1872 first used the term, pernicious anaemia. Vitamin B12 was first identified in the 1920’s by Minot and Murphy as being the extrinsic factor present in the liver which reverses the symptoms of pernicious anaemia, as reviewed by Markle and Okuda. The substance was isolated and named vitamin B12 in 1948. Hodgkin and coworkers determined the crystalline structure of vitamin B12 molecule in the 19507. Vitamin B12 is a term used for a group of physiologically active substances, which are chemically classified as cobalamin. The basic structure is a tetrapyrrole ring, acorrin, with a central cobalt atom and a purine nucleotide (5, 6-dimethylbenzimidazole) linked to it.

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A-33 The incidence of megaloblastic anaemiais increasing, althoughevidence for this may not be forthcoming easily. The diagnosis of vitamin B12 deficiency is not straightforward. There are no uniform diagnostic criteria and no single laboratory test constitutes a gold standard. Various criteria have been used in the literature for the diagnosis of this anaemia which includes macrocytosis of red cells, presence of megaloblastic changes in thebone marrow and subnormalmicronutrient levels. Gera et al shows an almost four fold rise in proportion of macrocytic anaemia cases over less than a decade at one center-2 % in1991 and 7.8% in 19998. A recent report reveals that 46.9% of non-anaemic adult subjects having subnormal levels of B12 or folate deficiency- vitamin B12 being five times more common than that of folate.A study from Mexico showed B12 deficiency in 19% - 41% of various population subgroups while no cases with folate deficiency were observed9. Another study on Guatemalan lactating mothers revealed B12 deficiency in 46% compared to 9% prevalence of folate deficiency10. Series on megaloblastic anaemia patients from Pakistan and Zimbabwe revealed B12 deficiency inover 50% cases while folate deficiency was seen in only 8% and 17% cases, respectively11. Sarode et al 12from Chandigarh reported B12 deficiency in nearly 85% cases of megaloblastic anemia. The same finding was also observed by Mukibi et al 13, Khanduri et al 14, 16 and various other workers. Gomber S et al in 199815 conducted a study on Prevalence &aetiology of nutritional anaemiaâ&#x20AC;&#x2122;s in early childhood in an urban slum. Randomly selected 300 children aged 3 months-3 year were analysed over a period of one year for estimating prevalence of nutritional anaemia. Pure iron deficiency anaemia (IDA) was detected in 41.4% of anaemic children. Vitamin B12 deficiency alone or in combination with iron was diagnosed in 14.4 and 22.2 per cent anaemic children respectively In the present study, microcytic anaemia was found to be the most common cause of anaemia in Hadoti region followed by megaloblastic anaemia.Cobalamin deficiency was responsible for megaloblastic anaemia in the majority of our patients (55% pure cobalamin deficiency and 10% combined deficiency) and pure folate deficiency in 8%. The serum assay of vitamin was not available in 27% of the patients. The MCV was found to be >90fl in 80% of the cases. In the rest of the cases, though MCV was normal, peripheral smear findings were characteristic. It includes Macro-ovalocytes, hyper segmented neutrophils, basophilic stippling. This could be because of concomitant iron deficiency.Pancytopenia was found in a few patients.

AABS; 4(1): 2017 anaemia. Bone marrow can also be done for the diagnosis. Since megaloblastic anaemia is a chronic disorder, most of the patients are well compensated. The symptoms arises when the haemoglobin is very low <4gm%. Blood transfusion is not urgency in such cases. The folate and vitamin B12 deficiency should be differentiated by the serum assay before the therapy is started. Most of the patients were found to be vegetarian.Even those who consider themselves as non-vegetarians usually consume meat only occasionally.Megaloblastic anaemia seen in infants and young children may be attributed to maternal deficiency which results in poor body stores at the time of birth.Cobalamincontent of breast milk is lower in vegetarian mothers and is directly proportional to serum cobalaminlevels. Brit et al alsoobserved higher prevalence of megaloblastic anaemia among vegetarian Indian settlers in UK17. The above findings suggest that most cases of megaloblastic anaemia are caused by nutritional deficiency of vitamin B12, folate or both. The other causes of deficiency account for minority of cases. Irrespective of the cause, vitamin B12 deficiency now appears to be more common than folatedeficiency in causing nutritional macrocytic anaemia.In India, the national nutritional program for anaemia control or prophylaxis gives only Iron and Folic acid. The various studies done earlier and the present study highlight the rise in incidences of vitamin B12 deficiency which needs to be elucidated. The government should acknowledge the burden of vitamin B12 deficiency and implement it in the anaemia control program. The vitamin B12 tablet should be distributed along with iron and folic acid. The other option which can be considered is fortifying foods with vitamin B12. This can help in achieving the target of decreasing the incidence of vitamin B12 deficiency and reduction of subclinical vitamin B12 deficiency. A few workers have suggested that fortification of foods with vitamin B12 may not be as effective due to malabsoption. But, in India inadequate diet is the main cause rather than absorption.

Conclusion

Complete blood count, peripheral smear and the serum assay of vitamins are the diagnostic tests of megaloblastic

The incidences of megaloblastic anaemia come next to iron deficiency anaemia. Data regarding the magnitude of the problem in Hadoti region of Rajasthan and the factors that might influence its incidence are lacking.Vitamin B12 deficiency causing megaloblastic anaemia is associated with other systemic manifestation and the morbidity is more. Our anaemia control programme only takes care of iron and folic acid deficiency. Vitamin B12 should be included in the national nutritional programme especially in the region where majority of the people are vegetarian. The Physician must keep in mind vitamin B12 deficiency in their differential diagnosis of anaemia.

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References

Sachdev HPS, Chandhary P. Nutrition in children: Developing country concerns Reprint. 1995; 1

Baker SJ, DeMaeyer EM. Nutritional anemia; its understanding and control with special reference to work of World Health Organization. Amer J ClinNutr 1979; 32: 368-417

10. Casterline JE, Allen LH, Ruel MT. Vitamin B12 deficiency is very prevalent in lactating Guatemalan women and their infants at three months postpartum. J Nutr 1997; 127: 1966-1970

Specker BL, Black A, Allen L et al. Vitamin B: Low milk concentration are related to low serum concentrations in vegetarian women and methylmalonicaciduria in their infants. Amer J ClinNutr 1990; 52: 1073-1076.

Chandra J. Megaloblastic anemia: Back in focus. Indian J Pediatr 2010; 77:795–9.

Antony AC. Megaloblastic anemia. In: Hoffman R, Benz EJ, Shattil SJ, FurieB,Cohen HJ, Silberstein LE, et al. (eds). Hematology. Basic principles and practice.4th ed. Edinburgh:Churchill Livingstone; 2005:519–56.

Addison T. Anaemia—disease of the suprarenal capsules. London Med Gazette1849;43:517–18.

G Richard Lee, John Foerster, John Lukens, FrixosParaskevas, John P Greer, George M Rodgers, Wintrobe’s Clinical Hematology 10th edition 1998, Williams &Wilkins A Waverly company volume 1 page 942.

Gera R, Singh ZN, Chaudhury P. Profile of nutritional anemia in hospitalized children over a decade. Conference Abstracts,38thNational conference of Indian academy of Pediatrics Patna 2001; HO-09, pp 60

Allen LH, Rosado JL, Casterline JE et al. Vitamin B12 deficiency and malabsorption are highly prevalent in Mexican communities. Amer J ClinNutr 1995; 65: 1013-1019.

11. Madood-ul-Mannan, Anwar M, Saleem M et al. Study of serum vitamin B12 and folate levels in patients of megaloblastic anemia in northern Pakistan. J Pak Med Assoc 1995; 45: 187-188. 12. Sarode R, Garewal G, Marwaha N, Marwaha RK, Varma S, Ghosh K, Pancytopenia in nutritional megaloblastic anemia: A study from north-west India. Trop Geogr Med 1989; 41:331–6. 13. Mukibi JM, Makumbi FA, Gwanzura C. Megaloblastic anemia in Zimbabwe: Spectrum of clinical and haematological manifestations. East Afr Med J 1992; 9: 8387. 14. Khanduri U, Sharma A, Joshi A. Occult cobalamin and folate deficiency in Indians. Natl Med J India 2005;18:182–3. 15. GomberS,KumarS,RusiaU,GuptaP,AgarwalKN,SharmaS. Prevalence and etiology nutritional anaemia in early childhood in an urban slum Indian J med Res.1998;73:107-269. 16. Khanduri U, Sharma A. Megaloblastic anaemia: Prevalence and causative factors. Natl Med J India 2007;20:172–5 17. Britt RP, Harper C, Spray GH. Pernicious anaemia in Indian immigrants in London area. Br J Haematol 1970; 18: 637-642.

*Corresponding author: Dr Lakshmi Agarwal, 34 A, Shreenathpuram, Near Ahinsha Circle, Kota, Rajasthan, India Phone: +91 9772464587 Email: drlaxmiagarwal@gmail.com Date of Submission : 05.02.2017 Date of Acceptance : 13.02.2017 Date of Publication : 21.02.2017

Acknowledgement : STS ICMR Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/AABS.2017.1339

A comparative Study of Clinicopathological Characteristics and Expression of Basal Markers (CK5/6 & EGFR) in Triple Negative and Non Triple Negative Breast Carcinomas in Kashmir Valley Subuh Parvez Khan, Syed Besina Yasin and Fiza Parvez Khan Department of Pathology, Sher e Kashmir Institute of Medical Sciences, Soura ,Srinagar, J&K, India

ABSTRACT Background: The aim of this study was to compare the clinicopathological characteristics of Triple Negative Breast Carcinomas with Non Triple Negative Breast Carcinomas (NTNBC). Evaluation of expression of Epidermal Growth Factor Receptor (EGFR), CK5/6 in TNBC and their comparison with NTNBC was done. Methods: 25 TNBC and 35 NTNBC were selected. The clinicopathological parameters of these two groups were compared. Each group was further immunostained for basal markers(CK5/6 and EGFR).The expression of markers in these two groups was studied and compared with each other. Results: The mean age of TNBC and NTNBC were 47 and 49 years respectively. The majority (48%) of cases from TNBC as well as NTNBC (45%)were in size range of 2-5 cm(T2 stage). IDC-NOS was predominant histological type seen in 92% of TNBC and 100% NTNBC.TNBCs had a significantly higher tumor grade than NTNBC at presentation. LVI was seen in 40% TNBC cases and 42% NTNBC cases. Majority (52%) of TNBC cases were node negative while majority (37.14%) of NTNBC cases belonged to N1 stage. IIA was the most common stage in 36% TNBC cases . In NTNBC, majority of the cases (34%) belonged to Stage IIIA.Expression of basal markers was significantly associated with triple negative breast cancers. Conclusion: TNBCs had a significantly higher tumor grade than NTNBCs at presentation. Expression of basal markers was significantly associated with TNBCs. Since EGFR was significantly associated with triple negative phenotype, TNBC could potentially benefit from EGFR targeted therapeutic strategies. Keywords: Cytokeratin, EGFR , Triple Negative Breast Neoplasms.

Introduction

Breast cancer is the second most common cancer in the world and, by far, the most frequent cancer among women1. Breast cancer is a heterogeneous disease and it encompasses a variety of entities with distinct morphological appearances and clinical behaviours. In recent years it has been evident that this diversity is the result of genetic alterations.2 Currently, the most widely used classification system of breast cancer combines histo-morphological information (such as histological subtype and grading) as well as TNM staging information.3,4 A new approach to classify breast tumours using molecular characteristics was first described by Sorlie et al.5 Triple-negative breast carcinomas lack the expression of estrogen receptor, progesterone receptor and Her2. Although patients with TNBC tend to have a poor prognosis, only chemotherapy is expected to be effective because no therapeutic targets have yet been established. Histological types of TNBCs mainly comprise of highgrade invasive ductal carcinoma, no special type [solid-

tubular carcinoma (or atypical medullary carcinoma), invasive ductal carcinoma with a large central acellular zone], typical medullary carcinoma, and metaplastic carcinomas. They can be classified into two subtypes: basal and nonbasal phenotype6. Basal type was defined as CK5-/6-positive and/or EGFR-positive, and nonbasal type was defined as having no expression of these two markers. Although the triple-negative phenotype has been considered as sufficient to identify the ‘basal-like’ tumours, increasing evidence has shown that the terms ‘basal-like’ and ‘triple-negative’ are not synonymous.2 The triple-negative group of breast cancer is not a homogeneous disease entity. However, a substantial fraction of these tumours belongs to the basallike tumour type, which does form a homogeneous group. The basal-like group comprises 8% to 20% of all breast cancers.7,5,8-15 The majority of these tumors are ductal of no special type, but occasionally are tubular mixed,14metaplastic,16 or medullary cancers.17They have common features including younger patient age, high

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histologic tumor grade, marked cellular pleomorphism, high nuclear-cytoplasmic ratio, lack of tubule formation, high mitotic index, frequent apoptotic cells, scant stromal content, a pushing border of invasion, central geographic or comedo-type necrosis. Basal-like cancer is associated with an aggressive clinical history, development of locoregional and distant metastasis (particularly in the first 5 years,12 shorter survival, and a relatively high mortality rate.13,14

Materials and Methods

The study was conducted in the Department of Pathology of Sher-e-Kashmir Institute of Medical Sciences (SKIMS), a tertiary care referral centre of Kashmir valley for a period of 7 years from December 2008 to November 2015.This study is approved by institutional review board.All the patients diagnosed with primary breast cancer enrolled in our hospital during this duration were analysed. 60 cases of invasive breast cancer were selected whose complete clinical profile and the paraffin block was available. Based on the hormonal status, the 60 cases were divided into 25 Triple Negative Breast Carcinomas(TNBC) and 35 Non Triple Negative Breast Carcinomas (NTNBC). The clinicopathological parameters of these two groups were compared. Triple negative category included those cases which were negative for ER, PR and Her 2. Non triple negative category included those cases which were ER/PR positive, Her 2 negative or ER/PR positive, Her 2 positive or ER/PR negative, Her 2 positive. Each group was further immunostained for CK5/6 and EGFR(Biocare antibodies,India).The expression of markers in these two groups was studied and compared with each other. Formalin fixed and paraffin embedded section were cut and placed on glass slides coated with 0.5% poly L lysine. Sections were kept in oven for half an hour at temp 55-60oC-section were deparaffinised by placing in xylene for 5 min. Slides were then dehydrated by transferring to absolute alcohol, 90% alcohol, and then to 70% alcohol followed by rehydrating the sections in running water for 10 min. Endogeneous peroxidase activity was blocked by placing slides in a mixture of methanol and hydrogen peroxide (9:1) for 20 minutes. For antigen retreival, either

heat treatment was given by transferring sections to citric acid buffer pH 6 (preheated) in an oven(for ER,PR,Her 2,CK 5/6)or enzyme treatment was given by using enzyme pronase(for EGFR). After cooling sections, sections were rinsed in distilled water and transferred to citric acid buffer for 5 min. Primary antibodies were added to the sections and left for overnight. In case of EGFR, primary antibody was kept for 30 minutes. Secondary antibody tagged with HRP was left for half an hour. Sections were then washed by citric acid buffer for 5 min. Few drops of 3,3 diaminobenzidine (DAB) were added to the sections for 5 minutes. Sections were washed and then counterstained with Haematoxylin. Slides were washed in water, dried and mounted in DPX. Positive and negative controls were used in all of the cases. Wherever there was a discrepancy, fresh slides were prepared and IHC was repeated with both positive and negative controls. ER and PR positivity was assessed using Allred Score system. Her 2 neu IHC score was calculated combining the intensity of stain and percentage of cells stained.18 Score of 0 and 1 were taken as negative, 2 as equivocal and 3 as positive. For CK5/6 and EGFR scoring, any weak or strong cytoplasmic and/or membranous invasive carcinoma cell staining was taken as positive.19 A statistical analysis was implemented by using the SPSS 16.0 version software. The Chi square test was conducted to assess the relationship between the immunohistochemical markers and other variables. The Fischer exact test was used when the expected cell counts were less than 5. A p-value less than 0.05 was considered significant.

Results and Observations:

The study was conducted on a total of 60 cases of breast carcinomas which included 25 cases of triple negative breast carcinomas (41.7%) and 35 cases of non triple negative breast carcinomas (58.3%) . All the patients were female (100%). Triple negative breast cancers had a significantly higher tumor grade than non triple negative cancers at presentation. Expression of basal markers was significantly associated with triple negative breast cancers. Comparision of TNBCs and NTNBCs is summarized in Table 1.

Table1: comparision of clinicopathological and immunohistochemical characteristics between TNBC and NTNBC group. CHARACTERISTIC â&#x2030;¤60 >60 Discharge Lump

TNBC (25 cases) Age 20 5 Symptoms 1 24

NTNBC (35 cases)

P value

24 10

>0.05

4 31

>0.05

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AABS; 4(1): 2017 CHARACTERISTIC T1 T2 T3 T4 IDC NOS IDC APO Medullary 1 2 3 present absent N0 N1 N2 N3 IA IIA IIB IIIA IIIB IIIC positive negative positive negative

TNBC (25 cases) Primary Tumor 10 12 1 2 Histopathological type 23 1 1 Tumor Grade 19 6 LVI 10 15 Nodal Stage 13 5 5 2 Tumor Stage 7 9 2 4 1 2 CK5/6 expression 10 15 EGFR expression 11 14

NTNBC (35 cases) 13 16 6 35 5 28 2 15 20 12 13 10 9 7 7 12 -

P value >0.05

>0.05

<0.05

>0.05

3 32

<0.05

6 29

<0.05

Fig. 1: Photomicrograph showing high power view of strong membranous immunoreactivity for CK 5/6 in breast carcinoma.(40X).

Fig. 2: Photomicrograph showing cytoplasmic and membranous immunoreactivity for EGFR in breast carcinoma.(40X).

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Discussion

Total number of cases in our study was 60.TNBC comprised 41.66% of total cases i.e. 25 cases and NTNBC comprised 58.33% of cases i.e. 35 cases. Age Distribution: The mean age of TNBC and NTNBC were 47 and 49 years respectively. Various studies show a significant relationship of TNBC with younger age. These include studies of Thike AA et al 20, Pillai SK et al21 and Rao C et al22 . Symptoms: Most common clinical presentation in our study was left sided lump . Left sided breast lump was the most common symptom in other studies as of Lakshmaiah KC et al23 and Suresh P et al.24 Primary Tumor: The majority (48%) of cases from TNBC were in size range of 2-5 cm (T2 stage) while as least number (8%) of cases belonged to T4 stage . Majority of cases (45%) from NTNBC were also in the T2 stage. No statistical significance was seen between primary tumor and tumor group. Our results are comparable to those of Yuan N et al25 and Pillai SK et al.21 Histological Type: Invasive Ductal carcinomasNOS(IDC-NOS) was the predominant type of tumor in TNBC comprising 23 out of 25 cases(92%).There was one case of IDC with apocrine differentiation and one case of medullary carcinoma. All the 35 (100%) cases of NTNBC were of IDC NOS type. There was no statistical correlation between histological type and tumor group. Gaopande V L et al26 and Qui J et al27 also found no statistically significant relation between histological type and tumor group. Tumor Grade: 19 out of 25 cases of TNBC were of Grade 2,hence the most common grade constituting 76% of cases. Grade 3 was seen in 6 cases(24%). In NTNBC group ,Grade 2 was seen in 28 cases accounting for 80% while it was followed by grade 1 seen in 5 cases(14.28%).Statistically significant association was found between tumor grade and tumor group with a p value of 0.037.Statistically significant association between tumor grade and tumor group was seen by other studies including. Li C Y et al. Li C Y et al28 conducted a study in Tianjin Medical University cancer Institute and Hospital, China in which they found 42.20% of TNBC cases belonged to Grade 2, and 34.47% cases belonged to Grade 3 versus 45.66% of NTNBC belonged to Grade 2 and 32.59% cases of NTNBC belonged to Grade 3.This association of tumor group with tumor grade was statistically significant. Some other studies which showed significant association between tumor group and tumor grade include those of Nabi MG et al29, Gaopande V L et al 26 and Albergaria A et al.30 Lymphovascular Invasion: The presence of Lymphovascular invasion (LVI) is an independent poor

prognostic factor.In our study,10 out of 25 (40%) TNBC cases and 15 out of 35(42%) of NTNBC cases had presence of LVI. No statistical significance was found between presence of LVI and tumor group. Our study is comparable to Tawfiq O et al 31 and Hashmi AA et al32 They also did not find any significant association of LVI with tumor group. Nodal Stage: 52% i.e. 13 out of 25 of TNBC cases were node negative .In NTNBC group, 37.14% i.e. 13 out of 35 cases belonged to N1 stage.No association was seen between nodal metastasis and tumor group. No statistical association was seen by Nabi MG29 ,Pillai SK21 . Tumor Stage: Tumor Stage is an important prognostic parameter. In our study IIA was the most common stage in TNBC seen in 9 out of 25 cases accounting for 36% .In NTNBC, majority of the cases i.e. 12 out of 35 cases(34%) belonged to Stage IIIA.No statistical correlation was seen between the tumor group and tumor stage. No association was also seen by Li C Y et al28 and Yuan N et al25 CK5/6 AND EGFR Expression: CK 5/6 was expressed in 10 (40%)cases from TNBC group(Figure 1)while as it was expressed in only 3(8%) cases from NTNBC group. The expression of CK5/6 was significantly higher in the TNBC group than the NTNBC group with a p value of 0.009. EGFR expression was present in 11 (44%) of TNBC cases(Figure 2) while as it was present in only 6(17%) of NTNBC cases. EGFR expression was significantly higher in TNBC group as compared to NTNBC group with a p value of 0.040. Our study found a significant correlation of basal markers with the negative hormone status. Our results are similar to many other studies carried out in reputable institution across the globe. Pintens S et al33 carried out a similar study in Belgium. They found basal markers expression in 88% of TNBC. On comparision with NTNBC, they found high association of basal markers with the TNBC than NTNBC. Similar significant correlation between basal like tumor and negative hormone status was seen by Pillai SK et al21 Choccalingam C et al32 ,Rakha EA et al35,Abulkhair O et al36 and Rao C et al22. Hence, in our study there was a significant overlap between triple negative carcinomas and basal like carcinomas. Oncologists may incorporate the use of targeted therapy in basal marker positive cancers as is currently being done in hormone receptor positive cancer. This may benefit a subgroup of patient population after carefully selecting them for this treatment.

Conclusion

In conclusion, triple negative breast cancers had a significantly higher tumor grade than non triple negative

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Acknowledgements

Dr. Kousar Sidiq, Senior Resident, Department of Community Medical Scienes, Government Medical College, Srinagar for helping in statistical analysis.

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35. 36.

breast cancers: an experience from Pakistan. Diagnostic Pathology.2014;9:43. Pintens S, Neven P, Drijkoningen M ,Van Belle V, Moerman P, Christiaens MR et al:Triple negative breast cancer: a study from the point of view of basal CK5/6 and HER-1: J Clin Pathol. 2009;62(7):624-8. Choccalingam C, Rao L, Rao S. Clinico-Pathological Characteristics of Triple Negative and Non Triple Negative High Grade Breast Carcinomas with and Without Basal Marker (CK5/6 and EGFR) Expression at a Rural Tertiary Hospital in India. Breast Cancer (Auckl). 2012; 6: 21–29. Rakha EA, Reis-Filho JS , Ellis IO. Basal-Like Breast Cancer: A Critical Review. Journal of clinical oncology. 2008; 26: 2568-2581. Abulkhair O, Moghraby JS, Badri M. Clinicopathologic features and prognosis of triple-negative breast cancer in patients 40 years of age and younger in Saudi Arabia. Hematology/Oncology and Stem Cell Therapy.2012;5(2):101-6.

*Corresponding author: Dr. Subuh Parvez Khan, Sher e Kashmir Institute of Medical Sciences, Soura, Srinagar. 190011, INDIA Phone: +91 9469343742 Email: khansubuh@gmail.com Date of Submission : 09.02.2017 Date of Acceptance : 19.02.2017 Date of Publication : 24.02.2017

Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/AABS.2017.1349

Cytological Diagnosis of Salivary Gland Lesions with Histopathological Correlation Ranbeer Singh1* and Pravin Sukhadeorao Pawane2 2

1 Dept. of Pathology, Government Medical College, Saharanpur UP, India. Department of Pathology, Maharaja Agrasen Medical College, Agroha, Haryana, India,

ABSTRACT Background: The aim of this study was to demonstrate the diagnostic accuracy of FNAC in various salivary gland lesions and itâ&#x20AC;&#x2122;s correlation with histopathology, which helps in the appropriate management of the patient. Methods: A total of 86 patients were included in this study that was to be investigated for the salivary gland lesions. FNACs were performed using 22 gauge needle. Surgical specimens were received from 68 cases only they were processed and slides were prepared. The cytological and histopathological stained slides were correlated clinically. Result: Out of total 86 cases 54 were male and 32 were female patients with male to female ratio of 1.7:1. Most common age of presentation was between 21-40 years and maximum patients (42) were in this age group. In this study the most common site of involvement was parotid gland, out of total 86 cases 65 occurred in parotid gland. Mucoepidermoid carcinoma was diagnosed in 3 (3.48%) cases. 2 (2.32%) cases found to have acinic cell carcinoma and both the patients (100%) were male. 2 (2.32%) cases found to have Ca. Ex. Pleomorphic adenoma. Metastatic deposit was found in 1(1.16%) patient. Conclusion: FNAC of the salivary gland is a safe and reliable technique in the primary diagnosis of salivary gland lesions and has a high diagnostic accuracy, though in case of some tumors due to variable cytomorphology, histological examination is must and proves to be accurate for diagnosis. This study concluded that FNAC of the salivary gland tumors is accurate, simple, rapid and cost-effective for the patient. Keywords: FNAC, Salivary Gland, Salivary Gland Tumors, Diagnostic Accuracy

Introduction

The technique of Fine Needle Aspiration was initially used to investigate lesions in salivary glands in 19201 with improvement and development in 1950 and 1960 and popularization in the seventies1. It is a minimally invasive diagnostic modality with minimum cost 1 used in diagnosis of neoplastic as well as non-neoplastic lesions and being able to differentiate between benign or malignant neoplasia with variable sensitivity and specificity. Salivary gland swellings can result from tumors, an inflammatory process, or cysts. Salivary gland tumors comprise less than 3% of all tumors of head and neck. About 80% are located in the parotids, 10% in the sub mandibular glands and the remainder being distributed between the sublingual and countless minor salivary glands (Nagarkar N et al 2004)2. Major salivary gland tumors tend to be benign whereas minor salivary gland tumors tend to be malignant (Epker B N et al 1969)3. Proper management of these tumors require an accurate diagnosis by the pathologist, correct interpretation by the surgeon, knowledge of surgical anatomy of salivary glands with a clear understanding of factors leading to recurrence and complications.

A review of the various recent studies shows that the diagnostic sensitivity of FNAC varied from 81-100%, that the specificity varied from 94-100% and that the diagnostic accuracy varied from 61- 80% 4-8. Hence, FNAC proves to be simple and accurate method for diagnosis and thus appropriate therapeutic management could be planned earlier. The present study was carried out to find out the prevalence of salivary gland neoplasms, to classify them as per WHO classification, to see associated conditions like sialadenitis, sialolithiasis coexisting with salivary gland neoplasms and to do cyto-histopathological correlation.

Materials and Methods:

The present study was carried out in the department of Pathology of Government Medical College and Hospital, Saharanpur, Uttar Pradesh. This study included 86 cases of salivary gland lesions that underwent FNAC over a period of 1 year (July 2015-June 2016). The clinical history and relevant clinical examination of all the patients was done. FNACs were performed using 22 gauge needles and 10 ml disposable syringes using Franzenâ&#x20AC;&#x2122;s syringe holder (Gun). The appearance, color, and texture of the aspirate was noted.

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.In each case, one -two wet fixed (Ether/Ethyl alcohol) smears for Papanicolaou stain, 3-4 dry fixed smears for Giemsa stain were prepared, and one-two smears were kept unstained for any further required stain. Only 68 cases were available for histopathology hence cytological and histopathological correlation was done in 68 cases only. We took histopathological diagnosis as gold standard. The diagnostic accuracy of FNAC was established. The detail clinical history and relevant clinical examination of all the patients has been done.

Result

Out of total 86 cases 54 (62.79%) were male and 32 (37.21%) were female patients with male to female ratio of 1.7:1. Most common age of presentation was between 21-40 years and maximum patients (42) (48.83%) were in this age group as shown in table no. 2. Out of total 42 cases in this age group 27 male and 15 were female patients. However lesions are present in any age group. The duration of lesions varied from few months to many years. Clinically the benign tumors presented as a painless swelling since many years while malignant tumors presented with persistent pain, fixity and rapid increase in size in a short duration. In this study the most common site of involvement was parotid gland, out of total 86 cases 65 (75.38%) occurred in parotid gland and 12 (13.95%) occurred in submandibular gland. Nine lesions were found in minor salivary glands of which 5 were in the palate, 3 in the floor of mouth and 1 in the lip as shown in the table 1. The most common gland involved is parotid gland.

On FNA out of total 86 cases, 62 (72.09%) were pleomrphic adenomas, and out of this 38 (61.29%) patients were male while 24 (38.71%) patients were females, 07 (8.14%) were of chronic sialadenitis, out of this 05 (71.42%) were male and 02 (28.58%) were female patients. Total 03(3.48%) were of sialadenosis out of which 01(33.33%) was male, 02(66.67%) were female patients. Benign parotid tumor was found in 1(100%) female patient that is (1.16%) of total 86 patients. Warthinâ&#x20AC;&#x2122;s tumor (Fig.1) was given in 2 (100%) male patients i.e. (2.32%) of total 86 patients, 3 cases were found to have cystic lesions out of which 2 (66.67%) were male and 1(33.33%) was a female patient shown in table no. 2. Mucoepidermoid carcinoma (Fig. 2, 3) was diagnosed in 3 (3.48%) cases out of which 2 (66.67%) were male and 1(33.33%) was a female patient. 2 (2.32%) cases found to have Acinic cell carcinoma (Fig. 4) and both the patients (100%) were male. 2 (2.32%) cases found to have Ca. Ex. Pleomorphic adenoma, out of which 1(50.00%) was male and 1(50%) was female patient. Metastatic deposit was found in 1(1.16%) patient and that was a (100%) male patient. On histological examination, out of total 86 cases histology was available in 68 cases. Out of 62 cases of Pleomorphic adenoma histology was available in 52 cases; in 10 cases we did not get histology. On histology the observations were as seen in table no. 3. Cytohistological correlation done. Out of total 86 cases diagnosed on cytology 55 (63.95%) were well correlated histologically while 13 (15.11%) cases not correlated and no histology available in 18 (20.93 %) cases as seen in table no. 4.

Table No.1: Shows site wise distribution of salivary gland lesions. Sr. No.

Glands Involved

No. of Cases

Case Percentage

Parotid gland

75.38%

Submandibular gland

13.95 %

Palate

5.81%

Lip

1.16%

Floor of Mouth

3.48%

Total Cases

100 %

Table No.2: Cytological Diagnosis. Sr.No.

FNA Diagnosis

Males

Females

Total Cases

Percentage

Pleomorphic adenoma

38 (61.29%)

24 (38.71%)

72.09 %

Chronic Sialadenitis

05 (71.42%)

02 (28.58)

8.14 %

Sialadenosis

01 (33.33%)

02 (66.67%)

3.48 %

Benign Parotid tumor

00 (00.00%)

01 (100%)

1.16 %

Warthinâ&#x20AC;&#x2122;s tumor

02 (100%)

00 (00.00%)

2.32 %

Cystic Lesions

02 (66.67%)

01 (33.33%)

3.48 %

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Sr.No.

FNA Diagnosis

Males

Females

Total Cases

Percentage

Mucoepidermoid Carcinoma

02 (66.67%)

01 (33.33%)

3.48 %

Acinic Cell Carcinoma

02 (100%)

00 (00.00%)

2.32 %

Ca Ex Pleomorphic adenoma

01 (50.00%)

2.32 %

10.

Metastatic Deposits

01 (100%)

00 (00.00%)

1.16%

Total

54 (62.79%)

32 (37.21%)

Table No. 3: Shows FNA diagnosis and their variations on histopathology Sr.No FNA Diagnosis Histopathology Diagnosis No.of cases Diagnosis 1. Pleomorphic Adenoma Basal cell adenoma Warthin’s tumor Myoepithelioma, Pleomorphic Adenoma 62 Benign lymphoepithelial cyst, Mucocele Sialadenosis Mucoepidermoid carcinoma.

100%

Cases 41 02 02 01 01 02 02 01

Histopathology not available

Benign parotid tumor

Pleomorphic adenoma

Warthin’s tumor

Cystic lesions

Mucocele

Ca. Ex. Pleomorphic adenoma

Mucoepidermoid carcinoma

Acinic cell carcinoma

Metastatic deposit

Chronic sialadinitis

10.

Sialoadenosis

Total

Table no. 4: shows Cytohistological correlation of salivary gland lesions. Sr.No. Cytology

No. of cases Histology correlated

Histology Histology not correlated

Histology not available

Pleomorphic Adenoma

41 (66.12%)

11 (17.74%)

Chronic sialadinitis

03 (42.85%)

Sialadenosis

02 (66.67%)

Benign parotid tumor

01 (100%)

Warthin’tumor

01 (50%)

Cystic lesion (Mucocele)

02 (66.67%)

Mucoepidermoid carcinoma.

02 (66.67%)

Ca. Ex. Pleomorphic adenoma

01 (50%)

Acinic cell carcinoma

02 (100%)

10.

Metastatic deposit

01 (100%)

55 (63.95%)

13 (15.11%)

18 (20.93%)

Total

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Fig. 1: FNAC, Cytomorphology of Warthinâ&#x20AC;&#x2122;s tumor showing cluster Of oxyphilic cells and scattered lymphocytes (Giemsa stain 40 X).

Fig. 2: Cytomorphology of mucoepidermoid Carcinoma showing intermediate cells with abundant vacuolated cytoplasm (Giesma stain 40 X).

Fig. 3: Histology of Mucoepidermoid Carcinoma showing mucin filled spaces and mucinous cells. (H&E Stain 40 X).

Fig. 4: Histology of Acinic cell Carcinoma showing papillary structures With Acinic cells (H&E Stain 40 X).

Discussion

be due to variation in the habits as tobacco, smoking, alcohol consumption as well as exposure to occupational hazards is more in males than females, in this study we found that most of the male patients were smoker and tobacco user. The commonest benign lesion in this study is pleomorphic adenoma and 41 (66.12%) cases of pleomorphic adenomas are correlated cytologically and histologically. The mucoepidermoid carcinoma is the most common malignant lesion reported in this study that is correlated with Nguansangiam et al9. The common nonneoplastic lesion was chronic sialadenitis, common in submandibular gland this is in concordance with study done by Atula T et al10. Cytomorphologically the diagnosis of mucoepidemoid carcinoma may have resemblance with pleomorphic adenoma, Ca. Ex. Pleomorphic adenoma and

In our study most common age of presentation was between 21-40 years and maximum patients 42 (48.83%) were in this age group. Out of total 42 cases in this age group 27 male and 15 were female patients. The duration of lesions varied from few months to many years. Clinically the benign tumors presented as a painless swelling since many years while malignant tumors presented with persistent pain, fixity and rapid increase in size in a short duration. In this study the most common site of involvement was parotid gland, out of total 86 cases 65 (75.38%) occurred in parotid gland and 12 (13.95%) occurred in submandibular gland. 9 lesions were found in minor salivary glands of which 5 were in the palate, 3 in the floor of mouth and 1 in the lip. We observed that tumors both benign as well as malignant are more in male than in females this may

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Conclusion

Pleomorphic adenoma was the commonest benign tumor while mucoepidermoid carcinoma was the commonest malignant tumor in the present study and the incidence of neoplastic lesions were high in major gland while nonneoplastic lesions were high in minor salivary glands. The overall accuracy in cytohistopathological correlation varied from 63.95% to more than 80% depending on the availability of histopathology. FNAC of salivary gland lesions can be more accurate but it has certain limitation in the form of limited availability of histology and limited histological architectural features. However multiple sampling and increasing experience help to minimize the errors12. Thus FNAC is safe and cost effective outpatient procedure. F NAC continues to be a reliable diagnostic technique. It is advantageous both for the patients and the clinicians because of its quick results, accuracy, costeffectiveness, and lack of complications to the patient.

Acknowledgements

Departmental head: Dr. Nausad Husain has given technical support.

Reference 1.

Mahendranath P, Vijay kumar K. Parotid Tumours: ACytohistopathological Correlation of 36 Cases in Khammam.IJSR, volume:4 Issue 7, July 2015.ISSN No. 2277-8178

Nagarkar N.M., Bansal S., Dass A. et al--Salivarygland tumors. Indian Journal of otolaryngology and head and neck surgery. 56(1): 31 – 34, 2004.

AABS; 4(1): 2017 3.

Epker B.N. and Henry F.A. — Clinical histopathologicand surgical aspects of Intraoral minor salivary glandtumors. Journal of oral surgery. 27: 792-804, 1969.

Jayaram G, Dashini M. Evaluation of fine needle aspiration cytology of salivary glands: an analysis of 141 cases. Malays J Pathol. 2001; 23: 93-100.

Stow N, Veivers D, Poole A. Fine–needle aspiration cytology in the management of salivary gland lesions: an Australian experience.Ear Nose Throat J. 2004; 83:128-31.

Rehman H, Khan MS, Wahid F, Ahmad I. A profile of parotid gland tumours from a tertiary care hospital in Peshawar. JPMI 2011; 25:158-62.

Lukas J, Duskova J. Fine needle aspiration biopsy in the diagnosis of tumors and non neoplastic lesions of salivary glands. Bratisl Lek Listy.

2006; 107: 12 - 5.

Piccioni LO, Fabiano B, Gemma M, Sarandria D, Bussi M. Fine needle aspiration cytology in diagnosis of parotid lesions. Acta Otorhinolarygol Ital. 2011; 31:1

10. NguansangiamS. JesdapatarakulS. DhanarakN., and Sosrisakorn K., “Accuracy of fine needle aspiration cytology of salivary lesions: routine diagnostic experience in Bangkok,Thailand,”Asian Pacific Journal of Cancer Prevention,2012; 13(4):1583–1588. 11. AtulaT. GrenmanR., and LaippalaP., “Fine-needle aspiration cytology of submandibular gland lesions,”The Journal of Laryngology & Otology, 1995; 109 (9):853–858. [7]. 12. Layfield L. J. and Glasgow B. J., “Diagnosis of salivary gland tumors by fine-needle aspiration cytology: a review of clinical utility and pitfalls.” Diagnostic Cytopathology, 1991; 7(3): 267–272. 13. Jaiswal K N,Johari S P, Shrivastav A C, Shrikhande AV.” Study of Salivary Gland Neoplasms. Indian Medical Gazette-March 2015, 96-100.

*Corresponding author: Dr Ranbeer Singh, H No: 2013, sector 16, 17, Hisar, Haryana 125005. India Phone: +91 091-01662-9034944345 Email: drpravinpawane@gmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 12.02.2017 Date of Acceptance : 21.02.2017 Date of Publication : 25.02.2017

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

Original Article DOI: 10.21276/AABS.2017.1351

Evaluation of Sympathetic Reactivity by Hand Grip Exercise Test in Children of Hypertensive Parents Ajay Kumar* and Sonu Ajmani Department of Physiology, Netaji Subhash Chandra Bose Medical college Jabalpur, M.P, India

ABSTRACT Background: Genetic factors likely play some role in causing a high blood pressure. About 40% of patients with primary hypertension have genetic predisposition. It is a well documented fact that sympathetic dominance increases heart rate and blood pressure and the same has been observed in young normotensive off springs of hypertensive parents. Aim: Our present study was focused to evaluate blood pressure responses to isometric hand grip exercise test in children of hypertensive and non- hypertensive parents. Methods: The present study was conducted in 50 healthy subjects doing MBBS, 25 of whom had a positive family history and 25 had no family history of hypertension. Isometric hand grip test for sympathetic evaluation was carried out in both the groups following measurement of resting blood pressure. Rise in diastolic blood pressure measured during isometric hand grip taken as a test response. Result: Results were tabulated and analysed using paired t test, p value <0.001 was considered as significant. Higher values were obtained in diastolic and systolic blood pressure in children of hypertensive parents at rest when compared to age matched controls while during isometric hand grip increase in diastolic blood pressure in offspring of hypertensives was statistically significant. Findings are suggestive of an increased sympathetic dominance in children of hypertensive. Conclusion: The present study suggests that healthy children of hypertensive parents have a higher sympathetic dominance when compared to healthy children of non hypertensive parents. Keywords: Isometric Hand Grip, Blood Pressure, Normotensive Offspring.

Introduction

Hypertension is one of the most important causes of premature deaths occurring worldwide and thus is claimed to be a silent killer by the World Health Organization. In India, hypertension is the third most important risk factor burdening both cardiovascular health and health care systems in the country. It is one of the major risk factor for cardiovascular mortality which accounts 20-50% of all deaths. There is also a duet relation between cardiovascular risk factors and blood pressure, the higher the blood pressure, the higher the risk of both stroke and coronary events. As per the studies conducted by Raghupati Anchala, thirty three percent of urban and twenty five percent of rural Indians are hypertensive1. Fortunately early diagnosis can prevent about 3 lakh deaths in India.2 increasing prevalence of the condition is blamed on life style factors, such as stress, physical inactivity, a salt rich diet created by processed , fatty food and alcohol and tobacco use.3 Genetic factors also play important role in causing a high blood pressure. About 40% of patients with primary hypertension have genetic predisposition.4 Several genes are implicated that encode for the onset of hypertension. Few of them are genes that encode for components of renin angiotensin and

aldosterone synthesis, Angiotensin converting enzyme, angiotensin receptor, atrial natriuretic peptide etc. Moreover there is a twofold increase in occurrence of cardiovascular disease which include coronary heart disease, congestive heart failure, stroke and renal failure in patients suffering with hypertension.5 Occurrence of high blood pressure before a person reaches 55 years of age occurs 3.8 times more frequently among persons with a family history of hypertension.5 Children with one hypertensive parent have a 25% probability of developing high blood pressure and in children with both the parents being hypertensive the probability of developing hypertension increases to 50%.6 It is likely that people with a family history of high blood pressure share common environments and other potential factors which might increase their risk of developing hypertension. Heredity combined with unhealthy life style choices, such as smoking cigarettes and eating junk food makes a person more susceptible to hypertension. It is a well documented fact that sympathetic reactivity increases heart rate and blood pressure and the same has been observed in young normotensive off springs of hypertensive parents.7,8They also manifest an elevated level

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of blood pressure during and after five minutes of exposure to physical stress.9 Certain other researchers found no sympathetic dominance in children born to hypertensive parents.10 Hence evaluating cardiac autonomic function may prove to be useful as a predictive tool in diagnosis of impending high blood pressure.

on three separate occasions to confirm that volunteers were normotensive. Only those subjects whose basal blood pressure in sitting posture was normal were included in the study. After the subjects rested for a period of five minutes blood pressure was recorded in them using auscultatory method and documented.

Isometric hand grip dynamometer is a simple tool which can be used with ease. Hence, using a dynamometer to demonstrate autonomic modulation is a simple, noninvasive and inexpensive method. While performing exercise using a dynamometer there is an increase in heart rate and blood pressure due to stimulation of efferent sympathetic pathway.11Also an increased tension produced in muscles due to isometric contraction increases the arterial pressure with a small increase in heart rate and cardiac output. In addition there is also a rise in peripheral vascular resistance which accounts for increase in diastolic blood pressure.12 The present study was undertaken to evaluate increase in sympathetic reactivity in children of hypertensive parents and compare them with children of non hypertensive parents.

Isometric hand grip dynamometer of the spring type was used in the study. Subjects were briefed on how to use the instrument before conducting the procedure. The paddles of the dynamometer were compressed with maximum effort with the dominant hand. The dynamometer has two needles, one remains at zero while the other needle portrays maximal voluntary contraction. The subjects were made to perform the same maneuver thrice with a brief interval of 30 seconds to prevent fatigue. The readings were documented respectively. Mean of the three readings was calculated and documented (Tmax). Thirty percent of the Tmax was calculated and subjects were asked to maintain the same for a period of two minutes. Blood pressure was recorded simultaneously in the non dominant arm during the procedure and after a rest of five minutes. Blood pressure in resting state and during the procedure was compared between groups.

Materials and Methods:

Result

The present study was undertaken in LLRM Medical College, Meerut after an ethical clearance from the institution and completed within a period of 12 months. The study group comprised of 50 volunteers both males and females in the age group of 18 to 24 years. Height and weight of the subjects were matched. Out of them 25(males & females) subjects had a positive family history of hypertension (FH+) and other 25(males & females) subjects belong to non-hypertensive parents (FH-). Selection criteria: The present study was conducted on healthy medical students doing MBBS in the same college. The medical students were considered as study group whoâ&#x20AC;&#x2122;s one or both the parents are hypertensive of varying duration and being treated with anti hypertensive drugs and other medical students of normotensive parents were considered as controls. All subjects included in the study were normotensive. All healthy subjects in age group of 18-24 years, non-smokers and having normal range of BMI ( 18.5-24.9 kg/mÂ˛) included in the study, while subjects not giving consent, suffering from any acute illness, Diabetes Mellitus, smokers and any direct metabolic disorder were excluded from the study.

Results were tabulated and analysed using paired t test. p value <0.001 was considered as significant. Table 1 gives an idea of distribution of body mass index of both control and study group. Average body mass index control was 21.52 kg/m2 and that of study group was 21.06 Kg/m2. There was not much significant difference in average body mass index in both the groups. The body mass index was found in normal range (normal range 20-25 kg/m2). Table 2, 3 shows that there was a significant increase in systolic blood pressure and diastolic blood pressure in children of hypertensives before performing isometric hand grip between control and study group. Table 4 shows the mean increase in Diastolic and Systolic blood pressure during hand grip test which was more in study group as compared to the age matched controls and statistically significant.

Discussion

Blood pressure was recorded using auscultatory method with a mercury type sphygmomanometer of Diamond make. Basal blood pressure in sitting posture was recorded

The present study shows blood pressure and mean arterial pressure were significantly higher in off springs of hypertensive parents when compared to children of normotensive parents. While performing isometric hand grip manoeuvre there was a significant increase in systolic blood pressure and diastolic blood pressure in the study group which was taken as a test response. Five minutes after performing isometric hand grip maneuver, parameters returned to normal in both the groups. Isometric exercise

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TABLE 1: Distribution of Body Mass Index (Bmi) In Offspring of Normotensive (Controls) and Hypertensive (Study Group) Parents. Controls

Study group

BMI ( kg/m²)

Mean

≤ 22

18.37

1.00

18.46

0.99

22-24

21.50

0.96

21.66

1.02

≥ 24

24.69

1.24

24.19

0.50

Total

21.52

2.61

21.06

2.25

Table 2: Mean ± Sd of Systolic Blood Pressure (Mm of Hg) in Controls and Study Group Before Hand Grip Test. controls

study group

Age (yrs)

Mean systolic BP

95% confidence

Mean systolic BP

95% confidence

19-20

114.36

4.17

111.9-116.82

119.63

7.78

115.03-124.23

21-22

116.66

5.00

113.39-119.93

119.00

4.78

115.69-122.31

23-24

121.60

2.60

119.32-123.88

120.00

6.06

115.15-124.85

Total

116.64

4.92

114.71-118.57

119.52

6.30

117.05-121.99

Table 3: Mean ± Sd of Diastolic Blood Pressure (Mm of Hg) in Controls and Study Group Before Hand Grip Test. Controls

study group

Age (yrs)

Mean Diastolic BP

95% confidence

Mean Diastolic BP

95% confidence

19-20

77.09

5.95

73.57-80.61

79.09

5.46

75.86-82.32

21-22

79.77

4.84

76.61-82.93

82.50

2.07

81.07-83.93

23-24

80.00

2.00

78.25-81.75

81.73

5.36

75.71-84.29

Total

78.64

5.02

76.67-80.61

80.40

4.69

78.56-82.24

Table 4: Mean ± Sd of Rise in Diastolic and Systolic Blood Pressure During Hand Grip Test. Control group (25)

Study group (25)

P value

Diastolic blood pressure

19.36 ± 2.05

26.72 ± 2.93

0.001

Systolic blood pressure

14.67 ± 12.88

17.07 ± 12.32

0.001

when performed causes an increase in intramuscular pressure which in turn compresses blood vessels within muscles. Compression of the vessels results in decreased muscle blood flow which in turn causes an accumulation of metabolites within the muscle that stimulate muscle chemo receptors resulting in an increase in sympathetic nerve activity.13 This in turn causes an increase in blood pressure during isometric exercise . Although systolic blood pressure increased in both groups, but it was more significant in cases as compared to age matched controls. Similar findings were reported by Schneider GM et al and Lopes et al.7, 14 The results obtained in this study are suggestive of a sympathovagal imbalance in off springs of hypertensive parents. This could be explained by findings obtained by Sherwood et al. They suggested that in the early stages there is increase in cardiac output following which peripheral resistance increases in children of hypertensive owing to rise in blood pressures.15 Nor- epinephrine when

infused in children of hypertensive have a greater pressor response was seen due to reduced threshold levels to nor epinephrine in them.16

Conclusion

The present study suggests that healthy children of hypertensive parents do have a sympathetic dominance when compared to healthy children of non hypertensive parents. The exaggerated sympathetic response seen in stress may be used as a marker to detect people who may develop hypertension in future. Simple and noninvasive tests are more useful for early detection of altered autonomic functions. The regular autonomic function testing might be beneficial to find hyper reactors who have positive family history. Such periodical analysis of autonomic function tests might help in early detection of future hypertension and may prove beneficial in reducing the global burden of hypertension on health care system.

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Acknowledgements

References

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P. K Budholia, Ajay Mishra, G.D Mehra 1.

Raghupathy Anchala, Nanda K. Kannuri, Hira Pant, et al. Hypertension in India: a systematic review and meta-analysis of prevalence, awareness and control of hypertension. Journal of Hypertension 2014, 32:1170-77.

Gupta R, Gupta VP. Hypertension epidemiology in India: lessons from Jaipur heart watch. Current Science.2009; 97:3.

Das SK, Sanyal K, Basu A. a study of urban community survey in India: growing trend of high prevalence of hypertension in a developing country: Int J Med Sci.2005; 2:70-8.

Stolraz K, Grodzicki T, Lubaszawski W et al. heart rate variability in offspring of hypertensive parents. Przegl Lek.2002; 59:892-94.

Kasper, Fauci, Hauser, Longo, Jameson, Loscalzo.In: Theodore A.Kotchen EDS. Hypertensive vascular disease.: Harrisons Principles of internal medicine. New York.Mc Graw Hill education.2012:1611-26.

Biagini M et al. Blood pressure response to exercise in young subjects with and without parental history of hypertension .J Hum Hypertens.1996; 10(3):81-3.

11. Mathias CJ, Bannister R. Investigation of autonomic disorders. In. Bannister R, Mathias C eds. Autonomic failure: A textbook of clinical disorders of the autonomic nervous system. Oxford. Oxford University Press.1992; 266. 12. Dos Santos Antonio AM, Cardoso MA, do Amaral JAT, de Abreulc, Valenti VE. Cardiac autonomic modulation adjustment in isometric exercises. Medical express.2015; 2(1):150102. 13. Friedman DB, Peel C, Mitchell JH. Cardiovascular responses to voluntary and non voluntary static exercise in humans. J Appl Physiol.1992; 73(5):1982-5. 14. Lopes HF and Cansolin FM. Increased sympathetic activity in normotensive offspring of malignant hypertensive parents compared to offspring of normotensive parents. Braz J Med Biol Res.2008;41:849-53.

Olson RP and Kroon JS. Behavioural treatment of essential hypertension. Biofeedback: A Practionerâ&#x20AC;&#x2122;s Guide edited by Schwartz MS (the Guilford Press: New York) 316-339.

Schneider GM, Jacobs DW, Gevirtz RN, et al. Cardiovascular hemodynamic response to repeated mental stress in normotensive subjects at genetic risk of hypertension. J human Hypertension.2003; 17:829-40.

15. Sherwood A, Allen MP, Obrist PA and Langer AW. Evaluation of beta- adrenergic influences on cardiovascular and metabolic influences on cardiovascular and metabolic adjustments to physical and physiological stress. Psychophysiology.1986; 23: 89-104.

Wilson MW,Sung BH, Pincomb GA, et al. Exaggerated pressor response to exercise in man at risk of hypertension. Am J Cardiol.1990;66:731.

16. De Lima JJ, Dias MM, Bernardes â&#x20AC;&#x201C;Silva H, Belloti G. Pressor response to nor-epinephrine in essential hypertension. A study in families. Hypertension.1990:15(2):137-139.

*Corresponding author: Ajay Kumar, house no 14 Sai colony Dhanwantri Nagar Jabalpur M.P 482003 Phone: +91 9516639699

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 12.02.2017 Date of Acceptance : 20.02.2017 Date of Publication : 25.02.2017

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

Original Article DOI: 10.21276/AABS.2017.1316

Characterization and Activity of Antimicrobial Polypeptide of Bacillus Spp from Wastelands of Kathmandu Valley Masna Rai1, Sagar Aryal2* and Pramila Parajuli1 1 Department of Microbiology, St. Xavierâ&#x20AC;&#x2122;s College, Kathmandu, Nepal Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal

ABSTRACT Background: Bacillus genus have been found to produce antimicrobial polypeptides that have a remarkable capability to combat the current increasing problem of antibiotic resistance. The aim of this cross sectional study was to determine the proportion and pattern of distribution of antibiotic producing and non-producing species of Bacillus in soil and also to analyse the activity of the antibiotic producers. Methods: A total of 40 soil samples were collected from the wastelands of Kathmandu Valley and 121 isolates were studied from them. They were identified according to Bergeyâ&#x20AC;&#x2122;s Manual. The antibiotic were extracted and the activity was analysed by agar- well diffusion method against the test organisms Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Result: Bacillus subtilis species were found to be in highest number (17.3%) and Bacillus macerans (0.82%) in lowest number. 18.33% of Bacillus isolates were found to produce antibiotics. The highest number of antibiotic producing isolate was B. subtilis (40.09%) and the least was B. circulans (4.54%). The number of antibiotic producing bacteria were not found to be affected by moisture content of soil and pH change. Among the antibiotic producers, 63.63% produced antibiotic against Gram positive bacteria, 27.27% against Gram negative bacteria and B. alvei (9.09%) had a broad spectrum activity. Some colonies were even found to produce antibiotic with a greater potential than the conventional antibiotics. Conclusion: Based on property of soil, moist-neutral soil had the highest number of bacilli isolates and dry-acidic and moist-alkaline had least number of isolates. Keywords: Bacillus, Characterization, Crude Antibiotic, Antimicrobial Activity, Kathmandu

Introduction

The current leading problem of the world is that the emergence of drug resistant strains of pathogens is more rapid than the rate of discovery of new drugs and antibiotics (1). In this regard, Anti-Microbial Polypeptides (AMPs), which are ubiquitous gene-encoded natural antibiotics, provide a promising alternative for a new generation of antibiotics (2, 3, 4).

The genus Bacillus is capable of producing a large number of AMPs and is therefore viewed as a promising starting point in the search for new inhibitory substances (8, 9). Several studies have shown that members of the genus Bacillus produce a wide arsenal of antimicrobial substances, including ribosomally and nonribosomally synthesized lipopeptides, bacteriocins, and other kinds of peptides (10, 11, 12).

Conventional antibiotics generally target metabolic enzymes that may selectively develop resistance, whereas AMPs kill microbes primarily through the generation of membrane pores, thus making it inherently more difficult for the organisms to develop resistance (5). Since AMPs are a natural part of the antimicrobial defense system, the possibility of developing pathogen resistance or unwanted side effects is less likely than with chemically synthesised conventional antibiotics. Moreover, compared with conventional antibiotics, which are mostly active against bacteria or fungi, AMPs are considered to be antibacterial, antifungal, and antiviral drugs that could be used in the treatment of infectious diseases and parasitic infections and may also be suitable for the treatment of cancer and HIV infection (2, 3, 6, 7).

The first class of AMPs comprises ribosomally synthesized peptides, including bacteriocins whereas the second class comprises small microbial peptides synthesized enzymatically by non-ribosomal pathways (13). The Bacteriocins and Bacteriocin-like inhibitory substances (BLIS) contain lanthionine and/or methyllanthionine residues employed to form a ring through intramolecular post-translational modifications like subtilin, a 32-aminoacid pentacyclic lantibiotic (3320 Da) produced by B. subtilis ATCC 6633. Mersacidin (1825 Da) is a lantibiotic, produced by Bacillus spp. strain HIL and includes globular and uncharged lantibiotics (14). Non-Ribosomal BioPeptides include cyclic lipopeptidides (iturin group) and macrolactones (surfactins, fengycins and plipastatins) (15).

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

A-51 Iturin A, bacillomycins and mycosubtilin form channels in bacterial cell membrane. Mycosubtilin alters the permeability of the plasma membrane, releasing nucleotides and proteins. Among small peptides secreted by B. subtilis, bacilysin contains an N- terminal alanine residue and L-anticapsin. The release of L-anticapsin irreversibly inhibits glucosamine synthase, involved in the synthesis of nucleotides, amino acids and coenzymes and resulting in the lysis of microbial cells such as S. aureus and Candida albicans (16). The non-ribosomal dodecapeptide bacitracin (1486 Da), released by some B. licheniformis and B. subtilis strains, proved to be an inhibitor of cell wall biosynthesis of Gram positive bacteria (17).

Materials and Methods

Materials and Reagents: The media used for culture were Nutrient Agar, Nutrient Broth and Mueller Hinton Agar. For the biochemical characterisation of the Bacillus genus, the biochemical media used were Simmon citrate media, Hugh and Leifson, Carbohydrate fermentation media (Glucose, Amylase, Mannitol and Arabinose), 6.5% NaCl media and Gelatin Agar. The reagents used included Gram staining reagent, Spore staining reagent, Catalase reagent, Methyl Red, Voges-Proskauer reagent (Barrit’s reagent) and HgCl2 solution. Isolation of Bacteria: Soil sample was collected from about 10cm below the surface of the earth. 1g of soil sample was added to 10ml of water, the contents of the tube were vortexed for 5 minutes and serial dilution was made up to 10-6 .From each dilution, 0.1ml was poured on NA and then spread on Nutrient Agar and incubated overnight at 28°C.The colonies forming a clear halo zone around them were taken as antibiotic producers. Other colonies of Bacillus were also identified by study of colony morphology followed by Gram Staining and Spore Staining. They were identified according to Bergey’s Manual of Determinative Bacteriology. Extraction of antibiotic by Tube Culture Method: All the colonies suspected to produce antibiotics were cultured in Nutrient Broth for about 3 days. The cultures tubes were checked every day for turbidity or visual change with periodic shaking of the tubes in between. When sufficient growth indicated by the formation of thick pellicle was observed, the tubes were centrifuged at 6000 rpm for 20 minutes. The pellet was discarded and to the supernatant, equal volume of ethyl acetate was added and again centrifuged at 6000 rpm for 20 minutes. The lower layer of liquid obtained was the crude antibiotic. The antibacterial activity of the obtained raw extract was evaluated using agar-well disc diffusion method. Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 Agar-well Diffusion Method for Antimicrobial Activity Testing: A standard inoculum of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) (with turbidity as 0.5 Mc Farland solution) was swabbed onto the surface of a Mueller Hinton Agar (MHA) plate. Wells were bored in the agar, where 100μl of antibiotic was allowed to diffuse in the well and allowed to diffuse. A well with 100μl of ethyl acetate was kept as negative control. After overnight incubation, the diameter of zone of inhibition was measured for each crude antibiotic diffused in the well. Disc Diffusion Method for Antimicrobial Susceptibility Testing: A standard inoculum of bacteria was swabbed onto the surface of a Mueller Hinton Agar (MHA) plate. Antibiotic discs were placed on the agar. After overnight incubation, the diameter of zone of inhibition was measured for each antibiotic disc.

Result

A Total of 40 soil samples were collected from wastelands of different locations during the study period, from which 121 bacterial colonies were isolated and studied further. 11.5%, 40.49%, 33.8% and 14.04% of the isolates belonged to Group I, II, III and IV Bacilli respectively. Group V and VI Bacilli were not isolated. Moist-neutral soil had the highest number of bacilli isolates (23.14%) and dry-acidic and moist-alkaline had equal number of isolates (9.09%). Out of the total 121 colonies studied, 22 (18.33%) of Bacillus isolates were found to produce antibiotics. Majority of the antibiotic producers belonged to the Group II Bacilli (59.09%) and none of the isolate from Group IV bacilli produced antibiotic. The highest number of antibiotic producing isolate was B. subtilis (40.09%) and the least number of antibiotic producing isolate belonged to the species B. circulans (4.54%). Among the 22 antibiotic producer isolates, 77.71% were from dry soil and 22.29% were from moist soil. Among the antibiotic producers B. licheniformis (18.18%) and B. alvei (9.09%) were only found in dry soil. Similarly, the antibiotic producing colony of B. circulans (4.54%) was only found in moist soil. The highest number of antibiotic producers were found in basic soil (45.45%) whereas the least were found in acidic soil (13.6%). B. subtilis was the only antibiotic producing species isolated from acidic soil. The highest zone of inhibition was by U2 against E. coli (22mm) and the lowest activity was by 2M2 against S. aureus (4mm). While studying the antimicrobial activity of the 22 antibiotic producing isolates, it was found that 63.63% colonies produced antibiotic against Gram positive bacteria 27.27% against Gram negative bacteria and 9.09% colonies had a broad spectrum activity. e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

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Table 1: Number wise distribution according to soil moisture and pH. Dry Soil

Bacilli Group Group I Group II Group III Group IV Total

Moist Soil

Total

0.82

2.47

3.3

0.82

3.3

0.82

11.57

4.95

6.61

11.5

5.78

7.43

4.13

40.49

1.65

9.91

7.43

4.13

8.26

2.47

33.88

1.65

0.82

5.78

4.13

1.65

14.04

121

9.09

19.83

22.31

16.52

23.14

9.09

100

(A= Acidic soil; N= Neutral soil; Al= Alkaline soil; N=Number of isolates)

Table 2: Isolation pattern of antibiotic producers. S.N

Antibiotic Producer

Number

% N=22

Group I

27.7

B. polymyxa

13.63

B. alvei

9.09

Group II

59.09

B. subtilis

40.09

B. licheniformis

18.18

Group III

18.18

B. brevis

13.63

B. circulans

4.54

Group IV

Total

100

Table 3: Zone of inhibition by the antibiotic producing isolates. S.N

Antibiotic Producer

Diameter of Zone of Inhibition (in mm) S. aureus

B. subtilis (B1)

B. subtilis (B4)

B. polymyxa (C1)

B. polymyxa (D2)

B. subtilis (E7)

B. brevis (H1)

B. licheniformis (H6)

B. subtilis (J1)

B. alvei (J2)

B. circulans (L2)

B. licheniformis (O5)

B. subtilis (R1)

E. coli

P. Aeruginosa

9 15 9 10

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A-53

AABS; 4(1): 2017 Diameter of Zone of Inhibition (in mm)

S.N

Antibiotic Producer

B. polymyxa (U2)

B. alvei (U3)

B. brevis (U4)

B. subtilis (V2)

B. licheniformis (W1)

B. licheniformis (X2)

B. brevis (Y2)

B. subtilis (Z1)

B. subtilis (2I2)

B. subtilis (2M2)

S. aureus

E. coli

P. Aeruginosa

15 15

Table 4: Classification of the antibiotic producers based on their antimicrobial activity. Antibiotic Producer Group I B. polymyxa B. alvei Group II B. subtilis B. licheniformis Group III B. brevis B. circulans Total

Gram Positive Inhibitor Number %

Antimicrobial Activity Gram Negative Inhibitor Number % 3

9 4

40.90 18.18

1 14

4.54 63.63

Broad Spectrum Inhibitor Number %

13.63

27.27

9.09

Table 5: Antimicrobial susceptibility testing of pathogenic test strains. S.N

Antibiotic

1 2 3 4 5

Penicillin Amikacin Bacitracin Erythromycin Methicillin

1 2 3 4 5

Ampicillin Nalidixic Acid Co-trimoxazole Ceftazidime Imipenem

1 2 3 4 5

Gentamicin Ampicillin Ciprofloxacin Cefazidine Co-trimoxazole

Zone of Inhibition (mm) Staphylococcus aureus 20 22 11 23 25 Escherichia coli 15 26 25 11 30 Pseudomonas aeruginosa 20 0 26 0 15

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Remarkable Zone of inhibition with extracted crude antibiotics (mm) 20 ( U2) 15 (J1) 15 ( J2) 15 (X2)

22 (U2) 15 (D2) 15 (U3) 15 (U4)

20 (U4) 18 (D2)

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

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Fig. 1: Antibiotic producing colony isolated on nutrient agar.

Fig. 2: Biochemical tests of Bacillus alvei (Amylase +ve, VP +ve, Citrate –ve, Mannitol –ve).

Fig. 3: Agar-well diffusion method for testing antimicrobial activity (Central well- Negative control with ethyl acetate).

Discussion

The present study was conducted in the Microbiology laboratory of St. Xavier’s College from February 2016 to June 2016. A total of 121 bacterial colonies were isolated from those samples and studied further. 11.5% colonies belonged to Group I Bacilli. However, the result of the study is different from a similar study carried out by Adamu et al (18), where they found that only 5.7% of the isolates belonged to Group I Bacilli. 40.49% colonies

belonged to Group II Bacilli. Similar results were observed in the experiments carried out as 30.8% belonging to Group II Bacilli (18). The Group III and IV are all strict aerobes and they have similar growth requirement (19). In this regard, the result of this study is contrasting as in this study 33.8% colonies belonged to Group III Bacilli and only 14.04% colonies belonged to Group IV Bacilli. The high frequency of Group III Bacilli is also different from a similar research where

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A-55 5.7% of the total isolates belonged to Group III (18). The Group V and VI comprise of thermophilic and acidophilic thermophiles respectively (19). Hence, the present study could not isolate these bacteria. Out of the different species isolated, B. subtilis species were found to be in highest number i.e 17.3% and the lowest number of species isolated was B. macerans (0.82%). In a similar experiment by Adamu et al (18), they identified only 3.8% of the isolates as B. subtilis. In their study, the species with the highest frequency among the isolated ones were B. lichenifomis (13.5%) and B. mycoides (13.5%). In this study B. lichenifomis (9.09%) and B. mycoides (9.91%) were isolated in approximately equal proportion. This result is similar to the study by Adamu et al (18) where it was found that the isolates of B. lichenifomis (13.5%) and B. mycoides (13.5%) to be in equal proportion. Out of 121 bacterial isolates, 62 (51.18%) were from dry soil and 59 (48.82%) were from moist soil. Among them the predominant species of dry soil was found to be B. subtilis (9.09%) and B. brevis (9.09%) and that of moist soil was B. subtilis (8.21%). The high number of these species of Bacillus isolated in this study matches with the feasibility of their isolation based on their growth requirement as presented by the study of Slepecky and Hemphill (20) which defines that B. subtilis group are non-fastidiuos among the other group of bacilli. Out of the total isolates, the species found uniquely found in acidic soil was B. marinus, in neutral soil was B. subtilis and that of moist soil was B. circulans. The isolation of B. marinus in high number in acidic soil and B. subtilis together with B. lichenifoemis was also reported by Slepecky and Hemphill (20). Out of the total 121 colonies studied, 22 (18.33%) of Bacillus isolates were found to produce antibiotics. The highest number of antibiotic producing isolate was B. subtilis (40.09%) and the least number of antibiotic producing isolate belonged to the species B. circulans (4.54%). This result is contrasting to a study carried out in soil of marine environment where antibiotic producers of genus Bacillus were all identified as B. subtilis (21). The highest number of antibiotic producers were found in basic soil (45.45%) whereas the least were found in acidic soil (13.6%). B. subtilis was the only antibiotic producing species isolated from acidic soil.

AABS; 4(1): 2017 bacteria, and also had good activity against MRSA (21). (Darabpour et al 2012). The frequency of the Gram positive inhibitors is higher in this study compared to the study of Darabpour et al (21). The largest zone of inhibition was obtained as 15 mm by B. subtilis (J2). In a similar study carried out by Darabapour et al (21), they isolated B. subtilis with a zone of inhibition 23 mm against MRSA. The present study found that 27.27% of the isolates produced antibiotic against Gram negative bacteria. Among them, the majority were B. brevis and B. polymyxa. Shandhya et al (22) performed a similar study where Bacillus strains were observed to inhibit growth of Gram negative test organisms such as E.coli, Klebsiella and Pseudomonas. 9.09% colonies had a broad spectrum activity. Abdulkadir and Waliyu (23) and Darabapour et al (21) also found Bacillus alvei inhibited the growth of both Gram positive and Gram negative bacteria and they were indicated as broad spectrum antibiotic producer. The specimen of Staphylococcus aureus ATCC 25923 used in this research was also tested against Penicillin, Amikacin, Bacitracin, Erythromycin and Methicillin together with the crude antibiotic extracted in this study. It was found that a colony of B. alvei (U3) gave a zone of inhibition 20 mm which was higher than the zone of inhibition by Bacitracin (11 mm) and equal to Penicllin (20mm). The Escherichia coli ATCC 25922 used was also tested with Ampicillin, Nalidixic acid, Co-trimoxazole, Ceftazimide, and Imipinem. A colony of B. polymyxa (U2) isolated in this study gave a zone of inhibition 22mm which was higher than the zone of inhibition by Ceftazimide (11 mm) and Ampicillin (15mm ) Similary, for Pseudomonas aeruginosa ATCC 27853, Gentamicin, Ciprofloxacin, Ampicillin, Ceftazidine and Co-trimoxazole were used. It was found that the Pseudomonas aeruginosa was Resistant to Ampicillin and Cefazidine whereas the zone of inhibition by Cotrimoxazole and Gentamicin were only 15 mm and 20 mm respectively. But, an isolate of B. brevis (U4) from this study gave a zone of inhibition 20mm for this test strain.

In the present study, 11.5% of the antibiotic producers produced antibiotic against Gram positive bacteria. In a study similar like this where the activity of the antibiotic producers was only tested against Gram positive bacteria, 5.88% showed antibacterial activity against Gram-positive

The antibiotics extracted from the Bacillus species in this study were only in a crude form. However, they gave an appreciable zone of inhibition and some even gave a zone of inhibition higher than the standard antibiotics used. This shows that these isolated strains produced antibiotics with a greater potential than the tested standard antibiotics. The standard antibiotics used in this study are the commonly used ones. Thus, we can say that some of these isolated strains have ability to produce antibiotics with a greater potential than the commonly used antibioitcs.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Original Article

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Conclusion

Based on property of soil, moist-neutral soil had the highest number of bacilli isolates and dry-acidic and moist-alkaline had least number of isolates. Out of the different species isolated, Bacillus subtilis species were found to be in highest number and the lowest number of species isolated was Bacillus macerans. Among them the predominant species of dry soil was B. subtilis and B. brevis and that of moist soil was B. subtilis. Among the species found uniquely in acidic soil was B. marinus in neutral soil was B. subtilis and that in alkaline soil was B. circulans. Out of the total colonies studied, 18.33% of Bacillus isolates were found to produce antibiotics. Majority of the antibiotic producers belonged to the Group II Bacilli and none of the isolate from Group IV bacilli produced antibiotic. The highest number of antibiotic producing isolate was B. subtilis and the least number of antibiotic producing isolate belonged to the species B. circulans. Among the antibiotic producers 77.71% were from dry soil and 22.29% were from moist soil. Also, the highest number of antibiotic producers were found in basic soil whereas the least were found in acidic soil. Furthermore, among the antibiotic producers, 63.63% produced antibiotic against Gram positive bacteria, 27.27% against Gram negative bacteria and 9.09% had a broad spectrum activity. Some colonies were even found to produce antibiotic with a greater potential than the commonly used antibiotics.

Acknowledgements

We heartily acknowledge Department of Microbiology, St. Xavier’s College, Maitighar, Kathmandu for providing us the opportunity to accomplish this task. We are also thankful to the staffs of National Health Research Council, Kathmandu for providing us the cultures needed in our study.

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*Corresponding author: Sagar Aryal, Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal. Phone: +977-9840052308 Email: sag.micro@gmail.com Date of Submission : 05.02.2017 Date of Acceptance : 23.02.2017 Date of Publication : 25.02.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Original Article DOI: 10.21276/AABS.2017.1358

Effectiveness of Teaching Aids in Medical Education Deepa Sanjeev Nair1*, Madhura Yogesh Bedekar1, Meena Jitendra Agrawal1 and Anagha Madan Gupte2 Department of Physiology, MIMER Medical College, Talegaon (D), Pune, India 2 School of Business Maharashtra Institute of Technology, Pune, India

ABSTRACT Background: Invention of computer and internet has revolutionized each & every field. Technology enhanced learning is very popular among students including medical students. Teaching aids have become an integral part of medical college teaching. The study was conducted to find out the effectiveness of teaching aids in medical college teaching and compare different teaching aid methods. Method: Study was conducted in the practical class of I MBBS. Students were divided in to three batches designated as A, B & C. Pre- test using multiple choice questions (MCQ) was conducted to analyze the knowledge level of the students. Different teaching aids were used for each batch .Chalk-board method, Power point presentation (PPT) with chalk-board, and PPT were the teaching aids used. Post- test with same MCQ was conducted. Effectiveness of different teaching aid was done by comparing pre-test and post-test marks using paired ‘t’ test. Different teaching aids were compared using ‘Z’ test. Result. Comparison between chalk-board with chalk-board plus PPT showed that gain in knowledge was more when chalk- board and PPT were combined and the difference was statistically significant (p<0.05). Chalk-board and PPT, when compared, there was no statistically significant difference between the two methods even though gain in knowledge was slightly higher when PPT was used. PPT and combine use of PPT plus chalk-board didn’t show any statically significant difference when compared, but the gain in knowledge was higher with combination of PPT and chalk-board method. Conclusion: Chalk- board method, when combined with PPT is a better teaching aid method than individual method when used alone. The reason for not getting the statically significant difference could be due to the less no. of MCQ in pre-test and post-test. Each teaching aid has got its own advantages and disadvantages. Teaching aid used should be appropriate to the topic Keywords: Teaching Aid, Chalk- Board Method, Power Point Presentation

Introduction

As they say, the change is inevitable. Invention of computer and internet has revolutionized each and every field. As the computer gained popularity, technology enhanced learning also became popular. Use of teaching aids is very common in present day teaching. Scenario in the medical colleges also is not different. Collins English dictionary defines teaching aid as ‘any device, object or machine used by a teacher to clarify or enliven a subject [1]. Various teaching aid methods include the traditional chalk and board method as well as the modern methods like over head projectors (OHP), PowerPoint presentations (PPT) and other audio visual aids. Power Point presentations are very effective in seminars and large group teaching. There is a view that traditional black board teaching method provides strong student teacher interaction but its effectiveness declines as the no. of student in the class increases [2]. Is it true? Or the power point presentations are replacing the conventional chalk and board method even when the no. of students is not so large. Nowadays there is too much of use of power point presentation especially among youngsters. The dependency on this method is so great to the extent that in

some class rooms there is not even provision for chalk and board. The modern teaching aids- Are they teacher’s aid or teaching aid? Is the modern power point presentation is beneficial to the students? Previous studies on the same subject showed the following finding. Seth V et al, in their study suggested that medical students preferred PowerPoint presentations over chalk and board [3] where as Vamshi KT et al suggested that students preferred the traditional chalk and talk over microsoft power point presentation [4]. The study conducted by Baxi SN et al suggested that equal no. of students’ preferred black board based or multimedia based lectures [5]. The results were different in different studies Most of the study conducted were using questionnaire and these were student’s perspective. Our study is an attempt to analyze the effectiveness of teaching aids in medical colleges in not so large groups, where teacher student interaction is possible. The study the effectiveness of teaching aids in medical college students and compare different teaching aid methods

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Material and Method

The following activity was done in the usual practical class of first MBBS. Three groups of students each were selected (Group A, Group B & Group C). A topic for practical demonstration, which was new to them, was selected. Topic selected was perimetry. The sequence was 1. Pre – test consisting of 10 Multiple Choice Questions (MCQ) Time given was 10 minutes for answering the MCQ Separate answer sheets were given to each student Answer sheets were collected after 10 minutes. 2. Briefing of practical aspect of perimetry along with necessary theory information 3. Practical demonstration 4. Post test with the same MCQ MCQ- Each correct answer carried 1 mark & no negative marking for wrong answer. For Briefing: For Group A- Briefing of the practical was done by Chalk - Board method Group B- PPT with LCD projector combined with chalkboard is used For Group C- PPT with LCD Projector was used Statistical Analysis Results of pre- test & post test of each teaching aid is compared with paired‘t’ test. . Effectiveness of different teaching aids was compared with ‘Z ‘test

Result

I. Comparison between before training and after training for 1. Chalk-Board method: Knowledge level of the students before and after training was compared. Paired‘t’ test was applied. It was observed that mean marks obtained by the students after training increased significantly as compared to mean marks before training. Mean difference in marks was 3.97, which was highly significant statistically. (p<0.001) (Table-1) 2. Chalk- Board +PPT with LCD projector method: Knowledge level of the students before and after training was compared. Paired ‘t’ test was applied It was observed that mean marks obtained by the students after training increased significantly as compared to mean marks before training. Mean difference in the marks, 4.68, was statistically highly significant.(p<0.001) ( Table-1)

AABS; 4(1): 2017 The difference between the knowledge level before training and after training by using PPT method was highly significant. Mean difference in the marks was 4.36 (p<0.001) ( Table-3) II. Comparison between Chalk-Board and PPT with chalk & Board Gain in knowledge was calculated by taking the difference between marks obtained before and after training in case of both the method- chalkboard (3.97) and PPT with chalk-board (4.68). ‘Z’ test was applied to test the mean increase in marks by both the methods. Though the training by both the methods improved the knowledge of the students, the training with the help of PPT with chalk- board produced better result. The difference between increases in marks was significant, means difference 0.71, and was statistically significant. (p<0.05) (Table-2) III. Comparison between Chalk-board & PPT The training by only chalk-board and then training by PPT method was compared. Difference between marks obtained before and after training in case of both the methods was calculated. ‘Z’ test was applied to test the mean increase in marks by both the methods. The average difference between increases in marks of the two methods were similar. There is no statistically significant difference between these two methods. Impact of these two methods is similar. Even if the average increase by chalk-board + PPT method (4.36) is more than only chalk-board method (3.97), the difference is not statistically significant. Difference in marks (0.39) was statistically not significant (p>0.05) ( Table-4). IV. Comparison between PPT and Chalk-board + PPT Gain in knowledge was found by taking the difference between marks obtained before and after training in case of both the methods. ‘Z’ test was applied to test the mean increase in marks by both the methods. . The average difference between increases in marks of the two methods is similar. There is no statistically significance between these two methods. (p>0.05) Chalk-board + PPT& PPT are giving similar result. But the increase in marks in PPT + Chalk- board training method is s higher (4.67 as compared to 4.36) even though it is not statistically significant (Table-5)

Discussion

3. PPT method: Knowledge level of the students before and after training was compared. Paired t’ test was applied.

In our study we observed that gain in knowledge occurred with all three teaching methods, that is, chalk – board teaching, PPT and combination of Chalk-board and PPT. The increase in the marks was statistically significant in all three teaching methods. [Table 1, & 3]. When we compared chalk-board with PPT plus chalk –board, we observed that gain in knowledge was more with the combined use

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Table 1: Comparison between before training and after training for Use of chalk-Board method Chalk- Board +Power point presentation (PPT) using LCD projector . Parameters

Chalk-Board method

Chalk – Board + PPT

Before training

After training

Before training

After training

No. of students evaluated

Mean marks obtained

3.13

7.10

3.32

8.0

1.53

1.06

1.17

0.98

Comparison between before and after training

Paired‘t’ test was applied. Mean difference = 3.97 t = 15.13; d.f. = 37; p <0.001; The difference between the knowledge level before training and after training by using chalk-board method is highly significant.

Paired‘t’ test was applied. Mean difference = 4.68 t = 23.23; d.f. = 33; p <0.001; The difference between the knowledge level before training and after training by using Chalkboard+PPT is highly significant.

Table 2: Comparison between Chalk-Board and PPT with chalk & Board Chalk-Board method

PPT with chalk & Board

Mean increase in marks

3.97

4.68

1.62

1.17

Comparison

Mean difference = 0.71; Z = 2.12; p <0.05, The difference is significant.

Table 3: Comparison between before training and after training for PPT . Parameters

PPT Before training

After training

No. of students evaluated

Mean marks obtained

3.38

7.74

1.77

1.07

Comparison between before and after training Paired‘t’ test was applied.

Mean difference = 4.36; t = 13.22; d.f. = 38; p <0.001; The difference between the knowledge level before training and after training by using PPT method is highly significant.

Table 4: Comparison between chalk-board method & PPT method.

Chalk-Board method

PPT method

Mean increase in marks

3.97

4.36

1.62

2.05

Comparison

Mean difference= 0.39; Z = 0.91; p > 0.05, The difference is not statistically significant.

Table 5: Comparison between PPT+ Chalk – Board & PPT. PPT + chalk & Board

PPT method

Mean increase in marks

4.67

4.36

1.17

2.05

Comparison

Mean difference= 0.31; Z = 0.79; p > 0.05, The difference is not statistically significant.

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A-61 of PPT & chalk-board and the difference was statistically significant (p<0.05) [Table-2]. The comparison between chalk-board with PPT showed that even though mean difference was more in PPT method, it was not statistically significant (p>0.05) [table-4]. As we compared the gain in knowledge of PPT with chalk-board plus PPT, we observed that increase in the marks after training was more in the latter compared to the former, but the increase was not statistically significant [Table-5]. Baxi et al conducted a study using questionnaire method in a medical college and they arrived at the conclusion that overall preference of the students was distributed equally between black board teaching and multimedia teaching [5]. Another study conducted by Sheikh S T arrived in a conclusion that medical student preferred a combination of teaching aid – chalk &board and power point presentation incorporating animation. The above study also used questionnaire method. Hall R suggested the importance of application of technology to medical education. The study was conducted in 1969 [6]. In our study when we compared the chalk-board method with PPT, we observed a higher increase in the marks in PPT compared to chalk-board even though it was not statistically significant. Topic chosen by us was ‘perimetry’. Here the three dimensional understanding of the instrument was required which cannot be done with chalk-board method. That might be the reason of slightly better result with PPT compared to chalk-board. Comparison between PPT & chalk-board plus PPT didn’t show statically significant difference, even though gain in knowledge was slightly higher in PPT plus chalk- board. The reason of not getting statically significant increase might be the no. of multiple choice questions used in pre test and post test. Only 10 questions were there as the topic was small. Studies with more no. of MCQ may produce a statistically significant increase in favour of combination method ( PPT plus chalk-board) The results of our study suggest that the combination method, Chalk- board plus PPT, as the best teaching method. This underlines the fact that we cannot ignore the importance of chalk-board method even in the present technology era. We can display a figure on a PPT presentation, but how to draw that figure

AABS; 4(1): 2017 can be better explained with chalk-board. Each teaching aid has got its own advantage and disadvantages. So selecting the teaching aid appropriate to the topic is very important. Combination of different teaching aids will be the best method to understand various concepts in medical subjects

Conclusion

Technology enhanced learning has gained popularity in medical colleges. Use of teaching aids is very common in present day teaching. In our study we compared the different teaching aid methods, chalk-board method, PPT and combination of chalk-board with PPT. Teaching aid used should be appropriate to the topic. Our study suggested that chalk-board and PPT when combined together resulted in better understanding of the topic in I MBBS students. It is true that we should adopt the new techniques which have benefit, at the same time we should preserve old methods which has got advantage and cannot be replaced by any other method.

Acknowledgement

Physiology department, MIMER Talegaon (D), Pune, India

Medical

College,

References 1.

Collins English Dictionary- Complete & Unabridged 12th edition 2014 , Harper Collin Publication

2. Shaikh S T. Teaching learning aids in medical education: The student perspective. Int J Clin Surg adv 2015 ; 3 (1): 32-37. 3.

Seth V, Upadhyaya P, Ahmed M, Moghe V. Powerpoint or Chalk and talk: Perception of medical students versus dental students in a medical college in India. Adv. Med Educ Pract 2010; 1: 11-16.

Vamshi K T, Datta V M, Kishan Y S S, Aditya V, Bhanuprakash G. Comparative study on the teaching effectiveness of chalk and talk and Microsoft Power Point presentation from student perspective. Int J Pharm Pharm Sci 2012; 4 (1) 191-193.

Baxi S N, Shah C J, Parmar R D, Parmar D, Tripathi C B. Student perception towards different teaching aids in a medical college. African Journal of health professions education 2009; 1(1):15.

Hall R. Audio Visual aids in medical education. BrMed J 1969; 4 (5674) 40-41

*Corresponding author: Dr. Deepa Sanjeev Nair, ‘Bhairavi’, Bungalow No.7, Sector-26, A.D.C, Behind L.I.C Training Centre, Pradhikaran, Nigdi, Pune, India, PIN- 411044 Phone: +91 9326026733 Email: drdeepasnair@rediffmail.com Date of Submission : 15.02.2076 Date of Acceptance : 23.02.2017 Date of Publication : 27.02.2017 Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

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

Original Article DOI: 10.21276/AABS.2017.1368

Evaluation of Metastatic Cutaneous Lesions on Fine Needle Aspiration Cytology: A Five Year Study Syed Besina Yasin, Naheena Bashir and Subuh Parvez Khan* Department of Pathology, Sher e Kashmir Institute of Medical Sciences, Soura, Srinagar, J &K India.

ABSTRACT Background: Cutaneous and subcutaneous metastatic deposits are rare and can be seen in 0.8-5% cases. This study was done to evaluate the role of Fine Needle Aspiration Cytology(FNAC) in the diagnosis of cutaneous and subcutaneous nodules in patients with known malignancy or as a primary manifestation of an unknown malignancy. Methods: It was a 5 year retrospective study and the record of patients subjected to FNAC during this period was reviewed. Results: Out of total number of 13572 patients 58(0.42%) were diagnosed as cutaneous /subcutaneous metastatic deposits .In 51 cases ,the primary was known and in 7 cases it was an initial manifestation of an unknown primary. There were 39 males and 19 females with age range of 27-82 years . Conclusion: Commonest site of metastases was chest wall and most common primary was lung. Adenocarcinoma was the commonest diagnosis on morphology. This study emphasizes the efficacy of FNAC on the accurate diagnosis of subcutaneous nodules especially in patients with known malignancy .It also offers clue to the underlying malignancy in occult primary. Keywords: FNAC ,Metastases, Cutaneous

Introduction

Cutaneous and subcutaneous metastates from an underlying malignancy is rare. The incidence of cutaneous metastases ranges from 0.8-9% [1,2,3,4]Typically they are associated with an advanced stage of diaease and a poorer prognosis .[4]Metastatic skin nodules may resemble other primary skin tumours or inflammatory lesions and needs histological or cytological confirmation.

were diagnosed.Cases suspected as primary skin tumours were excluded from the study. FNAC was performed using the conventional techniques. Both dried and alcohol fixed smears were prepared and stained with Giemsa and Pap respectively .Clinicopathologic correlation was done in every case .Slides were reviewed and data analysed.

Results

FNAC is a minimally invasive technique for early diagnosis of such cases and thus obviates the need for more invasive biopsies or surgeries. [1,2,5]It offers a clue to underlying malignancy in case of an occult primary[5]

Total number of 13572 patients were subjected to FNAC during a five year period (2011-2015). Out of these 58 cases (0.42%)were diagnosed as cutaneous /subcutaneous metastatic deposits from a known /unknown primary malignancy .The data was analysed [Table 1,2,3]

This study was done to evaluate the role of FNAC in the diagnoses of metastatic cutaneous and subcutaneous nodules in patients with known malignancy or as a primary manifestation of an unknown malignancy.

The age of patients ranged between 30-82 years in males and 27-70 years in females. The youngest patient was a 27 year female having a metastatic deposit in interscapular area from a primary epitheloid sarcoma left arm. The oldest patient was 82 year male with metastatic mucinous adenocarcinoma from ascending colon , lesion was on anterior abdominal wall.

Materials and Methods

This was a 5 year retrospective study conducted in the pathology department of SKIMS ,Srinagar ,Kashmir which is a tertiary care hospital and a Regional Cancer Centre.The record of all FNACs done from Jan 2011 to Dec 2015 were reviewed .A total of 13572 patients underwent FNAC during this period out of which 58 cases of cutaneous and subcutaneous metastatic deposits

The most common site of cutaneous metastases was chest wall in 16 cases(9.28%) and anterior abdominal wall in 14 cases (8.12%) followed by scalp (3.4%) and back (3.4%). In 48 cases (82.7%) there was a solitary metastatic nodule whereas they were multiple in 10 cases(17.2%).Size of the lesion varied from 0.5-6 cm .

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AABS; 4(1): 2017

The most common primary was lung (13 cases -25.4%) followed by esophagus (10 cases-19.6%).There were 51 cases(87,9%) with known primary and 7(12.06%) cases in whom the primary was unknown .On cytomorphology adenocarcinoma[Figure 1] was the commonest diagnoses (22 cases -37.9%) followed by Squamous cell carcinoma(SCC) (19 cases -32.7%)[Figure 2] which was compatible with the morphology of primary lesion.Some unusual cases were also seen in our study.3 cases in this series with subcutaneous deposits were known cases of sarcomas (1 epitheloid sarcoma, 1 leiomyosarcoma of soft tissue and 1 rhabdomyosarcoma nose).There was a case of hurthle cell carcinoma thyroid metastasizing to right chest wall .We had 2 cases of Non Hodgkin Lymphoma(NHL) with cutaneous metastates. A 75 year old male presented

with a soft jelly like solitary tumor in the chest wall. FNAC revealed features of Anaplastic Large Cell Lymphoma on subsequent examination and investigation .Patient also had generalised nodal involvement .Diagnosis was confirmed on Immunohistochemisrty. There was a case of small cell neurendocrine carcinoma of salivary gland with metastasis to scalp. Higher incidence was seen in males -39 cases (67.2%) than females -9 cases (37.2%).In 7 cases of unknown primary subsequent detailed work up revealed the primary site as colon and stomach in 3 cases and lung and esophagus in 2 cases of squamous cell carcinoma.1 case of metastatic neuroendocrine carcinoma had primary in prostate and there was a case of nodal NHL who initially presented as subcutaneous nodule.

Table 1: Site of metastasis

Males

Females

Total (% age)

Chest wall

16 (9.28%)

Anterior abdominal wall

14(8.12%)

Scalp

6(3.48%)

Peri-umblical

4(2.32%)

Upper limb

1(0.58%)

Lower limb

2(1.16%)

Back

6(3.48%)

Scar site

Groin

4(2.32%

Face

2(1.16%

1(0.58%)

Sacral

1(0.58%)

Neck

1(0.58%)

Total

38(65.5%)

20(34.48%)

58(100%)

Table 2: Primary Tumor

Morphological Diagnosis Males SCC -6

Lung

Females

Site of Metastasis

Number

Single

Multiple

chest wall (9)

Small cell carcinoma-3

Abdominal wall (2)

Adenocarcinoma-4 Esophagus

Back(2)

SCC

Stomach

Adenocarcinoma

Abdominal wall

Colon

Adenocarcinoma

Abdominal wall

GastroEsophageal junction

Adenocarcinoma

Para umblical

Abdominal wall

Abdominal wall +Paraumblical

scar site-1

Gall Bladder Ovary Rectum

Papillary Adenocarcinoma Papillary Adenocarcinoma Adenocarcinoma

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chest wall -2

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Original Article Primary Tumor

A-64 Morphological Diagnosis Males

Breast

Ductal Carcinoma

Prostate

Adenocarcinoma

Soft Tissue

Females

Site of Metastasis

Number

Single

Chest wall

Paraspinal

Epitheloid sarcoma

Interscapular

Leiomyosarcoma

Abdominal wall

Nose

Rhabdomyosarcoma

Right Cheek

Tonsil

NHL

Right Arm

Tongue

SCC

Abdominal wall

Thyroid

Hurthle Cell Carcinoma

Lymph Node Pancreas

Right chest

NHL

Abdominal wall

Papillary Adenocarcinoma

Scar site -2

Multiple

Parietal wall -1 SCC

Skin

Malignant Melanoma

Salivary Gland

Small cell Neuroendocrine

Right Thigh

Back

Groin

Scalp

1 1

Table 3: No.

FNAC diagnosis

Age

Sex

Site

No of Lesions

Small cell Neuro-endocrine Carcinoma

Sacral

Solitary

Adenocarcinoma

Parietal wall

Solitary

Non-Hodgkins Lymphoma

Chest wall

Solitary

Squamous cell carcinoma

Chest wall

Solitary

Adenocarcinoma(2)

35,55

Anterior Abdominal wall

Solitary

Squamous cell Carcinoma(Poorly differentiated )

Anterior Abdominal wall

Solitary

Fig. 1: Photomicrograph showing metastatic deposits of adenocarcinoma.(MGG stain,40X).

Fig. 2: Photomicrograph showing metastatic deposits of squamous cell carcinoma.(MGG stain ,40X).

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Discussion

Cutaneous and subcutaneous metastasis is rare.[1,2,3] It can occur due to direct spread from initial tumor or disseminate via vascular or lymphatic route.[6]Cutaneous metastasis are associated with advanced stage of the disease and a poor prognosis.[4]Infrequently it may be the first sign of malignancy [4,7]Clinically cutaneous metastasis manifests as nodules ,ulceration ,cellulitis like lesions or fibrotic processes. Lesions are solitary or multiple and mostly symptom free.[1,4,5]Histologically, they can be classified as adenocarcinoma ,squamous cell carcinoma, undifferentiated carcinoma and other miscellaneous types. [6]Skin metastases usually occurs close to the site of primary tumour –chest in lung carcinoma ,abdominal wall in GI malignancy and lower back in Renal cell carcinomas. Chest and abdomen is the commonest site of cutaneous metastases reported in the literature followed by head and neck.[1]The most common malignancies to metastasize to skin are lung followed by GI , melanoma ,RCC and carcinoma of oral cavity in males. Breast followed by colon ,melanoma ,lung ,ovary and sarcoma are the common primary sites in females.[1,4]Adenocarcinoma from various organs is the commonest to metastasize to skin.[1,5]Age ranges between 2-76 years.[1,8] Differential diagnoses includes primary adnexal tumors and primary squamous cell carcinoma of skin .Metastases is usually located in deeper dermis and subcutaneous tissue and overlying skin is free.[5] Cutaneous metastases also present as first sign of malignancy usually in cancers of lung ,kidney and ovary.[9]Sometimes ,primary sites have not been located even after autopsies.[10,11] Incidence of cutaneous metastases was 0.42% in our study. Age ranged between 27-82 years in our patients. Most common site of metastases was chest wall and mostly they were solitary. Lung was the commonest primary in males and gall bladder in females. Metastatic cutaneous lesions are more often found in women with breast cancer, than with other visceral malignancy with an incidence of upto 20%.However in our series gall bladder malignancy was the commonest primary. There was an unusual case of Hurthle cell carcinoma of the thyroid with cutaneous metastases to the right chest wall. Cutaneous metastases of thyroid carcinoma are infrequent [12,13,14] and FNA is an important method for rapid diagnosis of such lesions [12].There have been fewer than 30 case reports of cutaneous metastases of thyroid carcinoma in the literature with majority to head and neck.[12]

AABS; 4(1): 2017 cases of secondary NHL skin were seen in our study. Metastases of various types of sarcomas accounts for only 2-3% of all metastases. Skin lesions ,metastases of sarcoma occurs by hematogenous spread, so cutaneous metastases is an unusual finding.[15]3 cases of soft tissue sarcomas that had metastases to skin ans subcutaneous tissue were identified in 3 females .The histological types were epitheloid sarcoma, leiomyosarcoma and rhabdomyosarcoma .Metastases occured to interscapular area and abdominal wall .The diagnosis was established by FNAC.2 cases of metastaic neuroendocrine carcinoma were seen in our series .One patient had primary in salivary gland and the other presented with metastases in sacral area.Patient on follow up was found to have neuroendocrine carcinoma prostrate .Cutaneous metastases from neuroendocrine carcinoma of visceral organ has already been reported .When a neuroendocrine carcinoma is found in the skin ,it is importantto distinguish Merkel cell carcinoma from cutaneous metastases of visceral origin.Positive staining for CK20 is strongly diagnostic.[17]The diagnosis was confirmed by a complete IHC study of both primary and secondary lesions. Cutaneous metastases of an extracutaneous NHL seems to be poor prognostic factor.[18]

Conclusion

To conclude FNAC can diagnose a variety of tumours in the skin and support the diagnosis of a metastases in a case known primary and offer a clue to underlying malignancy in case of an occult primary .It is a minimally invasive rapid ,accurate and cost effective tool which helps in the prompt diagnosis of such cases .[19]All cases were correctly diagnosed in our study. Our study supports the utility of this technique in the evaluation of metastatic skin lesions and confirms its high degree of accuracy .

References 1.

Karki S, Pathak R, Manandhar U, Koirala S. Metastatic cutaneous and subcutaneous lesions: Analysis of cases diagnosed on fine needle aspiration cytology. Journal of Pathology of Nepal 2011;1:37-40.

Geramizadeh B, Marzban S, Karamifar N et al. Diagnosis of Subcutaneous Metastatic Deposits by Fine Needle Aspiration. J Cytol Histol 2012; 3:151.

Nava G, Greer K, Patterson J, Lin KY. Metastatic cutaneous breast carcinoma: A case report and review of the literature. The Canadian Journal of Plastic Surgery 2009;17(1):25-27.

Erdemır AT, Atılganoglu U, Onsun N, Somay A. cutaneous metastases from gastric adenocarcinoma. indian journal of dermatology 2011;56(2):236-237.

Cutaneous malignancies are less commonly in malignancies such as leukemia and lymphoma .3

5. Sharma, S., Kotru, M., Yadav, A., Chugh, M., Chawla, A. and Makhija, M. (2009), Role of fine-needle aspiration cytology in evaluation of cutaneous metastases. Diagn. Cytopathol., 37: 876–880.

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Fyrmpas G, Barbetakis N, Efstathiou A, Konstantinidis I, Tsilikas C. Cutaneous metastasis to the face from colon adenocarcinoma. Case report. Int Semin Surg Oncol 2006;3:2

13. Agarwal S, Rao S, Arya A, Gupta K, Arora R, Dhawan I. Follicular thyroid carcinoma with metastasis to skin diagnosed by fine needle aspiration cytology. Indian J Pathol Microbiol 2008;51:430-1

Molina Garrido MJ, Mora Rufete A, Guillen Ponce C, Macia Escalante S, Carrato Mena A. Skin metastases as first manifestation of lung cancer. Clin Transl Oncol. 2006;8:616–617.

14. Caron Ph, Moreau-Cabarrot A, Gorguet B & Bazex J. Cutaneous metastasis from follicular carcinoma of the thyroid gland.Thyroid 1993;3:235–237.

Bansal R, Patel T, Sarin J, Parikh B, Ohri A, Trivedi P. Cutaneous and subcutaneous metastasis from internal malignancy. An analysis of cases diagnosed by fine needle aspiration. Diagn Cytopathol 2011;39(12) 882-7 Schwartz RA. Cutaneous metastatic disease. J Am Acad Dermatol 1995;33:161-82

10. Didolkar MS, Fanous N, Elias EG, Moore RH. Metastatic Carcinomas from Occult Primary Tumors: A Study of 254 Patients. Annals of Surgery 1977;186(5):625-630.

15. Corcoran S, Hogan AM, Nemeth T, et al. Isolated cutaneous metastasis of uterine leiomyosarcoma: case report and review of literature. Diagnostic Pathology 2012;7:85. 16. Rao UM, Hanan AH, Karakousis CP. et al. Distant skin and soft tissue metastases from sarcomas. J Surg Oncol. 1998;69:94–98. 17. Fluehler C, Quaranta L, di Meo N, Ulessi B, Trevisan G. Cutaneous Metastasis of Neuroendocrine Carcinoma. Indian Journal of Dermatology 2013;58(3):247.

11. Osteen RT, Kopf G, Wilson RE. In pursuit of the unknown primary. Am J Surg. 1978 Apr;135(4):494–497.

18. Reich A, Wróbel G, Kazanowska B et al: Skin involvement in highly malignant non-Hodgkin lymphomas of childhood and adolescence. Acta Dermatovenerol Alp Panonica Adriat 2006; 15(4):158-68.

12. Rahman G A, Abdulkadir A Y, Olatoke S A, Yusuf I F, Braimoh K T. Unusual cutaneous metastatic follicular thyroid carcinoma. J Surg Tech Case Report 2010;2:35-8

19. Rodrigues LK, Leong SP, Ljung BM, et al.Fine needle aspiration in the diagnosis of metastatic melanoma. J Am Acad Dermatol. 2000;42:735–740.

*Corresponding author: Dr. Subuh Parvez Khan, Department of Pathology, Sher e Kashmir Institute of Medical Sciences, Soura, Srinagar, J&K, India.190011 Phone: +91 9469343742 E-mail: khansubuh@gmail.com Date of Submission : 17.02.2017 Date of Acceptance : 25.02.2017 Date of Publication : 27.02.2017 Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/AABS.2017.1369

Observational Study of Blood Groups Distribution Among Medical Students in Central India Ajay Kumar*, Sonu Ajmani and Priyanka Chouhan Department of Physiology, Netaji Subhash Chandra Bose Medical college Jabalpur, M.P, India

ABSTRACT Background: The ABO and Rhesus (Rh) blood group systems are clinically most important compare to other systems. Acquaintance of the geographical and ethnicity wise distribution of ABO and Rh blood group is necessary for operative management of blood banks, transfusion medicine and genetic research. Aims: This Study was designed to collect data on ABO and Rh distribution among medical students who are natives of central India and its comparison with similar Indian studies. Materials and Methods: Study was accomplished on 192 first and second year medical students belongs to central India (Madhya Pradesh) in the Department of Physiology NSCB Medical College Jabalpur, M.P. The blood samples were collected by finger prick method. ABO blood grouping and Rhesus factors (Rh) typing were decided by glass slide method. Results: Among these medical students the maximum prevalent blood group was B (35.33%) followed by O (34.95%), A (21%) and AB (08.72%). Rh positive prevalence among these students was 96.32%. Conclusion: The study endorsed that blood group B is the commonest of the ABO blood group system in central Indian population and the AB blood group is the least. Rhesus positive is much commoner than Rhesus negative among them. Keywords: ABO Blood Groups, Rh, central India, Medical Students.

Introduction

ABO and Rhesus (Rh) blood group systems till today remain clinically most important in spite of being identification of around twenty nine blood group systems, enumerated by International Society of Blood Transfusion. In 1900, Karl Landsteiner detected the human ABO blood group.1 The Rh blood group system was discovered during 1939â&#x20AC;&#x201C;1940 by Landsteiner, Weiner, Levine and Stetson, clarifying the basis of many unpredicted transfusion reactions. In 1945, Coombs, Mourant and Race described the use of antihuman globulin (Coombs test) for incomplete antibodies.2 Later these two systems have substantiated to be the most important in transfusion medicine. Today, the requirement for blood group frequency and its prevalence is multiuse, besides their importance in relation to blood transfusion and organ transplantation. Blood group antigens can also be applied in genetic research, forensic pathology, anthropology and ancestral evolution of human.3 Nowadays, the ABO blood groups display an extensive geographical variation and vary noticeably, both within and among ethnic groups. Hence, the knowledge of blood group distribution in diverse population is for utmost importance in health care and transfusion practices.4

Knowledge of blood group distribution is vital for clinical studies as it provides information of dependable geographical region and it will aid a lot in genetic studies of such population. Knowledge of blood grouping provides access to safe supply of blood which will help significantly in reducing the preventable deaths. Therefore, it is relevant to have statistics on the frequency distribution of these blood groups in any population group.

Material and Methods

This cross sectional observational study conducted over students of central India at the Department of Physiology, NSCB Medical College, Jabalpur in the Period of January to March, 2016. The entire students included in the study group belong to Madhya Pradesh only. After prior informed consent and institutional ethical clearance samples were collected from one hundred ninety two medical students, studying first and second professional MBBS. All medical students, were from various areas of Madhya Pradesh i.e. belongs to regions of Indore, Gwalior, Bhopal, Rewa, Jabalpur, Chindwara, Balaghat and Shadol. The students in the same batches which were from other states of India were excluded from this study. Under a septic preventive

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Results

measure by finger prick method non haemolysed blood samples were collected, and non-greasy glass slides was used for identification of ABO blood grouping and Rhesus factors (Rh) typing. Commercially available Tulip diagnostics Eryscreen anti sera A, anti sera B and anti sera D kits were used for this study. Blood samples were treated with anti-A, anti-B and anti-D anti sera over glass slides and mixed uniformly over an area of 2.5 cm by mixing stick. After two minutes of gentle rocking samples were observed for agglutination, both macroscopically and microscopically.5

Out of 192 medical students 132 (68.75%) were males and 60 (31.25) % were females with meanÂą SD age of 19.95Âą0.85 years. In our study, among all students most prevalent blood group was B, 70 cases (35.33%) followed by O in 66 cases (34.95%), A in 41(21%) cases and AB in 15 cases (08.72%). Among 192 students 187 (97.39%) showed Rh positive blood group. In Rh-positive cases, among boys AB and O blood group all were Rh positive, while in girls A and O blood groups were Rh positive. Among these students only 3 males and 2 females having Rh negative blood group.

Following Landsteiner laws blood groups and IgG antibodies of Rh system of those students who were identified and their relevant data was entered into Microsoft Excel sheet 2013 and tabulated with graphical representation.

It was also seen that blood group B was more prevalent among Muslims followed by O. All Muslim students showed Rh positive blood group

Table 1: Frequency distribution of different blood groups among medical students. Blood

Male (n=132)

Rh pos

Rh neg

Female (n=60)

Rh pos

Rh neg

Total

groups B

Table 2: Frequency distribution of different blood groups among Muslim medical students. Blood groups

Total no (n=18)

Percentage

Male

Female

11.11

44.44

11.11

33.34

Discussion

Variables in ABO and Rh D phenotypes are noted broadly across races and geographical boundaries.6 Limited studies on the prevalence of ABO and Rh blood groups have been conducted in the Indian population and common studies are limited to particular regions of the country. Very few such studies have yet been reported from central India. In India, we have many types of ultra-rare phenotypes of blood groups like Bombay (Oh), -D -/-D-, In (a+b-), Colton-null phenotype, CdE/CdE (ryry) phenotypes; those phenotypes are potential enough to pose problems in providing blood to the recipients having these phenotypes.7 The present study revealed among medical students of central India having prevalence of blood group B>O>A>AB. A study conducted by Talukdar et al, outside the State in Guwahati revealed blood group O was the

commonest at 37.23% followed by blood groups B at 31.0%, A at 24.83% and AB at 6.93%.8 Studies done in Northern parts of India by Chandra et al at Lucknow and Sindhu et al at Punjab showed blood group B was the commonest, followed by O, A and AB which have similar finding as seen in our study. In western Ahmedabad, Patel et al carried a hospital based study predominantly among male donors and reported the commonest ABO blood was B (39.40 %) followed by O (30.79 %), A (21.94 %) and AB (7.86 %) . In this present study among student similar findings were seen with B as most prevalent (35.33%) blood group followed by O (34.95%), A (21%) and AB (08.72%). A study done in Durgapur of West Bengal by Nag et al found that commonest blood group was O followed by B, A and AB, different from our study.9,10,11,12 The present study pointed that blood group B and O were also common in distribution among the female study group

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A-69 followed by the A and AB. Study done in North India by Chandra et al displayed among female blood donors the B positive blood group was most prevalent.9 A study carried on the distribution of blood groups among south Indian medical students of the Great Eastern Medical School, Andhra Pradesh by Swamy et al showed O was the commonest blood group in both sexes, but in this study among male students the commonest blood group was A and female students the maximum prevalent blood groups were A and B. 13 The Rhesus blood group is the most polymorphic and its medical implication in transfusion medicine is only next to the ABO blood group system. Multicentric study in India by Agrawal et al revealed 94.61% of the donor population was Rh positive and the rest were Rh negative, similar to present study where 97.39% students were Rh positive and rest2.61% were Rh negative. Study conducted by Parmanik et al over Nepalese medical students, in Nepal medical college, Kathmandu exhibited 96.66% Rh positive cases which was consistent finding with our study.14, 15 A study conducted by Hussain et al among North Indian Muslim population highlighted the most frequent blood group was found to be group O 29.97% followed by A 26.52% , B 20.03% and AB 19.34% 16. An epidemiological study in relation to Muslim and Christian communities of Kheda, Gujarat by Pant et al showed Blood group B was dominant in both the communities, having similar to our present study which shows that in medical students both B and O blood group were prevalent.17 A study done over 152 medical students in Davangere, Karnataka by Hemlatha et al published the most common blood group was O (41.5 %) followed by B group (32.2 %), A group (19%) and least being AB group (7.2% ). Among the Rh blood group, 94% students were Rh positive, however only 6% were Rh negative. In this study among all one hundred medical students A, B and O blood groups were seen in equal proportion, each in 30 % cases and least being AB group (10%) and Ninety seven percentage students were Rh positive.18

Conclusion

We noted that ABO and Rhesus ‘D’ blood group distribution diverged in different parts of India. The study highlights similar frequency of distribution of A, B and O blood group among students and B is the most prevalent blood group among central Indian population. Rhesus positive is much more common than Rhesus negative among this population as well as in respect to Indian population. Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 The knowledge of blood group distribution is important for clinical studies, for reliable geographical information and blood bank management. This study would help in developing India-specific reagent cell-panels for antibody screening and identification, which would further aid in the improvement of transfusion services in the country.

Acknowledgements

P. K Budholia, Ajay Mishra, G.D Mehra

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Garratty G, Dzik W, Issitt PD, Lublin DM, Reid ME, Zelinski T. Terminology for blood group antigens and geneshistorical origins and guidelines in the new millennium. Transfusion. 2000;40(4):477-89.

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Dar NJ, Srivastava A, Dar FA. Distribution of ABO blood groups and Rh(D) factor among the Brahmin and Kushwaha populations of Jhansi District UP. Nature Precedings.2011;hdl:10:1038/npre.6362.1.

“Racial and Ethnic Distribution of ABO Blood Types bloodbook.com, Blood information for life,” 2006. http:// www.bloodbook.com

Makroo RN. ABO blood group system. In: Makroo BN (ed.) Compendium of transfusion medicine. New Delhi: Kongposh Publications Pvt Ltd; 2009.p.42.

Joshi SR, Vasantha K.A profile of rare bloods in India and its impact in blood transfusion service. Asian J Transfus Sci 2012;6(1):42–3.

Talukdar L, Sarma U. Frequency of Major Blood Group Antigens among Blood Donors at a Tertiary Level Hospital in North East India. IJSR 2014;3(9):2130-2.

Tulika C, GuptaA. Frequency of ABO and Rhesus blood groups in blood donors. Asian J Transfus Sci 2012;6(1):52-3.

10. Sidhu S. Distribution of the ABO blood groups and Rh(D) factor among the scheduled caste population of Punjab. Anthropologist2003;5:203-4. 11. A Patel Pyush, Patel Sangeeta P, V Shah Jigesh, V OzaHaren. Frequency and Distribution of Blood Groups in Blood Donors in Western Ahmedabad – A Hospital Based Study. Nat l J Med Res 2012;2(2):202-206. 12. Nag I, Das SS. ABO and Rhesus blood groups in potential blood donars at Durgapur Steel City of the district of Burdwan, West Bengal. Asian J Transfus Sci 2012;6(1):54-5. 13. Swamy GG, Chandrasekhar B, Parameswari J, Madhuravani S.Frequency and distribution of blood groups among medical students of Great Eastern Medical School, Srikakulam, Andhra Pradesh, India. Int J Med Pharm Sci 2013;3(9):26-33.

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14. Agrawal A, Tiwari AK, Mehta N, Bhattacharya P, Wankhede R, Tulsiani S, et al. ABO and Rh (D) group distribution and gene frequency; the first multicentric study in India. Asian J Transfus Sci 2014;8:121-5. 15. Parmanik T, Parmanik S. Distribution of ABO and Rh blood groups in Nepalese medical students: a report. East Mediterr Health J 2000;6(1):156-8. 16. Hussain R, Fareed M, Shah A, Afzal M. Prevalence and gene frequencies of A1A2BO and Rh(D) blood group alleles

among some Muslim populationsof North India. The Egyptian Journal of Medical Human Genetics 2013;14:69-76. 17. Pant CS, Gupta DK, Sharma RC, Gautam AS, Bhatt RM.Frequency of ABO blood groups, sickle-cell haemoglobin, G-6-PD deficiency and their relation with malaria in scheduled castes and scheduled tribes of Kheda District, Gujarat. Indian J Malariol 1992;29(4):235-9. 18. Hemalatha N R, Bhagya V, Frequency and Distribution of Blood Groups Among Medical Students in Davanagere. J Pub Health Med Res, 2015;3(1):1-4.

*Corresponding author: Dr Ajay Kumar, House no 14 Sai colony Dhanwantri Nagar Jabalpur M.P 482003 India Phone: +91 9516639699 Email: acalculia007@gmail.com Date of Submission : 20.02.2017 Date of Acceptance : 25.02.2017 Date of Publication : 27.02.2017

Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/AABS.2017.1331

Fine Needle Aspiration Cytology of Parapharyngeal Tumors Kuldeep Singh* and Rahim Gousia Dept. of Pathology, GMC Jammu. J & K, India

ABSTRACT Parapharyngeal space tumors (PPS) are rare, representing only 0.5% of head and neck neoplasm. 7 patients diagnosed with PPS tumors which presented to us within a span of two years were included. Cytopathologic evaluation was done by fine needle aspiration cytology, computed tomography and ultrasonography guidance wherever necessary. Histopathology confirmation was available in all the cases. The age of the patient ranged from 15-55 years with male to female ratio 1:1. The commonest clinical presentation was a slowly increasing painless mass in neck. Out of 7 cases the tumors encountered were pleomorphic adenoma (02), neurofibroma (01), Schwannoma (01), Paraganglioma (01), Lipoma (01), Angiofibroma (01). Two false negative cases were encountered. Overall diagnostic accuracy being 71.4%. With proper clinical and radiological assessment, FNAC can be extremely useful in diagnosing most of these lesions except a few which need histopathological confirmation. Keywords: Parapharyngeal Space, Computed Tomography, Magnetic Resonance Imaging, Parapharyngeal Tumors, Cytology

Introduction

PPS is a rare location for head and neck tumors (1). It is a space in the supra hyoid neck that contain fat and is surrounded by several other spaces defined by the fascial layers of neck (15). Para pharyngeal space tumors often present therapeutic and preoperative diagnostic problems due to variable nonspecific symptoms and complex anatomy of the region. Most of the lesions (70-80%) are benign (3) comprising of salivary gland tumors, neurogenic tumors and paragangliomas in descending order of frequency (17). Other rare tumors include lipoma, hemangioma, aneurysm, branchial cleft cyst, meningioma, chordoma, and sarcoma (2). On FNAC cytomorphology of meningioma mimics that of other head and neck tumours like acinic cell carcinoma and paraganglioma etc.(18). Therefore, the possibility of meningioma should be kept in the differential diagnosis of PPS tumour. The most common malignant tumour in this region is adenoid cystic carcinoma by Spiro et al. (14). Malignant peripheral nerve sheath tumour is rare at this site and have grave prognosis (4). Occurrence of metastatic papillary thyroid carcinoma in the PPS is extremely rare (13). The patient usually present with swelling of two specific sites namely oropharynx and lateral neck (cervical or submandibular mass), there may be oral cavity bulge, serious otitis, headache, syncope or even cranical nerve palsies involving vagus and hypoglossal nerves, leading to disorders of swallowing or speech(21). The diagnosis is done by physical examination, radiological imaging, and pathological examination. Fine

needle aspiration cytology (FNAC) can be an easy, rapid, and effective method of diagnosing these myriad of lesions of a specific anatomic space(9). Diagnostic difficulty persists due to their similar mode of presentations and at times morphological overlap. Hence, some cases can only be confirmed by histopath-ological examination. It is very important to diagnose the nature of tumor whether benign or malignant prior to therapy, because treatment protocol is variable and early diagnosis can give better survival rate.

Material and Methods

The present study was conducted on seven patients mainly presenting with painless mass in the neck. Other symptoms seen included foreign body sensation, dysphagia, hoarseness of voice and impaired hearing.Cytomorphological patterns were evaluated by doing FNAC with 10cc syringes and 22 gauge/24 gauge needle following the standard procedure (11). FNAC was also done under guidance (Computed tomography/ ultrasonography) as and when necessary. The smears were prepared in the usual manner. The wet smears were immediately fixed in isopropyl alcohol and latter stained by Papanicolaou and Hematoxylin& Eosin. Air-dried smears were stained by May-GrunwaldGiemsa. Confirmation was done by histopathology (routine hematoxylin and eosin stain).

Result

Total seven cases of parapharyngeal space tumors were selected of which all were benign. The age ranged from 15-55 years with Male: Female ratio was 1:1. Maran et al. (8) and Pang et al.(12) also reported no sex predominance in their studies. The most common mode of presentation

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was gradually increasing painless mass in neck, the other symptoms being foreign body sensation, dysphagia, hoarseness of voice and impaired hearing (Table I). Radiological investigation revealed parapharyngeal space mass lesions in all the cases. All cases underwent surgery with histopathological examination to confirm the diagnosis. The most common surgical approach was transcervical (5cases), followed by transparotid and combined transcervicaltransparotid approach (1 case each)(Table II). Out of 7 cases, 5 gave a satisfactory cytological material, in one case of paraganglioma only blood was aspirated, whereas case of angiofibroma cellularity was very scanty to diagnose. Histopathological confirmation of both the lesions was done after cytological detection. Pleomorphic salivary adenoma (28.6%) showed maximum incidence followed by neurofibroma (14.3%), Schwannoma (14.3%)(Fig.1), Paraganglioma (14.3%), Lipoma (14.3%), angiofibroma (14.3%)( Table III). Table I: Clinical Features of patients of PPS tumor. Clinical Features

No. of patients

Painless mass in neck

Dysphagia

Hoarseness of voice

Impaired Hearing

Total

Table II: Surgical approaches in PPS Tumors Surgical Approach

Number of Cases

Trans cervical

Trans parotid

Combined Trans cervical- arotid

Total

Table III: Diagnosis of Parapharyngeal space tumors Diagnosis

Number

Percentage

Pleomorphic adenoma

28.6%

Neurofibroma

14.3%

Schwannoma

14.3%

Paraganglioma

14.3%

Lipoma

14.3%

Angiofibroma

14.3%

Total

Discussion

Tumors of PPS have been of interest to head and neck surgeons because of wide variety of histologic tumors that occur in this space. Parapharyngeal tumors are difficult to diagnose early due to their location and plethora of presentation. They are usually benign, pleomorphic adenoma arising from the deep lobe of parotid has been found to be the most common tumor of parapharyngeal region by several authors(1) and also by us. Studies have shown that there is no difference in the prognosis of pleomorphic adenoma even if they are cellular and show cytologicatypia in the form of scattered hyperchromaticnuclei(19). Neurofibroma and Schwannomas are rather common tumors of parapharyngealspace(1). But their occurrence at this site, slow growth and associated neurological manifestations can mimic other entities(16). The paragangliomas occurring in head and neck comprise 3% of all parangangliomas and almost all located in PPS and arise in carotid body(5). Most follow a benign course, may be bilateral, multicentric or a component of multiple endocrine (MEN) syndrome. Histopathological confirmation was done to confirm the diagnosis.

Fig. 1: Case Diagnosed as Schwannoma A. FNAC Show Spindle Cells

B. Histological Section with palisading (MGG x 400).

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A-73 Angiofibroma can also arise in the site(6), but we failed to diagnose them on cytology alone and most of these smears were grossly hemorrhagic with few spindle cells only. Cytological diagnosis was almost corroborative with final histopathological diagnosis in all cases. True positive cases comprised 5(71.4%), whereas there were only two false negative cases due to paucity of materials of difficulty in approaching the lesions. Thus, diagnostic accuracy in this series was approximately 71.4% which corroborated with the findings of earlier studies(10). Parapharyngeal masses sometimes fail to be detected early due to their location and overalapping symptoms with other common illnesses. Their important lies in the fact that even benign lesions, if left untreated, may be fatal due to encroachment of vital structures. Clinical examination alone may not be sufficient to diagnose a mass or parapharyngeal lesion. High resolution CT scan has facilitated and contributed significantly to the preoperative assessment of these lesions. It helps in determining the size and extent of tumor origin, demonstrate the degree of tumor vascularity, differentiates benign from malignant lesions and its relation to major vessels(20). This assist in planning the management of tumor. MRI has also become a very useful diagnostic tool. With proper clinical and radiological assessment, FNAC can be extremely useful in diagnosing most of these lesions except a few which need histopathological confirmation.

References 1. 2. 3.

4. 5.

Ahmed F, Waqar-Uddin, Khan MY, Khawar A, Bangush W, Aslam, J. Management of parapharyngeal space tumors. J Coll Physicians Surg Pak 2006; 7-10. Chu W, Strawitz JG. Parapharyngeal growth of parotid tumor: Report of two cases. Arch Surg 1977; 709-11. Fernandez Ferro M, Fernandez Sanroman J, Costas Lopez A, Sandoval Gutierrez J, Lopez de Sanchez A. Surgical treatment of benign parapharyngeal space tumors: Presentation of two clinical cases and revision of literature. Med Oral Pathol Oral Cir Bucal 2008: 61-4. Graccion JG, Enzinger FM. Malignant schwannoma associated with von Recklinghausen neurofibromatosis. Virch- ows Arch (A) 1979: 43-57. Lack E. Tumors of adrenal gland and extra adrenal paraganglioma: Atlas of tumor pathology 1997; Series 3, Washington DC: Fasc 19.

AABS; 4(1): 2017 6. 7.

8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Laryngology and Head and Neck Surgery. In: Hibbert J, editor. Scott-Brownâ&#x20AC;&#x2122;s otolaryngology. 6th ed. Oxford: Butter-worth-Heinemann; 2005. Luna-Ortiz, Navarrte-Aleman JE, Grana-dos-Gracia M, Herrea-Gomez A. Primary parapharyngeal space tumors in Mexican cancer center. Otolaryngol Head Neck Surg 2005; 587-91. Maran AG, Mackenzie IJ, Murray JA. The parapharyngeal space. J. Laryngol Otol. 1984 Apr; 98(4): 371-80. Mondol A, Raychoudhury BK. Peroral fine needle aspiration cytology of parapharyngeal lesions. ActaCytol 1993; 694-8. Mondol, A, Gupta S. The role of preoral fine needle aspiration cytology (FNAC) in the diagnosis of parapharyngeal lesions: A study of 51 cases: Indian J PatholMicrobiol 1993: 253-9. Orell SR, Sterrett GF, Whitaker D. Techniques of FNA cytology. In: Orell SR, Sterrett GF, Whitaker D. editors. Fine needle aspiration cytology. 4thed. New York; Churchill Livingstone; 2005, P. 9-30. Pang, KP, Goh CH, Tan HM. Parapharyngeal space tumors: an 18-year review. J Larngol Otol. 2002 Mar; 116 (3); 170-5. Saydam L, Kalcioglu T, Demirkiran A, Gurer M. Occult Papillary thyroid carcinoma presenting as a parapharyngeal metastasis. Am J otolaryngol. 1999 May-June 20(3): 166-8. Spiro RH, Huvos AG, Strong EW. Adenoid cystic carcinoma of salivary gland: A clinicopathologic study of 242 cases. Am J Surg 1974; 512-20. Stambuk HE, Patel SG. Imaging of the parapharyngeal space.OtolayngolClin North Am 2008: 77-101. Szmeja Z, Sefter W, Szymice E, Wierzbicka M, Zdanowicz E. Neurofi-broma of the parapharyngeal space: A case report. Otolaryngol Pol 1998: 215-7. Tak B, Bhuie HS, Gupta, A.K. Paraphar-yngeal space tumors: An overview. Indian Journal of Otolaryngology and Head and neck surgery 2001, 53 (2) 173-75. Tan LH. Meningioma presenting as a parapharyngeal tumor: report of a case with fine needle aspiration cytology. Actacytol.2001 Nov-Dec; 45(6) 1053-9. Thunnissen FB, Peterse JL, Buchholtz R, Van der Beek JM, Bosman FT. Polyploidy in pleomorphic adenomas with cytologicatypia. Cytopathology 1992; 101-9. Whyte AM, Hourihan MD. The diagnosis of tumours involving the parapharyngeal space by computed tomography.Br J Radiol. 1989 Jun; 62(738): 526-31. Zhi KO, Ren WH, Zhang L, Wen YM, Zhang YC. Diagnosis and surgical approach of a parapharyngeal space neoplasms. Shanghai Kou Qiang Yi Zue 2007: 547-50.

*Corresponding author: Dr. Kuldeep Singh, Associate professor; Dept. of Pathology, GMC Jammu. J & K, India Email: conifer1@indialines.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 08.02.2017 Date of Acceptance : 03.03.2017 Date of Publication : 11.03.2017

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Original Article DOI: 10.21276/AABS.2017.1383

MicroRNA-155 in Patients with Chronic Stable Angina Mohamed M Elshafae1, Jehan H Sabry1, Mohamed A Salem2, Hanan M Elshafee Clinical and Chemical Pathology Department, Faculty of Medicine, Benha University, Egypt. 2 Cardiology Department, Faculty of Medicine, Benha Universityz, Egypt.

ABSTRACT Background: Atherosclerotic cardiovascular disease is a chronic inflammatory disease and one of the major causes of death worldwide. MicroRNAs are associated with many physiological and pathological situations as inflammation and cardiovascular disease. The communication between microRNAs, inflammation, and atherosclerosis, drive attention to the possibility that inflammation-related microRNAs, miRNA-155, could have a role in atherosclerosis progression. Objectives: We aimed to determine the levels of circulating miRNA-155 in chronic stable angina patients and to study the impact of microRNA-155 on coronary artery disease severity and extent. Methods: MicroRNA was extracted and assessed from plasma of 50 subjects (20 normal controls and 30 patients with chronic stable angina) using quantitative reverse-transcription PCR (RT-PCR). Levels were compared in the two groups and correlated with Gensini score in the group with chronic stable angina (CSA). Results: Plasma levels of microRNA 155 were significantly lower in CSA patients (0.59±0.48, 1.94±0.76, in patients and control group respectively (P<0.001).The expression of miR-155 correlated negatively with Gensini scores (P<0.001), total cholesterol (P <0.001), LDL-c (P= 0.002) and triglycerides (P= 0.03).There was a significant difference in miRNA-155 levels among the quartiles of the CSA group (P < 0.001) denoting negative correlation between microRNA 155 and coronary artery disease extent. ROC curve showed that microRNA-155 sensitivity and specificity for the prediction of severity of atherosclerosis were 86.67% and 80% respectively. Conclusion: Plasma miRNA-155 is significantly lower in CSA angina patients and levels significantly decreases with the progression of atherosclerosis. Keywords: Micro RNA 155, CSA, Gensini score, Atherosclerosis, Reverse-Transcription PCR.

Introduction

Coronary artery disease (CAD), secondary to coronary atherosclerosis, is the most common type of cardiovascular disease and leading cause of death globally. Understanding the underlying molecular and cellular mechanisms may contribute to the prevention of cardiovascular diseases 1. Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the vessel wall. Inflammation and immune responses play an axial function in all atheroscleroticphases starting from preface of the fatty streak ending in acute coronary syndromes (ACS) eruption2. MicroRNAs (miRNA-155) are small noncoding RNA molecules that negatively regulate gene expression by triggering either translation repression or RNA degradation 3. They are leading components for many cellular processes. MicroRNA expression was described to betissue-specific, firmly adjusted during embryogenesis, and overexpressed/underexpressed in different pathologies, including cardiovasculardiseases4. To date, about 2000 microRNAs have been detected. Human genome consist of large number of microRNA

genes accounting for 1–5 % of all predicted human genes and mammalian microRNAs are known to regulate approximately 30 % of all protein- coding genes5. MicroRNA155 is widely expressed in various cells, such as T cells, Bcells, mononuclear cells and endothelial cells. As a multifunctionmicro RNA, it is extensively involved with the differentiation, proliferation and apoptosis of many cells, and the development of many tissues. MiRNA-155 can inhibit inflammatory response and affect lipid uptake in macrophages through regulating othertargets6. In this study the levels of circulating microRNA-155 in patients with chronic stable angina were measured. In addition, the impact of microRNA155 on the severity and extent of coronary artery disease was evaluated.

Material and methods

Study Design: This observational cross-sectional study included fifty persons who were enrolled for coronary angiography due to suspicion of coronary artery disease at Cardiac Catheterization Unit, Department of cardiology, Benha University Hospital from february 2016 until august

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2016. All investigations and research work were done in the Clinical and Chemical pathology departement, Benha University Hospital. Subjects were categorized into the following groups; patient group (30 patients who had chronic stable angina based on clinical history and results of coronary angiography) and control group (20 persons who had normal coronary angiography).

the left main coronary artery, 2.5 for the proximal left anterior descending artery (before 1stseptal branch) or proximal left circumflex artery (before 1stobtuse marginal branch), 1.5 for the mid-region of left descending artery or left circumflex artery,1 for the distal left anterior descending artery, and 1 for the mid or distal region of the left circumflex artery or right coronary artery]8.

Inclusion Criteria Were: Patients with suspicion of stable angina who were referred for coronary angiography based on one or more of the following indications: 1- Patients who have high pretest probability and severe symptoms. 2-Patients with reduced LVEF < 50% and typical angina. 3- Patients with positive findings at non-invasive risk stratification studies.

Blood samples: Five milliliters of venous blood samples were withdrawn from each participant via antecubitalvenipuncturing into EDTA- containing tubes. Samples were centrifuged at 1,200 X g for 10 minutes at 4°C. Plasma were collected and centrifuged again at 12,000 X g for 10 minutes at 4°C. The clear plasma obtained was aliquoted and frozen at -80oC until used for lipid profile analysis and miRNA extraction.

Exclusion Criteria Were: Evidence of acute coronary syndrome, left ventricular ejection fraction ≤ 30%, subject receiving or scheduled to receive chemotherapy of malignancy, subject receiving immunosuppressant therapy and/or has known immunosuppressive or autoimmune disease, patients with acute or chronic inflammatory disorders, subject with documented or suspected liver disease, known renal insufficiency. The study was approved by the Local Ethics Committee and all study participants gave a written informed consent before enrollment . I Baseline Evaluation: Baseline evaluation included review of medical history including demographic data (age, sex) and risk factors of CAD (diabetes mellitus, hypertension and smoking), analysis of cardiac symptoms, cardiac medications and comorbidities, clinical examination and 12- lead ECG were done. II Coronary angiography (All patients underwent elective coronary angiography using standard Judkin’s technique by femoral approach7. Each major epicardial vessel was visualized in at least two orthogonal projections to detect the degree of luminal stenosis; percent diameter stenosis was determined by using quantitative coronary angiography (QCA).

Detection of miRNA-155 by real time reverse transcription polymerase chain reaction (RT-PCR) was performed as follows 1- RNA extraction:- Using the miRNeasy Mini Kit Qiagen according to the manufacturer’s instruction RNAs were extracted from plasma samples. 200 μL of splasma were mixed with 1000 μL Qiazollysis reagent and incubated for 5 minutes at room temperature. Then 200 μL of chloroform were added and the tube was vortexed vigorously for 15 seconds. After 3 min incubation at room temperature, the tube was centrifuged at 12,000 X g for 15 min, in which the sample separated into 3 phases. 350 μL of the upper aqueous phase was transferred into new collection tube and mixed with 900 μL of 100% ethanol. About 700 μL of the sample were pipetted into an RNeasy Mini Spin column in a 2 ml collection tubes and centrifuged at 800 X g for I5 Sec. at room temperature. Then the flow through was discarded. Specimen was washed three times with 700 μl of RWT buffer and 500μl of RPE Buffer respectively through centrifugation at 8000X g for 15 sec. Finally RNA was eluted using 50 μL R Nase-free water after centrifugation for 1 min (≥ 8000 X g).

Ginsini score (GS) calculation: The Gensini score (GS) was used in the present study to assess the burden of coronary atherosclerosis. This was done as described by Gensini GG in (1983): [The GS system yields a qualitative and quantitative evaluation of the coronary angiogram. The score grades the narrowing of the lumen of the coronary artery as: 1 for ≤25% narrowing, 2 for 26–50% narrowing, 4 for 51–75% narrowing, 8 for 76– 90% narrowing, 16 for 91–99% narrowing and 32 for total occlusion. Next, this primary score was multiplied by a factor that took into account the importance of the position of the lesion in the coronary arterial tree: 5 for

2- Reverse transcription:- Using the miScript II RT Kit according to the manufacturer’s instruction extracted RNA was reverse-transcribed into cloned DNA. The reverse transcription master mix were prepared on ice as follows: 2 μL of 10 x mi Script Nucleics, 4μL of 5 x mi Script Hispec buffer, 7μL of RNase-free water, 2 μL of mi Script Reverse Transcriptase Mix and 5 μL of Template RNA. The total volume was 20 μL/reaction. Template RNA were Added to each tube containing reverse transcription master mix. Then mixed gently, briefly centrifuged,, incubated for 60 min at 37°C, then incubated for 5 min at 95°C to inactivate miScript reverse transcriptase mix in Veriti thermal cycler applied biosystem.

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3- RNA quantification: PCR quantification was performed with PCR (Step One real time PCR, Applied Biosystem ) PCR was performed using miScript SYBR Green PCR Kit supplied by (QIAGEN) according to manufacturer instructions.The primers for micro RNA155 and housekeeping gene were supplied by Qiagene. Fluorescene measurements were made in every cycle. The cycling conditions used were as follows: PCR initial active stepat 95°C for 15 min, then 40 cycles which includes denaturation at 94°C for 15s, annealing at 55°C for 30s then extension at 70°C for 30s. The normalization control used was SNORD 68.StepOne software was used for quantification and expression of miRNA 155 as relative miRNA level compared with a SNORD68 gene according to the 2−ΔΔCt method 9. Statistical analysis : The collected data were summarized in terms of mean ± Standard Deviation (SD) and range for quantitative data and frequency and percentage for qualitative data. Comparisons among the different study groups were performed using the Chi-square test (χ2) and Fisher’s Exact Test (FET) to compare proportions as appropriate. The Student t-test (t) was used to detect mean difference between two groups regarding parametric data and the Mann-Whitney test (z) was used to compare two non-parametric data. Comparisons between more than two groups were carried out using the Kruskal Wallis test (χ2) regarding non-parametric data. The Spearman correlation coefficient (rho; ρ) was used to test the correlation between micro RNA-155 levels and studied parameters. Receiver Operator Curve (ROC) analysis of micro RNA-155 as a diagnostic test for stable angina patients was carried out and the Area Under the Curve (AUC), the best cut off point and the corresponding sensitivity and specificity were determined. The corresponding P-values were obtained. A P-value < 0.05 was considered statistically significant (S), a P-value < 0.001 was considered statistically highly significant (HS), while a P-value > 0.05 was considered statistically non-significant. The statistical analysis was conducted using STATA/SE 11.2 for Windows (STATA Corporation, College Station, Texas).

Results

Study Population: The mean age was 54.56±10.26 years (57.57±9.35, 53.05±8.01, in patients and control group respectively. P= 0.08). 54% of the study population were males (63% vs 54% in patients’ and control groups respectively, P= 0.10) and 46% were females (37% vs 60% in patients’ and control groups respectively, P= 0.10). 50% of the study population were hypertensive (57% vs 40% in patients’ and control groups respectively, P= 0.25). Persons

with history of diabetes mellitus represented 50% of the study population (47% vs 55% in patients’ and control groups respectively, P= 0.56). 18% of the study population were smokers (23% vs 10% in patients’ and control groups respectively, P= 0.12)[Table 1]. II Baseline investigations: The mean total cholesterol was 163.5 ± 30.18 mg/dl (166.93 ± 31.27mg/dl, 158.35 ± 28.74 mg/dl in patients’ and control groups respectively. P= 0.33). Mean triglycerides level was 146.04 ± 45.46 mg/dl (142.83 ± 45.2 mg/dl, 150.85 ± 46.6 mg/dl in patients’ and control groups respectively, P= 0.55). The LDL-c was 86.4 ± 29.97 mg/dl (91.45 ± 29.39 mg/dl, 78.83 ± 29.95 mg/ dl in patients’ and control groups respectively. P= 0.15). HDL-c was 47.86 ± 7.84 mg/dl (46.87 ± 7.88 mg/dl, 49.36 ± 7.73 mg/dl in patients’ and control groups respectively. P= 0.28). The ejection fraction was 59.94 ± 8.4% (60.97 ± 8.26%, 58.4 ± 8.57% in patients’ and control groups respectively. P= 0.29)[Table 2]. III Coronary angiography in patients with chronic stable angina: The mean Gensini score was 36.23 ± 26.86 IV MicroRNA 155 in study population: The mean level was 1.13 ± 0.9 (0.59 ± 0.48, 1.94 ± 0.67 in patients’ and control groups Respectively, P< 0.001) [Figure 1]. V Micro RNA 155 and Gensini score: We reported a strong negative correlation between the level of microRNA 155 and the Gensini score (r= - 0.87, P < 0.001)[figure 2]. On dividing the case study group into four quartiles according to Gensini score there was a statistically significant gradual downregulation in microRNA 155 (P value < 0.001) with significant decrease in the third and fourth quartiles compared with the first quartile group (p<0.001) and significant decrease in the fourth quartile compared with the second quartile group[Table 3]. VI Correlation between microRNA 155 and study variables: When analyzing the correlation between proatherogenic risk factors and microRNA 155, we found that microRNA 155 correlated negatively with total cholesterol levels (r= -0.60, P< 0.001), LDL-c (r= -55, P= 0.002) and triglycerides (r= -0.39, P= 0.03) [table 4]. VII Diagnostic performance of Micro RNA 155 for atherosclerosis: Roc curve analysis of microRNA 155 for the prediction of severity of coronary atherosclerosis shows 86.67% sensitivity and 80% specificity with 87% positive predictive value and 80% negative predictive value and the area under the curve was 0.9567 and best cutoff point value of 1.43 suggesting that microRNA 155 may act as a marker of severity of atherosclerosis [Figure 3].

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Table 1: Base line characteristics of the study population. Cases (no.=30)

Variable No. Age (years)

Mean ±SD; range

HTN DM Smoking

No.

57.57±9.35 (40-77)

Females Males Positive Negative Positive Negative Positive Negative Ex-smoker

Sex

Controls (no.=20)

11 19 17 13 14 16 7 17 6

Total (no=50) No.

53.05±8.01 (30-61)

36.67 63.33 56.67 43.33 46.67 53.33 23.33 56.67 20.0

12 8 8 12 11 9 2 17 1

54.56±10.26 (30-77)

60.0 40.0 40.0 60.0 55.0 45.0 10.0 85.0 5.0

23 27 25 25 25 25 9 34 7

46 54.0 50.0 50.0 50.0 50.0 18.0 68.0 14.0

Test

t= 1.77

0.08

χ2= 2.63

0.10

1.33

0.25

0.33

0.56

FET

0.12

Table 2: Baseline investigations in both groups: Cases (no.=30)

Variable

Controls (no.=20)

Total (no.=50)

Mean

±SD

Range

Mean

±SD

Range

Cholesterol

166.93

31.27

118-223

158.35

28.47

142.83

45.2

78-211

150.85

46.6

LDL

91.45

29.39

36.4141.4

78.83

HDL

46.87

7.88

32-58

60.97

8.26

41-75

114-223

0.98

0.33

78-262

0.61

0.55

29.97

30142.6

1.47

0.15

47.86

7.84

32-60

1.10

0.28

59.94

8.4

41-75

1.06

0.29

Test

χ2= 21.58

<0.001 (HS)

Mean

±SD

Range

114-212

163.5

30.18

88-262

146.04

45.46

29.95

30-142.6

86.4

49.35

7.73

36-60

58.4

8.57

41-71

Table 3: Comparison of miRNA-155 expressions between the four quartiles

GENSINI score <18 (no.=7)

Variable

18(no.=8)

31(no=7)

Mean ±SD Range Mean ±SD Range Mean RQ Micro RNA

1.24

0.47

0.581.67

0.66

0.2

0.410.97

†0.32

46-126 (no.=8)

±SD Range 0.17

Mean

0.18†‡0.21 0.63

±SD

Range

0.09

0.130.38

†Significant differences compared to GENSINI score <18 ‡Significant differences compared to GENSINI score 18-<31 Table 4: Correlation between micro RNA 155 and estimated parameters in case group. Variable (no.=30)

Spearman correlation coefficient (rho; ρ)

Age (years)

0.27

0.15

Cholesterol

-0.60

<0.001 (HS)

-0.39

0.03 (S)

LDL

-0.55

0.002 (S)

HDL

0.15

0.41

-0.02

0.89

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Fig. 1: MicroRNA 155 levels in patients and control groups.

Fig. 2: Correlation between micro RNA 155 and genesis score

Fig.1 : Roc curve analysis of microRNA 155 for the prediction of severity of coronary atherosclerosis.Â

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Discussion

Atherosclerosis is a chronic inflammatory disease characterized by lipids and inflammatory cells deposition in the vessels’ walls. It is one of the major causes of death worldwide. Inflammation and immune responses play an axial function in all atherosclerotic phases starting from preface of the fatty streak ending in acute coronary syndromes (ACS) eruption2. Micro RNAs (miRNAs) are small non coding RNA molecules linkedto many physiological and pathological conditions, as most of inflammatory processes thorough cardiovascular disease. Through enhancing translation repression or RNA degradation, miRNAs regulate gene expression negatively9. As a multifunctional miRNA, miRNA-155, was implicated in cardiovascular diseases, viral infections, and various types of cancers as it is able to regulate complex pathophysiological processes and to be involved in cardiovascular remodeling, which results in cardiovascular diseases such as coronary artery disease, abdominal aortic aneurysm and heart failure 10. Besides, being highly expressed in multiple autoimmune inflammatory diseases and in activated immune cells, miRNA-155 is expressed in different cells, smooth muscle cells, monocyte and macrophages are some examples. In myeloid cells, miRNA-155 expression can be induced by various inflammatory signals, aslipopolysaccharide, tumor necrosis factor and interferon-b. Moreover,miRNA-155 expression in THP-1 macrophages was induced byoxidized low-density lipoproteins (oxLDLs).Increase in the inflammatory stimuli (oxLDLs) uptake and foam cell formation in these cells occured when miRNA-155 was inhibited. As a result, miRNA-155 is considered to be an inflammation-related miRNA11. Aiming to studythe impact of miRNA-155 levels on both the severity and extent of coronary artery stenosis assessed by coronary angiography and the Gensini score, wecompared the relative expression of miRNA-155 in a group of chronic stable angina patients with a normal matched group proved by coronary angiography. In the present work, we dealt with two groups of patients, a normal control group (20 individuals) and a group of chronic stable angina patients (30 patients). The two groups were matched with no significant difference regarding age, sex, history of hypertension, history of diabetes and history of smoking. Also there was no statistically significant difference between the two studied groups in levels of cholesterol, triglycerides, HDL, and LDL.

AABS; 4(1): 2017 fraction between the control group and the stable angina group with a P value 0.29. In accordance with our aim, the micro RNA 155 relative quantity was significantly lower in the stable angina group than in the control group. These results coincides with Fichtlscher et al.12 who performed a study designed to assess the suspected prognostic role of micro-RNAsin stable CAD. They studied the miRNA profiles in 16 subjects,only 8 of them were stable CAD patients. All participants received the indicated treatment. The results of the studyrevealed elevation inthe cardiomyocyte-enrichedmi RNAs(miRNA-133 and miRNA-208a) in the patients’ group. On the other hand, significant decrease in plasma levels of endothelial cell-enriched (miRNA-126, miRNA-17 and miRNA-92a),vascular smooth muscle cells associated (miRNA-145) and inflammatory cell-enriched (miRNA-155) were observed in the patients’ group. Larger study by the sameauthor confirmed those results. The results of the present study are also in accordance with that of Zhu et al.13 who performed a study on 110 patients. In that study, the expression patterns of miRNA-155 in Peripheral blood mononuclear cells PBMCs of the CAD group was significantly lower than that of the non-CAD group and the plasma expression pattern of microRNA-155 were in accordance with the pattern in PBMCs; that it was significantly lower than that of the non-CAD (P<0.05). The plasma level of miRNA-155 was lower in patients with SAP, UAP, and AMI than in patients with CPS, whereas no statistically significant difference was observed between patients with SAP and CPS. These results go in line with Zhang et al. 6 who performed a study to explore the action mechanism of microRNA 155 in atherosclerosis and to study the relationship between miRNA-155 and the severity of CAD and plaque stability. They isolated (PBMC) form blood samples from patients with acute myocardial infarction (AMI), unstable angina (UAP), stable angina (SAP) and chest pain syndrome (CPS) and they found that the expression level of miRNA-155 in blood samples from coronary heart disease patients was much lower than in the blood samples of non-coronary heart disease (P<0.05) and the miR-155 level in the CPS group was higher than in the SAP, UAP, and AMI groups; the differences were statistically significant. This result was also approved by D’Alessandro and colleagues,14 who found that circulating levels of endothelial-enriched inflammation-associated miRNA-155 are significantly reduced in CAD compared with controls.

In addition, there was no significant difference in ejection

As miRNA-155 can be activated by the stimulation of transcription factors in traditional signaling pathways and

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some related factors in other signaling pathways, a feedback mechanism that controls the over-reactivation of immune cells could bethe reason ofmiRNA-155 downregulation15. Also miRNA-155 serves as a negative feedback regulator in OxLDL-stimulated THP-1 inflammatory responses and lipid uptake 16. Zhang et al.6 examined the effort of OxLDL on RAW264.7 macrophage and miRNA-155 overexpression RAW264.7 macrophage. They found that the viability of the cell in miRNA-155overexpression group was much higher than the control groupand the difference was statistically significant (P<0.05). These results further confirmed that miRNA-155levels declines as CAD progresses and the overexpression of miRNA-155 can inhibit the apoptosis of RAW264.7 macrophage induced by OxLDL. The important miRNA-155 has multiple functions in endothelial cells, not only in the regulation of inflammation, but also in the inhibition of EC migration in response to Ang II 17. Also it has been found to directly target endothelial nitric oxide synthase (eNOS) mRNA. Collectively, these data highlight the importance of miR-155 in regulating the inflammatory signaling pathways of the macrophage lineage 18. However, no difference in miRNA-155 expression in patients with CAD could be detected by Hoekstra et al.19, who didn’t use coronary angiographies in the assessment of coronary arteries conditionin their study. On the contrary, Wei et al. 20 performed a study on early stages of atherosclerotic plaques in mice and claimed that upregulation of miRNA-155 is the way to atherosclerosis and the inflammatory stimulation of macrophages via miR342-5p. Variation inmethods used to analyze the atherosclerotic plaque and different animal models, may be the cause of the conflicting results that obtained by Nazari et al.21 who assumed that miRNA-155 enhances atherosclerosis by repressing Bcl6 in macrophages. And they claimed that there is a harmful effect of miRNA-155 on the atherosclerosis process and plaque stability. Correlation studies revealed highly significant negative correlation between miRNA-155 levels and Gensini scores (representing the severity and extent of coronary stenotic lesions) in patients with chronic stable angina (-0.87, P value < 0.001). We further divided all patients into four quartiles according to the Gensini score. It was found that there was significant downregulation in microRNA 155 (P value <0.001) among the four quartiles with significant decrease in quartile three and four when compared with the first quartile group and significant decrease in quartile four compared with the second quartile group.

Finding a negative correlation between miRNA-155levels in PBMCs and Gensini score byZhu et al. 13who analyzed the correlation between miR-155 levels and Gensini scores(– 0.663, P<0.001) in all coronary artery disease patients goes in line with our results. And when dividing the coronary artery disease patients into 4 groups according to the number of diseased vessels, they stated that miRNA-155 levels were significantly lower in the patients with two or more diseased vessels compared with those with no diseased vessel (P<0.001) . The relationship between Gensini scores and miRNA-155 in the present study and in other studies suggests that miRNA-155 might have a defensive functionversus atherosclerosisprogression. These results can be indicative of using micro RNA 155 as a marker of severity of atherosclerosis. In the present study, we applied the Gensini scoring system, instead of number of diseased vessels, to assess the extent and severity of coronary stenotic lesions, because in this system, the coronary trees are divided into 15 segments and even mild lesions are also calculated in the final score 22. Onpredicting the severity of coronary atherosclerosis using ROC curve analysis, miRNA-155 shows 86.67% sensitivity and 80% specificity with 86.67% positive predictive value and 80% negative predictive value and the area under the curve was 0.9567 at the best cutoff point value of 1.43 suggesting that microRNA 155 can act as a marker of severity of atherosclerosis. When levels of microRNA 155 were correlated with different study variables, it was found that miRNA-155 levels showed significant negative correlation with cholesterol (-0.60, P<0.001), triglycerides (-0.39, P=0.03) and LDL levels (-0.55, P=0.002). These results were in accordance with Zhu et al.13 who found that, miR-155 levels in all patients were negatively correlated with LDL cholesterol (r= – 0.315) although showing positive correlation with total cholesterol. Among many studies investigated the relation between miRNA-155 and coronary atherosclerosis, Zhu et al.23 reported that miRNA-155 adjusts the endothelial inflammation and migrationinduced by angiotensin II. On transplanting bone marrow from a mouse with miRNA-155 -deficiency to a hyperlipidemic one, Donners et al.24discovered that the deficiency of miRNA-155 inhematopoietic cells promoted plaque formation and decreased its stability declaringanatheroprotectiverole of miRNA-155 on atherosclerosis. Zhu et al.25also supported this protective function of miRNA-155 in atherosclerosis and plaque stability. MiRNA-155 was found to be either upregulated or downregulated in coronary artery disease patients and thus might promote or prevent CAD. These

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A-83 conflicting results of miRNA-155 in the pathophysiology of atherosclerosis related ischemic heart diseases indicate the complexity of this multifunctional molecule in regulation of cardiovascular remodeling induced by atherogenesis26. The cause of this inconsistence might be due to different pathological stages of the disease. Therefore, more studies of underlying mechanisms of miRNA-155 involvement in CAD are needed 1. MicroRNAs, these tiny epigenetic silencing phenomena only recently discovered, are moving close to become a realizable diagnostic and therapeutic focus for coronary artery disease (CAD), and the results presented in this study just may have provided supportive data allowing that work to begin in intent on achieving that goal. Wei and colleagues27 performed a study on the hypothesis that the effect of miR-155 in macrophages is stage dependent. They found that in early atherosclerosis, miR-155 suppressed macrophage proliferation by targeting colony-stimulating factor-1 receptor and in advanced atherosclerosis, it impaired efferocytosis by downregulating B-cell leukemia/lymphoma 6. They concluded that targeting the relation between miRNA-155 and B-cell leukemia/ lymphoma 6 might be a promising approach to inhibit the atherosclerosisprogression. Targetingvariant genes implicated in the same pathway process gives miRNA sapotential therapeutic advantage over traditional therapies as they target a single protein, while unaffected others canpreserve the pathological mechanism 28. Haveing specific target in the disease pathogenetic mechanism gives miRNAsa high specificity of treatment. Even with weaker power of inhibition,this high specificity, and the long-lasting effect and widespread of action make miRNA modulation a more effective therapy compared to traditional therapy29. The present study supported the protective role for miRNA-155 against the progression of atherosclerosis. Increasing miRNA level sthrough a mimic approach could be now recommended as a considerable therapeutic target. Mimics are double-stranded oligonucleotides containing a (guide strand)identical to the mature miRNA, and a (passenger strand) complementary, or partially complementary strand. The guide strand,loading into the RNA induced silencing complex, act as the endogenous targeted miRNA and block gene expression 30. In conclusion, MiRNA-155 was found to be clearly downregulated in studied patients with chronic stable angina. Negative correlation evidenced between miRNA-155 expression and coronarystenotic lesions Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 severityassessed by Gensini scores, boostedthedefensive role of miRNA-155 against atherosclerosis progression.

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*Corresponding author: Jehan H Sabry, Asisstant professur of Clinical and Chemical Pathology , Faculty of Medicine, Benha University, Egypt Email: jehanrayan@yahoo.com

Date of Submission : 01.03.2017 Date of Acceptance : 10.03.2017 Date of Publication : 15.03.2017

Financial or other Competing Interests: None.

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Case Report DOI: 10.21276/AABS.2017.1279

Pigmented Dermatofibrosarcoma Protuberans (Bednar Tumor) Masquerading Clinically as Malignant Melanoma: A Case Report Ira Mondal*, Arnab Chaudhuri, Aparajita Samaddar, Nandini Das, Dipanwita Nag Department of Pathology, Medical College, Kolkata. India

ABSTRACT Bednar tumor is a rare neoplasm of intermediate malignant potential, which account for less than 5% of dermatofibrosarcoma protuberans (DFSP).This tumor is a pigmented variant of DFSP. The diagnosis is commonly made in early to mid adult life except in cases with melanin containing cells. The case report presents a 19 years old female who presented with a painless slow-growing 3.5Ă&#x2014;3Ă&#x2014;1.2 cm blackish mass on the dorsal aspect of her right foot. Histopathological examination of the biopsy specimen revealed typical features of a Bednar tumor, the diagnosis was confirmed through immunohistochemical study. Keywords: Dermatofibrosarcoma Protuberance (DFSP), Bednar Tumour,Immunohistochemistry

Introduction

Bednar tumor, a variant of dermatofibrosarcoma protuberance (DFSP), is a rare neoplasm of intermediate malignant potential described by Bednar in 1957. It accounts for less than 5% of all cases of dermatofibrosarcoma protuberans (DFSP).1,2 The lesion comprised of heavily melanin pigmented dendritic spindle cells arranged in a storiform pattern with elongated nuclei and a scant to moderate amount of cytoplasm.3 Mitotic activity was sparse. The lesion has an infiltrating margin and involved the sub-cutaneous tissue. Immunohistochemical stains in most cases show positivity for vimentin and CD34.4,5,6 We report a rare case of a Bednar tumor in a 19-year-old patient.

Case Report

A 19-year-old female presented with a slow growing painless soft tissue mass on the dorsum of the right foot which was present since childhood as single black spot. Gross examination revealed single irregular globular

partially skin covered tissue piece, measuring 3.5 cm in the greatest dimension. Light microscopic examination of hematoxylin-eosin stained sections, of biopsy specimen, showed spindle cell tumor with moderate cellular pleomorphism occupying the dermis and subcutaneous tissue. There is grenz zone between overlying epidermis and tumor mass. The tumor cells show intracytoplasmic brown pigments of melanin. The basal layer of epidermis does not show any juctional activity. However there is increase melanin pigment in the dermis and presence of melanophages in the papillary dermis. Fatty tissue and dermal appendages are entrapped within the tumor. (Fig.1A, B, C) Immunohistochemically, the spindle tumor cells were diffusely positive for vimentin and CD34 antigen. (Fig.2 A, B). The melanin containing pigmented cells were positive for S-100 protein (Fig. 2C) where as tumor cells show negativity for HMB-45 (Fig. 2D).Thus, the diagnosis of Bednar tumor (pigmented DFSP) was confirmed.

Fig. 1: A-Photomicrograph showing spindle cell tumor occupying dermis and subcutaneous tissue and entrapped adipose tissue (X40).B, C- Photomicrograph showing intracytoplasmic brown pigment within spindle tumor cells (X200). This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Fig. 2: A. Immunohistochemistry for CD34 show strong diffuse nuclear staining (x100). B. Tumor cell show diffuse positivity of vimentin in the cytoplasm of the non-pigmented spindle cells (x100). C. The pigmented dendritic cells show cytoplasmic and nuclear S-100 protein immunoreactivity. (x200) D. tumor cell show negativity for HMB-45. (x200).

Discussion

Initially designated â&#x20AC;&#x153;storiform neurofibromaâ&#x20AC;? by Bednar in 1957, this variant of dermatofibrosarcoma protuberans contains abundant melanotic pigment. It usually occurs after 20 years of age, but it is also found in the children, and called as congenital Bednar tumor.1,2 Some reports suggest a slight male predominance, others demonstrate an equal distribution between the sexes. It has been described in all ethnic groups; however it is generally more prevalent in blacks.7The lesions present as slow growth, over a period of months to years. The most frequent location is in the trunk, also involved upper and lower extremities, or the head and neck region.7 Bednar tumors differ from the typical cases of DFSP by the presence of heavily melanin pigmented dendritic cells, the histiogenesis of which continues to be debated. Several investigators have suggested that this tumor is derived from neuroectodermal cells according to the ultrastructural and immunohistochemical findings as well as the presence of melanosome containing cells.2 In immunohistochemical studies most of the tumor cells exhibit a positive reaction to CD 34 and vimentin, and are negative for HMB-45 and protein S-100. However, those cells containing melanin may react positively to protein S-100.6 However, to date it is uncertain whether Bednar tumors are simply colonized by the melanin bearing cells or the tumor is derived from putative neuromesenchyme.

The differential diagnoses include other benign or malignant cutaneous pigmented neoplasms such as pigmented (melanotic) neurofibroma, psammomatous melanotic schwannoma, and desmoplastic (neurotrophic) melanoma.8 A pigmented neurofibroma can be confused with a Bednar tumor because the melanin laden cells of both processes are similar. However, the Bednar tumor exhibits a more extensive storiform growth pattern, has greater immunoreactivity for CD34 and lacks diffuse proliferation of S-100 protein positive Schwann cells. Psammomatous melanotic schwannoma is rather circumscribed, heavily pigmented with psammomatous bodies and diffusely positive for S-100 protein, whereas the Bednar tumor is poorly circumscribed and composed of CD34 positive spindle shaped cells with scattered pigmented cells. Desmoplastic (neurotrophic) melanoma shows a neurotropism, focal melanocytic junctional activity, and diffuse and strong S-100 protein, HMB 45 immunoreactivity, CD34 negativity, whereas the Bednar tumor does not show any juctional activity, diffuse and strong CD 34 immunoreactivity. In addition to a careful histology examination, immunohistochemical study for CD34 is the most useful marker for differentiating a Bednar tumor from other cutaneous pigmented tumors.

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The typical DFSP has a rate of recurrence ranging from 20-50% among reports with a long-term follow up. The biologic behaviour of Bednar tumor is less aggressive than typical DFSP. However the rare case of metastatic Bednar tumor has been reported in literature.9 Mochzuchi cited a recurrence rate of 17% among reported cases of Bednar tumors. In addition, they reported an average interval of 9 years for recurrence (range, 9 months to 23 years).10 Although it is difficult to assess the biologic behavior of this tumor in pediatric patients because of the rarity of cases.

Fletcher C D M, Theaker J M, Flanagan A et al. Pigmented dermatofibrosarcoma protuberans (Bednar tumour): melanocytic colonization or neuroectodermal differentiation? A clinicopathological and immunohistochemical study. Histopathology1988;13:631-43

Aiba S, Tabata N, Ishi H et al. Dermatofibrosarcoma protuberans is a unique fibrohistiocytic tumor expressing CD34.Br J Dermatol 1992;127: 79-84

Brathwaite C, Suster S Dermatofibrosarcoma protuberans:a critical reappraisal of the role of immunohistochemical stains for diagnosis. Appl Immunohistochem 1994;2: 36-41

The recommended treatment for DFSP or Bednar tumors in the adult or pediatric patient is Mohsâ&#x20AC;&#x2122; micrographic surgery, wide excision with more than 2-3 cm margins of visibly uninvolved tissue and inclusion of the superficial fascia.9

Kagoura M, Toyoda M, Nagahori H, Makino T, Morohashi M. An ultrastructural and immunohistochemical study of pigmented dermatofibrosarcoma protuberans (Bednar tumor). Eur J Dermatol. 1999; 9: 366-9.

Vandeweyer E, Deraemaecker R, Somerhausen ND,Geledan L, Gebhart M. Bednar tumor of the foot: a case report. Foot Ankle Int. 2001; 22:339-41.

Reis-Filho JS, Milanezi F, Ferro J, Schmitt FC. Pediatric pigmented dermatofibrosarcoma protuberans (Bednar tumor): case report and review of the literature with emphasis on the differential diagnosis. Pathol Res Pract 2002; 198: 621-6.

Marcus JR, Few JW, Senger C, Reynolds M. Dermatofibrosarcoma protuberans and the Bednar tumor: treatment in the pediatric population. J Pediatr Surg 1998; 33: 1811-4.

Mohsâ&#x20AC;&#x2122; surgery is one of the many methods of obtaining complete margin control during removal of a skin cancer (CCPDMA- Complete circumferential peripheral and deep margin assessment using frozen section histology).

References 1.

Kaburagi Y, Hatta N, Kawara S, Takehara K. Pigmented dermatofibrosarcoma protuberans (Bednar tumor) occurring in a Japanese infant. Dermatology 1998; 197: 48-51.

Dupree WB, Langloss JM, Weiss SW. Pigmented dermatofibrosarcoma protuberans (Bednar tumor). A pathologic, ultrastructural, and immunohistochemical study. Am J Surg Pathol 1985; 9: 630-9.

10. Mochizuki Y, Narisawa Y, Kohda H. A case of Bednar tumor recurring after 23 years. J Dermatol 1996; 23: 614-8.

*Corresponding author: Ira Mondal, Post graduate trainee, Department of Pathology, Medical College, Kolkata. India Email: dr.iramondal@gmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 12.01.2016 Date of Acceptance : 12.01.2016 Date of Publication : 25.01.2017

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

Case Report DOI: 10.21276/AABS.2017.1314

Two Point Fixation in Zygomatic Complex Fractures Sourav Sharma1* and Vandana D2 1

Oral and maxillofacial surgeon. Superspeciality Dental and Maxillofacial centre. Jammu. J&K. India. 2 Superspeciality Dental and Maxillofacial centre. Jammu. J&K. India.

ABSTRACT Zygomatic bone along with its adjacent articulating bones is known as zygomatic complex.The zygomatic complex fractures are one of the frequently occurring maxillofacial injuries due to its prominence and facial contour. Personal altercations, Assaults, Road traffic accidents are the common causes leading to fracture of the zygomatic bone.The zygomatico-maxillary complex is one of the principle buttress of the face and helps in transmitting the occlusal forces to the skull base.Fracture of zygomatic bone may lead to aesthetic and functional deformity.There are various surgical techniques and approaches to reduce and fix the fractured zygomatic complex. Expertise of the Maxillofacial surgeon and thorough anatomical knowledge is of advantage in managing these fractures. Keywords: Mini Plates, Open Reduction and Internal Fixation, Surgical Approach, Two Pont Fixation.

Introduction Zygomatic complex fractures are common maxillofacial inuries.It is always challenging for maxillofacial surgeons who are specialized in treating this part of the human body to achieve the lost functions and regain proper facial contour. [1] Zygomatic bone due to its convex external surface provides prominence to the cheek region. Lateral prominence and articulation of zygoma with adjacent facial bones causes this bone to be frequently fractured.[2,3]Proximity of the zygomatic bone with the orbit and maxilla often leads to ocular or visual disturbances and disturbed occlusion. This necessitates surgical intervention in order to achieve satisfactory stability and best post operative results. Closed reduction has been found to be of no use in providing stability and fracture reduction in zygomatic complex fractures.[4] The fractured zygoma biomechanically has six possible directions of motion: rotation around X,Y and Z axis; translation across X,Y and Z axis. Wire fixation of zygomatic complex fracture has shown unsatisfactory results in terms of accurate reduction and fixation of small fractured fragments.[5,6]Mini plates have revolutionized

the standards of management of maxillofacial fractures by providing rigid internal fixation and ultimately better stability and less complications. Zygomatic bone is attached to adjacent bones and has four suture lines; which are the areas of anatomical weakness . Depending upon the velocity and impact of the trauma; one or many of these sutures are involved in the fracture. The topic of controversy here has been the amount of fixation which is required to precisely stabilize and fix the fractured zygomatic complex. We present a report of patient with zygomatic complex fracture who was treated by fixation of mini plates at infra orbital rim and frontozygomatic suture region. Case Report: A 40 Year old male reported to our centre with history of road traffic accident three days back. Patient had restricted mouth opening, depressed zygomatic bone and paresthesia of the infra orbital nerve. Radographic investigations revealed fractured zygomatic complex. Opthalmic opinion was taken .Treatment was explained to the patient and informed written consent was obtained prior to surgery (fig 1). Keenâ&#x20AC;&#x2122;s intraoral technique was used to elevate and reduce the displaced zygomatic complex.

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A small 1cm incision in the mucobuccal fold beneath the zygomaticomaxillary buttress was placed. Roweâ&#x20AC;&#x2122;s zygomatic elevator was inserted behind the infratemporal surface of the zygoma and bone was reduced into correct anatomical location. Head of the patient should be held firmly by the assisting surgeon during this maneuver.

AABS; 4(1): 2017

Local anaesthesia with adrenaline 1:80,000 was infiltrated and lateral eyebrow incision approximately 1.5cm in length was placed over fronto-zygomatic suture region (fig 2). Skin over the supra orbital rim region should be supported using two fingers. Periosteal elevator should be used while drilling to protect the eyeball from any injury.

Infraorbital incision was then placed and fracture site was exposed and reduction was achieved. First hole should always be drilled in the unstable segment of the fracture. Finally, all the screws were tightened (fig 3).Wound was closed in layers using 3-0 vicryl and 5-0 ethilon sutures. Post operative medications were advised for atleast a week. Suture removal was done on 8th postoperative day. Healing was uneventful .Post operative PNS view was taken after surgery (fig 4). Patient was on periodic follow up and Infra orbital paresthesia was resolved. Patient had an almost imperceptible scar.

Fig.1: Pns View Showing Left Zmc Fracture.

Fig. 2: Mini Plate Fixation At Fronto Zygomatic Suture

Fig. 3: Mini Plate At Infra Orbital Rim.

Fig. 4: Post Op Pns View Showing Two Point Fixation and Stabilization.

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Discussion Facial asymmetry due to facial trauma may lead to mental agony and social discomfort. Progressive advent and gradual increase in number of high speed vehicles has led to increased cases of facial trauma. The integrity of the zygomatic complex is critical in maintaining normal facial width and cheek prominence. Zygomatic bone alone rarely gets fractured; its articulating surfaces: maxilla, temporal, frontal and sphenoid are also involved. It serves as the buttress between the face and the skull which absorbs the distortion forces and gets fractured at the sutures; thereby reducing the impact on the brain. Peter B Grey et al[7] advocated ophthalmic opinion is mandatory in most of the zygomatic complex fractures; as delayed retrobulbar haemorrhage may lead to blindness. Infra orbital and zygomatico frontal suture approach was used to treat the displaced zygomatic complex fractures.[8]Six mm screws can be safely placed above the frontozygomatic suture. Infra orbital rim incision provided adequate exposure of the fracture site; compressed infra orbital nerve was released from the fractured fragments. Fractured bony segments were accurately aligned and fixed using mini plate. Studies have also shown higher incidence of recovery of infra orbital nerve paresthesia using mini plate osteosynthesis.

Conclusion

We conclude that two point fixation in Zygomatic complex fractures yields satisfactory post operative stability and aesthetics in addition to resolution of infra orbital paresthesia. Severely displaced zygomatic complex fractures may require additional points of fixation.

Reference 1.

Zingg M, Laedrach K,Chen J et al.Classification and treatment of zygomatic fractures:A review of 1025 cases.J Oral Maxillofac Surg 1992; 50:778-790.

Covington D.S , D.J Wainwright, J.F Teichgraeber and D.H Parks.Changing patterns in the epidemiology and treatment of zygoma fractures:10 year review. J Trauma 1994;37(2):243-248.

Poswillo D.Reduction of the fractured malar by a traction hook. British Journal of Oral Surgery1976;14:76-79.

Kovacs A.F, Ghahremani M. Minimization of zygomatic complex fracture treatment. J Oral Maxillofac Surg 2001;30(5):380-383.

Lund K .Fractures of the zygoma:a follow up study on 62 patients. J Oral Surg 1971;29:557-560.

Pozatek ZW, Kaban LB, Guralnick WC.Fractures of the zygomatic complex:an evaluation of surgical management with special emphasis on the eyebrow approach. J Oral Surg 1973;31:141-148.

Gray B.P,Leen MM and Loftus M.J.Late retrobulbar hemorrhage and blindness following malar fracture complicated by factor XI deficiency.Journal of Oral and Maxillofacial Surgery 1993;51:699-702.

8. Olate,Monteiro L,Renato S,Roger W F,Marcio M.Surgical Approaches and Fixation Patterns in Zygomatic Complex Fractures. Journal of Craniofacial Surgery2010; 21(4): 1213-1217.

*Corresponding author: SOURAV SHARMA, 102/A. Main Road Roop Nagar. Near Janipur Police Station. Jammu. J&K.180013. India Email: dr.souravsurgeon@gmail.com Date of Submission : 05.02.2017 Date of Acceptance : 13.02.2017 Date of Publication : 21.02.2017

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Case Report DOI: 10.21276/AABS.2017.1320

Osteolipoma of Knee: A Rare Case Report Arnab Chaudhuri*, Ira Mondal, Aparajita Samaddar, Nandini Das and Dipanwita Nag Department of Pathology, Medical College, Kolkata. India

ABSTRACT Lipomas are the most common benign soft tissue tumors and appear in any part of the body. They typically consist of mature adipose tissue. Osteolipoma is an extremely rare histologic variant of lipoma that contains mature lamellar bone within the tumor and osteolipoma independent of bone tissue are very rare. We report a case of histologically confirmed osteolipoma independent of bone located just above left knee. A 61 year old female presented with medial sided left knee pain of 6 month duration. X-ray demonstrated a partially calcified 3.5 cm diameter mass located in anterior to the distal medial femur. Histologic examination of an excisional biopsy showed the lesion to be an osteolipoma. Osteolipoma is a rare histologic variant of lipoma with osseous metaplasia, but should be considered in the differential of a fat-containing neoplasm with ossification. Keywords: Osteolipoma; Ossifying lipoma; Knee joint.

Introduction

Lipoma is a common benign soft tissue neoplasm that sometimes may have mixed tissue components. Lipomas with mixed components are named according to the type of tissue. Ossification of a lipoma was first described in 1959, and it is rarely reported [1, 2]. Several names have been used to describe ossification of lipomas, including secondary calcification, ossified lipoma, ossifying lipoma, or osteolipoma, and some authors have used these terms interchangeably. As with classic lipomas, lipomas with ossifications may be found in any part of the body, but are usually found in the head, neck region. Only 6 cases of osteolipoma arising in connection with knee joint have been described [3]. Here, we present a case of an osteolipoma in the left knee region.

Case Report

A 61-year-old female presented with a proximal left knee mass. The patient reported pain along the medial aspect of the knee for the past 6 month, with recent notice of a palpable mass. The pain was described as a constant, moderate throbbing, exacerbated by activity. Conservative treatments, including physical therapy and various other pain medications had failed to provide symptomatic relief. Radiographs revealed a calcified mass anterior to the distal left medial femur in the region of the suprapatellar fossa. The mass abutted the anteromedial femur and medial patellar facet without evidence of osseous involvement. (Fig.1A). Provided initial differential included parosteal osteosarcoma, chondrosarcoma, and myositis ossificans and excision biopsy was considered for management. Following excision biopsy, the mass was sent to our department for histopathological examination. Grossly, the

resected specimen demonstrated a 3.5 cm diameter mass. The mass was tan-brown, firm, ovoid, and surrounded by fibro fatty soft tissue. (Fig.1B) Histologic examination revealed mature adipose tissue in which a large fragment of cortical-type bone was embedded. No atypia was seen either in adipose tissue or bony component (Fig.1C,D). The pattern was consistent with an osteolipoma.

Discussion

Lipomas are the most common benign soft-tissue tumors composed of only mature adipose cells without cellular atypia [4]. However, other mesenchymal elements such as smooth muscle, fibrous, chondral or osseous tissue may occasionally be found in addition to adipocytes. Variants of lipoma have been named according to the type of tissue present such as fibrolipoma, myelolipoma, leiomyolipoma, chondrolipoma, osteolipoma and angiolipoma [4]. A lipoma containing mature osseous elements is called osteolipoma. The terms ossifying lipoma, osseous lipoma and lipoma with osseous metaplasia have been used interchangeably with osteolipoma [5]. Osteolipoma is the rarest subtype of lipoma, with the first case being reported in 1959 [1]. An osteolipoma is defined as a lesion with mature adipose tissue and randomly distributed trabeculae of laminated bone [5]. They have been found at various sites, with the highest frequency in the head and neck regions [6, 7, 8]. Osteolipoma is very rare in distal femur and knee region and only 6 other such cases were reported previously. The age of these 6 patients ranged from 21 to 64 years (mean of 41.2 years), involving 4 men and 2 women. Symptoms were described in all 6 cases, with 4 reporting joint pain ranging from 3-36 months in duration, exacerbated by activity, and causing difficulty while performing simple tasks such as walking [3, 9]. Two of the 4 patients reported

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Fig. 1A: Frontal radiograph of left knee demonstrate an area of ossification (arrow) anteromedial to the medial femoral condyle. No underlying osseous involvement is identified. 1B. Grossly, the tumor consists largely of fat and calcified area. 1C. H&E, x10, 1D. H&E, x20, both photomicrographs show mature trabecular bone within mature adipose tissue. No atypia was seen in both adipose and bony component.

joint pain at restÂ [3, 10]. Our case was a 61 year old female with similar symptomatology. The radiographic differential diagnosis for an ossified mass is dependent on many factors, including age and location. Considering the intra/juxta articular location of tumor, a broad radiographic differential diagnosis of a calcified and/or ossified mass including benign entities, such as a hemangioma, synovial chondromatosis, calcified synovitis, myositis ossificans, or a loose body or even, malignancies such as conventional or surface-based osteosarcoma, or soft tissue sarcomas such as a synovial sarcoma, liposarcoma may also enter the differentialÂ [11].Â So considering the wide range of differential, a wide local excision biopsy becomes the preferred treatment. But Definitive diagnosis of osteolipoma can easily be done with histopathologic examination. A histopathologic appearance of diffuse, mature ossification within fatty tissue clinches the diagnosis. The adipose component is usually predominant and the mature bone tissue is irregular in distribution. Bone spicules may be surrounded by fibrous tissue bands [10, 12].

Histologically confirmed osteolipomas are benign neoplasms, as with classic lipomas, and do not recur, so have excellent prognosis.

Conclusion

In conclusion, osteolipomas are a rare occurrence. When arising in a juxta and/or intra-articular location, they result in a broad differential diagnosis radiologically. Because of the absence of specific radiologic findings, the differential diagnosis for lesions with fatty and osseous components should include not only malignant entities such as liposarcoma but also heterologous differentiation of rather benign lipomas such as an osteolipoma, especially in the setting of internal mature bony formation. Osteolipoma has a same prognosis as simple lipoma and surgical excision is the recommended treatment. No recurrences have been reported. So to summarize, although osteolipomas are very rare, it is important to keep them in mind when a lesion with adipose tissue in combination with ossification is encountered.

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Reference 1.

Plaut GS, Salm R, Truscott DE. Three cases of ossifying lipoma. J Pathol Bacteriol. 1959,78:292-5

Adebiyi KE, Ugboko VI, Maaji SM, Ndubuizu G. Osteolipoma of the palate: report of a case and review of the literature. Niger J Clin Pract. 2011,14:242-4.

Huynh TPV, Cipriano CA, Hagemann IS, Friedman MV. Osteolipoma of the knee. Radiology Case Reports. in press.

Murphey MD, Carroll JF, Flemming DJ, Pope TL, Gannon FH, Kransdorf MJ: From the archives of the AFIP: Benign musculoskeletal lipomatous lesions. Radiographics. 2004,24:1433-66

Obermann EC, Bele S, Brawanski A, Knuechel R, Hofstaedter F: Ossifying lipoma. Virchows Arch.1999,434:181-3.

Durmaz A, Tosun F, Kurt B, Gerek M, Birkent H. Osteolipoma of the nasopharynx. J Craniofac Surg. 2007,18(5):1176-9.

Kameyama K, Akasaka Y, Miyazaki H, Hata J. Ossifying lipoma independent of bone tissue. ORL J Otorhinolaryngol Relat Spec. 2000,62(3):170-2.

Amaral MB, Borges CF, de Freitas JB, Capistrano HM, Mesquita RA. Osteolipoma of the oral cavity: a case report. J Maxillofac Oral Surg. 2015,14(Suppl.1):195-9.

Hashmi AA, Malik B, Edhi MM, Faridi N, Ashraful M. A large parosteal ossifying lipoma of lower limb encircling the femur. Int Arch Med. 2014,7(1):5

10. Pudlowski RM, Gilula LA, Kyriakos M. Intraarticular lipoma with osseous metaplasia: radiographic-pathologic correlation. Am J Roentgenol. 1979.132(3):471-3 11. Friedman MV, Kyriakos M, Matava MJ, McDonald DJ, Jennings JW, Wessell DE. Intra-articular synovial sarcoma. Skeletal Radiol. 2013,42(6):859-67. 12. Val-Bernal JF, Val D, Garijo MF, Vega A, Gonzalez-Vela MC. Subcutaneous ossifying lipoma: Case report and review of the literature. J Cutan Pathol. 2007, 34: 788-92.

*Corresponding author: Arnab Chaudhuri, 175, Laxmi Narayan Colony, PO. Nabapally, Barasat, Dist. 24 Parganas (North) Pin: 700126. Phone: +91 9051663572 Email: dr.arnab1234@gmail.com

Financial or other Competing Interests: None.

Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

Date of Submission : 06.02.2017 Date of Acceptance : 13.02.2017 Date of Publication : 21.02.2017

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

Case Report DOI: 10.21276/AABS.2017.1332

Schistosomiasis of Appendix with Review of Literature Omaia Mahdy1, Nazima Haider2* and Sohaila Fatima2 Department of Histopathology, Aseer central Hospital, Abha KSA 2 Department of Pathology, King Khalid University, Abha KSA

ABSTRACT Schistosomiasis a widespread parasitic disease is caused by a typical trematode with vertebrate-invertebrate lifecycle and occurs in welldefined geographical areas. The development of water resources and migration of populations are responsible for the introduction of the disease into new regions of the world. Schistosomiasis is an unusual cause of appendicitis. We present a case of appendicitis in a 60 year old Saudi male and discuss its review of literature. Keywords: Schistosoma, Appendix

Introduction

Schistosomiasis is one of the most widespread yet neglected parasitic diseases in the world. They have a typical trematode vertebrate-invertebrate lifecycle, with humans being the definitive host. Environmental changes that result from the development of water resources and migration of populations are responsible for the introduction of the disease into new geographic areas. Schistosomiasis of the appendix is a well-recognized disease but does not frequently cause appendicitis. Thus the condition is uncommon even in endemic regions. We hereby present a case of appendicitis in a 60 year old Saudi male to highlight this unusual cause of appendicitis and discuss its review of literature.

Case report

A 60 year old Saudi male presented with a history of on and off right iliac fossa pain for 2 years. He was seen by

Fig. 1: Granulomatous inflammation (arrow) in appendix. Hematoxylin and eosin (10X).

clinical practitioner who did an ultrasound which was unremarkable and he was treated with analgesics, antacids and anti spasmodic but there was no improvement. Besides the right iliac fossa pain, there were no other bowel or bladder complaints. On examination, the abdomen was soft but there was slight tenderness in the right lower quadrant on deep palpation. The rest of the abdominal examination was normal. The repeat ultrasound examination was also unremarkable. The full blood count was within normal range. A diagnosis of recurrent acute appendicitis was made and appendicectomy was performed. The appendix was slightly thickened and congested grossly. Histopathology of the appendix showed schistosoma eggs with lateral spine and granulomatous inflammation (Fig 1 & 2) without any evidence of acute inflammation. Serum anti-bilharzias antibodies were positive but no schistosomal ova were found in urine and stool. He was given a single dose of Praziquantel 40Â mg/kg.

Fig. 2: Schistosoma eggs (arrow) with lateral spine. Hematoxylin and eosin (40X), Inset: Hematoxylin and eosin (40X), PAS (20X)

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Discussion

Schistosomiasis is a water-borne trematode infestation, affecting approximately 240 million people with 800 million are at risk. It occurs in well-defined geographical areas in tropics and subtropics particularly Africa, China and Middle East. [1] Schistosomiasis was first described by Theodor Bilharz, hence also called bilharziasis , in 1851 but it was in 1908 when Pirajá da Silva first described the entire disease cycle. Cercariae, which escape from the snail (intermediate host) to the freshwater actively swim toward a human (definitive host) where they penetrate the skin directly, sheds tail, transform into a blood vessel inhabiting form termed the schistosomulum. These circulate with the blood as they develop to sexually mature blood flukes until they inhabit the portal hepatic tract (S. mansoni and S. japonicum) or the pelvic organs (S. haematobium). Eggs are released into the bloodstream and many embolize in smaller blood vessels and capillaries of diverse organs, inducing the characteristic granulomatous reaction of schistosomiasis. Passage of the eggs through the wall of bowel or bladder to the lumen from where they exit to the external environment in faeces or urine. [2] Despite major advances in control and substantial decreases in morbidity and mortality, schistosomiasis continues to spread to new geographic areas. [2] Two species S. mansoni and S. haematobium, are endemic in Saudi Arabia. Infection with S. mansoni was reported mainly in the highlands of the western areas and some parts of the Central and Northern Regions, whereas S. haematobium was reported mainly in lowland coastal plain in the southern areas. Biomphalaria pfeifferi and Bulinus beccarii are the species of fresh water snails incriminated in transmission of S. Mansoni and S. hematobium in Saudi Arabia, respectively. The hamadryas baboon (Papio hamadryas) is a species of baboon from the Old World monkey family. In Saudi Arabia, they inhabit an extensive range from the Yemen border in south to Akhal near Madinah in the northern Saudi Arabia. They use the same water sources in their habitat where human schistosomes have been recorded and proved to be natural reservoir hosts of many zoonotic diseases including S. mansoni infection. [3] Schistosomiasis of the appendix was first described by Turner in 1909. [4] The incidence of schistosomal appendicitis is 0.02-6% in endemic areas but its very rare in developed countries. [5, 6] Studies where schistosomiasis is endemic, have shown appendicitis to be relatively uncommon and the cause is Annals of Applied Bio-Sciences, Vol. 4; Issue 1: 2017

AABS; 4(1): 2017 not usually attributable to a schistosomal infestation of the appendix. The most common hypothesis is that eggs in the appendix wall stimulate inflammation and thus fibrosis and narrowing of the appendiceal lumen. Other hypotheses include schistosomal egg emboli causing ischaemia and granulomatous inflammation of the serosa and periappendicular tissue causing fibrosis. [6, 7] The patients may present with abdominal pain, vomiting and fever or altered bowel motion. All the three species deposit eggs in the appendix. Gross examinations may show acute appendicitis with fibrinopurulent exudates, serosal congestion and thickened wall, fecaliths or it may be unremarkable. Confirmation of appendicular schistosomiasis is a purely histological diagnosis which shows the eggs, granulomatous inflammation with tissue eosinophilia and infiltrates of acute and chronic inflammatory cells. Morphologically, S. haematobium ova have a terminal spine while S. mansoni have lateral spine.[5,6,7] Some eggs are surrounded by a layer of eosinophilic material, the Splendore–Hoeppli phenomenon. [8] Physicians must be aware of the possibility of seeing atypical presentations of parasitic diseases, especially considering the worldwide increase in immigration and tourism. Migrants, tourists and expatriates with chronic abdominal symptoms should also be asked about possible exposure. If an unexplained eosinophilia is detected, the respective parasitic and serologic tests should be performed. The detection of circulating antigens i.e. circulating cathodic antigen (CCA) and circulating anodic antigen (CAA) and schistosome DNA and RNA is proving to be a highly sensitive diagnostic approach. [9] Antihelmintic Praziquantel which acts on adult worms is in most common drug treating both urinary and gastrointestinal forms of the disease, and administered as a single dose. However, it’s not effective against the immature, early schistosome stages. Artemisinin derivatives, such as artemether, is one such option which has the potential to kill the immature stages. [10, 11]

Conclusion

Schistosomal appendicitis is an unusual presentation and histopathological examination is essential for its diagnosis. Such atypical presentations of parasitic diseases become more significant considering the worldwide increase in immigration and tourism.

References 1.

WHO. 2014. Schistosomiasis fact sheet 115. World Health Organization, Geneva, Switzerland.

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Case Report

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Khan GM, Grillo IA, Abu-Eshy SA, Khan AR, Mubarak J, Jastaniah S. Pathology of appendix. J Natl Med Assoc. 2001;92: 533-5.

Lofty W M, Alsaqabi S M. Human schistosomiasis in the kingdom of Saudi Arabia: a review. Journal of the Medical Research Institute. 2010; 31: (1-6).

Edington GM, Gilles HM, editors. Pathology in the Tropics. London: Edward Arnold Publishers Ltd; 1976. pp. 149–79.

Turner SA. Bilharziasis of the appendix. Trans Mrd J. 1909; 5:210.

Badmos KB, Komolafe AO, Rotimi O. Schistosomiasis presenting as acute appendicitis. East Afr Med J. 2006. Oct;83(10):528–32.

Weerakoon K G A D, Gobert G N, Cai P, McManus D P. Advances in the Diagnosis of Human Schistosomiasis. Clin. Microbiol. Rev. 2015 ; 28(4): 29 939-967. doi:10.1128/ CMR.00137-14

Ahmed S A, Mohammed U, Sanda R B, Makama J, Shehu M S, Ameh E A, Mayun A A. Schistosomiasis of the Appendix in a Tertiary Hospital in Northern Nigeria: A 22-Year Review. Lab Physicians. 2014;6(1): 18–21. doi: 10.4103/0974-2727.129085.

10. Bierman W F W, Wetsteyn J C F M, van Gool T. Presentation and diagnosis of imported schistosomiasis: relevance of eosinophilia, microscopy for ova, and serology. Journal of Travel Medicine. 2005; 12, (1): 9–13. 11. Liu Y, Wu W, Liang Y, Jie Z, Wang H, Wang W, Huang Y. New uses for old drugs: the tale of artemisinin derivatives in the elimination of schistosomiasis japonica in China. Molecules.2014. 19:15058–15074.

*Corresponding author: Nazima Haider, Department of Pathology, King Khalid University, Abha KSA Phone: +91 966-559103778 Email: nazima_haider@yahoo.com

Financial or other Competing Interests: None.

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Date of Submission : 08.02.2017 Date of Acceptance : 18.02.2017 Date of Publication : 24.02.2017

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