AABS 4.4 (2017) by PaGe

eISSN: 2349-6991 pISSN: 2455-0396

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

Oct-Dec. 2017; Vol. 4, Issue 4

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

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

Annals of Applied Bio-Sciences Disclaimer

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

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

Publication Frequency

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

•

Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work’s authorship and initial publication in this journal.

3. 4.

Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal’s published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.

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

Thoughts, Language and examples in published research papers are entirely of Authors of Research Papers. It is not necessary that both Editor and Editorial Board are satisfied by the research paper. The responsibility of the matter of the research paper/ article is entirely of author. Scientific knowledge is ever changing. As new research and experiences broaden our knowledge, old concepts may be required to be looked into afresh. The Authors and editors of the material herein have consulted sources believed to be reliable in their efforts to provide information that is complete and in accord with the standards accepted at the time of publication. However, in view of the possibility of human error by the authors, editors, or publisher, nor any other who has been involved in the preparation of the work warrants that the information contained herein in every aspect accurate or complete, and they are not responsible for any errors or omissions or the results obtained from the use of such information. Readers are advised to confirm the information contained herein with other sources. In any condition and due to any reason, if any Educational/Research Institution (College or University) denies accepting the research paper published in AABS, it will not be the responsibility of the Editor, Publisher or the Management. Only the First author is entitled to receive the print copies of all co-authors.

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

Table of Contents Review Articles Efficacy of cyanoacrylate and black braided silk for the closure of incision after surgical removal of impacted third molars: A Systematic Review Divya James, Uma Maheswari G

R38-R46

Original Article Assessment Of Peripheral Blood Smear Preparation Technique In Laboratories A150-A156 With High Sample Load Sumanashree Mallappa, Sachin Kolte, Nimisha Sharma, Indrani Dhawan

Case Reports

Syphilis in Blood Donors in Tertiary Care Teaching Hospital Ravi Jain, Ashok Yadav

A157-A162

Hematological profile of pregnant and non-pregnant females: A comparative study in a tertiary care hospital in Haryana, India Sujata Raychaudhuri, Shveta Lukhmana, Deepsikha Rana, Mukta Pujani, Nimisha Sharma, Rashmi Ahuja

A163-A170

Laboratory parameters in clinically suspected dengue cases in tertiary care teaching hospital in North Maharashtra Madhuri Magan Suryawanshi, Shubhangi Chandrashekhar Dange, M N Dravid, Sunil P Lilani, Pooja Shah

A171-A175

An integrated approach to detection of Mycobacterium tuberculosis using BD MGIT 320 system and other diagnostic modalities. Gargi Choudhury, Partha Pratim Das, Lahari Saikia

A176-A181

Transfusion Transmitted Diseases Among Blood Donors In Tertiary Care Teaching Hospital Of Central India Ravi Jain, Ashok Yadav

A182-A187

Histomorphology Of Upper GI Endoscpic Biopsies: A Study In Urban Care Centre Puvitha R Duraisamy, Lalitha chithambram., Shifa Seyed Ibrahim, Kavitha Madasamy, Lavanya Krishnagiri Balan, Pavithra Thandavarayan

A188-A194

Tender Coconut (Cocos nucifera L.) husk anaerobic leachate as a potent antifungal and antibacterial agent Praveen Krishnakumar, Treesamol Antony

A195-A200

Paramedian forehead flap in post Radiotherapy fistula Manish Munjal, Japneet Kaur, Venus Tilavat, Amanjot Kaur, Shubham Munjal

Letter to Editor Metanephric Adenoma

Aneeta Singh Malhotra, Urvashi Andotra, Harminder K Rai, Arvind Khajuria, Kuldeep Chander Goswami

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India)

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

E-mail: contactus.aabs@pacificejournals.com

III

C34-C37 L1-L2

Review Article DOI: 10.21276/AABS.2017.1685

Efficacy of Cyanoacrylate and Black Braided Silk for The Closure of Incision After Surgical Removal of Impacted Third Molars: A Systematic Review Divya James* and Uma Maheswari G. Department of Oral and Maxillofacial Surgery, Saveetha Dental College and Hospital, Chennai, India

ABSTRACT Introduction: Wound closure is a part of any surgical procedure and the objective of laceration repair or incision closure is to approximate the edges of a wound so that natural healing process takes place. Over the years new biomaterials have been used as an alternate to conventional suture materials. Cyanoacrylate bio adhesives are one among them. They carry the advantage of rapid application, patient comfort, resistance to infection, hemostatic properties, and no suture removal anxiety. Hence this study was undertaken to study the effect of long chain cyanoacrylate as an adhesive for intraoral wound closure and also to explore its hemostatic and antibacterial effects.The aim of this study is to compare the efficacy of cyanoacrylate and black braided silk for the closure of surgical incision after removal of impacted third molars. Search Strategy: Databases searched: PubMed CENTRAL, Google Scholar, Cochrane, Bibliographies of Clinical Studies and Reviews. Hand search done from 1988 till October 2016. Selection Criteria: Clinical trials evaluating the severity of pain and hemostasis in the closure of incisions after surgically removed third molars. Results: The reviews found some clinical evidence that, there is significant difference between sutures and cyanoacrylate on postoperative pain following mandibular third molar surgery. There is also significant difference between sutures and cyanoacrylate on haemostasis following mandibular third molar surgery. Conclusion: The clinical evidence in this review is adequate to state that, there is a difference in postoperative pain and haemostasis following mandibular third molar surgery between black braided silk and cyanoacrylate. Keywords: Cyanoacrylate, Black Braided Silk, Impacted Third Molars, Pain, Haemostasis

Introduction

The removal of mandibular third molar is a common procedure in a dental clinic. After removal of an impacted third molar the conventional method of suturing the surgical wound is by using black braided silk and leaving the wound to heal by primary intention. Suturing in the most posterior area of the oral cavity is not easy, it takes time and good suturing skills. Besides the difficulty faced during operative procedure, the patient has to come for a second visit after seven days for suture removal. Moreover, incompatibility with the tissues may cause foreign body reactions and fistula formation. Over – tight sutures may lead to necrosis and ischemia. Black braided silk has an effect known as ‘wicking’ which makes the operated site to retain bacteria which is followed by secondary infection, because the knots become a favorable place for bacterial colonies. To overcome these difficulties more effective method of surgical wound closure has been found with better efficiency and fewer complications.

Tissue adhesives as adopted the idea of suture less wound closure. Plastic adhesives were discovered in 1949 and 10 years later COOVER et al. reported their use in surgical procedures. Cyanoacrylate glue is the general term used for quick super bonding glues used as two separate liquids, one for pouring into the mould and other used as a hardener.

Aim

Aim of this systematic review is to compare the efficacy of cyanoacrylate and black braided silk for the closure of incision after surgical removal of impacted third molars.

Structured Question

Does cyanoacrylate has better efficacy than black braided silk in the closure of incision after surgical removal of impacted third molars?

Pico Analysis

Populations: Patients who are undergoing third molar surgery

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

R-39

AABS; 4(4): 2017

Intervention: Cyanoacrylate

Results

Comparison: Black braided silk Outcome: Severity of pain and hemostasis Null Hypothesis: There is no difference in cyanoacrylate and black braided silk. Alternative Hypothesis: There is cyanoacrylate and black braided silk.

difference

Materials and Methods

Sources used for identification of studies included or considered for this review, detailed search strategies were carried out on the following databases. –

PubMed

–

PubMed Advanced search

–

MEDLINE

Language: There was no language restriction during the electronic search. Hand Searching: The following journals were hand searched: International Journal of Oral and Maxillofacial Surgery, British Journal of Oral and Maxillofacial Surgery, Journal of Oral and Maxillofacial Surgery, Oral Surg Oral Med Oral Pathol Oral Radiol Endod Inclusion Criteria: Criteria for considering studies for this review. Types of Studies: Randomized controlled trial or clinical trials evaluating and comparing the effectiveness of cyanoacrylate and black braided silk for the closure of incision after surgical removal of impacted third molars. Types of Participants: All participants undergoing third molar surgery.

Type of Intervention Cyanoacrylate

Types of Outcome Measures

To evaluate severity of pain and hemostasis of cyanoacrylate and black braided silk for the closure of incision after surgical removal of impacted third molars.

Exclusion Criteria –

The following studies were excluded,

–

Case reports / case series

–

Animal studies

–

In vitro studies

–

Studies involving primary teeth

–

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

Description Of Studies: The search identified 1 article. Full articles were obtained. 3 hand searched article fulfilled the inclusion criteria. Therefore, a total of 4 articles fulfilled all criteria for inclusion.

Quality Assessment

The quality assessment of included trials was undertaken independently as a part of data extraction process. Four main quality criteria were examined. 1. 2. 3. 4.

Method of Randomization , recorded as a) Yes – adequate as described in the text b) No – inadequate as described in the text c) Unclear in the text Allocation Concealment , recorded as a) Yes – adequate as described in the text b) No – inadequate as described in the text c) Unclear in the text Outcome assessor blinded to intervention, recorded as a) Yes – adequate as described in the text b) No – inadequate as described in the text c) Unclear in the text Completeness of follow-up (was there a clear explanation for withdrawals and dropouts in each treatment group) assessed as : a) Yes – dropouts were explained b) No – dropouts were not explained c) None – no dropouts or withdrawals

Other methodological criteria examined included: 1. 2. 3. 4.

Presence or absence of sample size calculation Comparability of groups at the start Clear inclusion / exclusion criteria Presence/absence of estimate of measurement error. The validity and reproducibility of the method of assessment.

Risk of Bias In Included Studies: The assessments for the four main methodological quality items are shown in table. The study was assessed by to have a “high risk” of bias if it did not record a “yes” in 3 or more of the 4 main categories , “moderate” if 2 out of 4 categories did not record a “yes” and “low” if randomization , assessor blinding and completeness of follow up were considered adequate.

Discussion

Interpretation of Results: According to Sneha Sethiya et al.1 , the severity of pain was significantly (p < 0.05) less in e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article

R-40

TABLE 1: VARIABLES OF INTEREST Sr. No.

Variables of interest

Severity of pain

2. TABLE 2: RESULTS AND SUMMATION Sr. Author No.

Hemostasis

Year

Country Study design Sample Age size

Set - up

Technique used

Method of evaluation

2014

India

Prospective controlled clinical study

18 – 35 University Silk sutures Pain and bleeding years and were measured cyanoacrylate using visual analogue scale.

Sneha Setiya, Rajshekhar Halli, Anand Shah, Gaurav Chabbaria, Tarun Singh

Mehdi Ghoreishian, 2009 Rasoul Gheisari, Maasoumeh Fayazi

Iran

Controlled clinical trial

19 – 24 University Silk sutures Pain and bleeding years and were measured cyanoacrylate using visual analogue scale.

M. Gogulanathan, 2015 P. Elavenil, A. Gnanam, V. B. Krishnakumar Raja

India

Prospective, randomized controlled clinical trial

Ajit D. Joshi, Harish Saluja, Uma Mahindra, Rajshekhar Halli

India

Randomized controlled clinical trial

20 – 32 University Silk sutures Pain and bleeding years and were measured cyanoacrylate using visual analogue scale.

Table 3: RESULTS Sr. Author and No. Year 1. Sneha Setiya, 2014

Mehdi Ghoreishian, 2009

2011

Materials used Silk sutures and cyanoacrylate

Silk sutures and cyanoacrylate

Method of evaluation Pain and bleeding were measured using visual analogue scale.

College

Mean Values

Pain Group 1 (Sutures) Day 1 – 2.42 Day 2 – 1.98 Day 7 – 0.26 Group 2 (Cyanoacrylate) Day 1 - 2.02 Day 2 – 1.2 Day 7 – 0 Hemostasis Group 1 (Sutures) Day 1 - 1 Day 2 – 0 Day 7 – 0 Group 2 (Cyanoacrylate) Day 1 – 0.2 Day 2 – 0 Day 7 – 0 Pain and Pain bleeding were Group 1 (Sutures) measured Day 1 – 4.38 using visual Day 2 – 3.63 analogue scale. Day 3 – 2.47 Day 4 – 0.71 Day 5 – 0.59

Silk sutures Pain and bleeding and were measured cyanoacrylate using numerical rating scale.

Outcomes Day 1 - There was significant difference in pain and hemostasis between the two groups Day 2 - There was significant difference in pain but there was no significant difference in hemostasis between the two groups. Day 7 - There was significant difference in pain but there was no significant difference in hemostasis between the two groups.

Day 1 - There was significant difference in pain and hemostasis between the two groups. Day 2 - There was significant difference in pain and hemostasis between the two groups.

Table Cont.

http://www.pacificejournals.com/aabs

R-41

AABS; 4(4): 2017

Cont. Table Sr. Author and No. Year

Materials used

M. Gogulanathan, 2015

Silk sutures and cyanoacrylate

Ajit D. Joshi, 2011

Silk sutures and cyanoacrylate

Method of evaluation

Mean Values

Outcomes

Group 2 (Cyanoacrylate) Day 1 – 4.19 Day 2 – 3.09 Day 3 – 2.47 Day 4 – 1.34 Day 5 – 1.31 Hemostasis Group 1 (Sutures) Day 1 - 3 Day 2 – 1.50 Day 3 – 0 Group 2 (Cyanoacrylate) Day 1 - 2 Day 2 – 1 Day 3 – 0 Pain and Pain bleeding were Group 1 (Sutures) measured 3.5 ± 1.6 using numerical Group 2 (Cyanoacrylate) rating scale. 2.0 ± 1.5 Hemostasis Group 1 (Sutures) 251.9 ± 67.9 Group 2 (Cyanoacrylate) 1.2 ± 0.4 Pain and Pain bleeding were Group 1 (Sutures) measured Day 1 – 1.43 using visual Day 2 – 1.37 analogue scale. Day 3 – 1.32 Day 4 – 1.2 Day 5 – 1.2 Group 2 (Cyanoacrylate) Day 1 – 1.3 Day 2 – 1.2 Day 3 – 1.3 Day 4 – 1.2 Day 5 – 1.2 Hemostasis Group 1 (Sutures) Day 1 – 0.17 Day 2 – 0 Day 3 – 0 Group 2 (Cyanoacrylate) Day 1 – 0 Day 2 – 0 Day 3 – 0

Day 3 - There was no significant difference in pain and hemostasis between the two groups. Day 4 - There was significant difference in pain between the two groups. Day 5 - There was significant difference in pain between the two groups.

There was significant difference in pain and hemostasis between the two groups.

Day 1 - There was significant difference in pain and hemostasis between the two groups. Day 2 - There was significant difference in pain but there was no significant difference in hemostasis between the two groups. Day 3 - There was significant difference in pain but there was no significant difference in hemostasis between the two groups. Day 4 - There was no significant difference in pain between the two groups. Day 5 - There was no significant difference in pain between the two groups.

Table 5: Summation table for individual parameters Sr. No. Author

Year

Evaluation period Outcome

Sneha Setiya et al.

2014

1st, 2nd and 7th day

The severity of pain and the bleeding was less in study group as compared to control group.

Mehdi Ghoreishian et al.

2009

Pain: 1st to 5th day Hemostasis: 1st to 3rd day

There was no significant difference in severity of pain in study group and control group. There was less significant difference in the bleeding in study group and control group on the 1st and 2nd postoperative days. There was no significant difference between the two methods on the third day

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

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

Review Article

R-42

Sr. No. Author

Year

Evaluation period Outcome

M. Gogulanathan et al.

2015

Pain: 1st and 7th day Hemostasis: Time noted in seconds for bleeding to stop after closure.

The postoperative pain was significantly lower in the control group as compared to study group. The mean time taken for hemostasis in study group is 200 times faster than the control group.

Ajit D. Joshi et al.

2011

Pain: 1st to 5th day Hemostasis: 1st to 3rd day

There was significant difference in severity of pain in study group and control group. The postoperative bleeding was less significant in the control group as compared to study group on the 1st and 2nd days. Patients were unable to make any significant remarks over the bleeding on the 3rd postoperative day

CHART 1: SEARCH FLOWCHART

http://www.pacificejournals.com/aabs

R-43

AABS; 4(4): 2017 Pain (1.2)

Pain (3)

Pain (4)

GRAPH 1: PRIMARY OUTCOME (PAIN).

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

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

Review Article

R-44 Hemostasis (1,2)

Hemostasis (3)

GRAPH 2: SECONDARY OUTCOME (HEMOSTASIS)

GRAPH 3: SAMPLE SIZE

http://www.pacificejournals.com/aabs

R-45 the study group as compared to the control group on the1st, 2nd, and7th day postoperatively. Mannâ&#x20AC;&#x201C;Whitney U test was used to compare pain scores in both the groups. First cross-tabulation test was used to compare postoperative bleeding in both the groups and statistical analysis showed that there was significant difference in bleeding in the study group when comparison was done between the two groups on the1st postoperative day (p < 0.05). According to Mehdi Ghoreishian et al.2, there was no significant difference in the severity of pain between the 2 methods on the right and left sides of the mandible at all times recorded (P > 0.05). The data analysis showed that postoperative bleeding with the cyanoacrylate adhesive method was less significant than with suturing on the first and second days after surgery (P < 0.05); however, the bleeding index showed no significant difference (P > 0.05) between the 2 methods on the third day. According to M. Gogulanathan et al.3, compared to the control group, the study group showed a statistically significant reduction in the durations for achieving local haemostasis. The mean time for achieving haemostasis in the study group was 200 times faster than that in the control group (1.2 s vs. 251.9 s). The postoperative pain score assessed on the first postoperative day was also significantly lower for the study group patients (2.0) as compared with the control group (3.5). According to Ajit D. Joshi et al.4, there was significant difference in the severity of pain between the two methods on the right and left sides of the mandible at all times recorded (P < 0.05). The analysis showed that the severity of pain in closure with suture was more in first 3 days and later on it became same but marked elevation in pain have been noted on the second day. The data analysis showed that postoperative bleeding with the cyanoacrylate adhesive method was less significant than with suturing on the first and second days after surgery (P < 0.05); however, the bleeding index showed no significant difference (P < 0.05) between the two methods on the third day. Patients were unable to make any significant remarks over the bleeding on 3rd postoperative day. Defending The Result: All the four studies discussed, compare the closure of surgical incisions after surgical removal of impacted third molars with silk sutures and cyanoacrylate. The two parameters assessed were pain and hemostasis. In the first and third study, the severity of pain and the bleeding was less in cyanoacrylate group as compared to control group. In the second study, there was no significant difference in severity of pain in both the groups. There was less significant difference in the Annals of Applied Bio-Sciences, Vol. 4; Issue 4: 2017

AABS; 4(4): 2017 bleeding in study group and control group on the 1st and 2nd postoperative days. In the fourth study, there was significant difference in severity of pain in both the groups. The postoperative bleeding was less significant in the control group as compared to study group on the 1st and 2nd days. Quality of Evidence: All studies included in the review were randomized controlled studies. All the four studies included in this review had a level of evidence 2. Risk of bias in one study showed a low risk of bias. Hence the interpretation obtained from these studies are reliable. Inference: Use of cyanoacrylate adhesive had certain advantages over conventional suturing technique like it was hemostatic, reduced pain and avoids second visit for suture removal. The cost of tissue adhesive was the only limitation. Thus we can conclude that cyanoacrylate glue is a better alternative to conventional suturing for closure of intraoral minor surgical wound. Surindar et al.5 studied the efficacy of cyanoacrylate in patients undergoing various minor intraoral procedures and found it to be a hemostatic adhesive that polymerizes almost immediately on contact with cut oral tissue. The same was noted by Kulkarni et al.6 after the use of cyanoacrylate in periodontal flap surgery. Immediate hemostasis in bleeding pulp took place in the study conducted by Milton et al.7 . Fauad and Maged8 described the hemostatic effect of cyanoacrylate glue on warfarin- treated patients undergoing oral surgery. Implication for Practice: Cyanoacrylate is an effective means of mechanical as well as biological closure of intraoral wounds. This tissue adhesive markedly hastens haemostasis, reduces the operating time, and demonstrates less postoperative pain, thereby enhancing patient comfort significantly. Also it avoids second visit for suture removal Implication of Research: All Studies carried out are randomized control trial. So the results were reliable and further research is not required. Summary Aim of this systematic review is to compare the efficacy of cyanoacrylate and black braided silk for the closure of incision after surgical removal of impacted third molars. Randomized controlled trial or clinical trials evaluating and comparing the effectiveness cyanoacrylate and black braided silk for the closure of incision after surgical removal of impacted third molars. Trials were selected if they met the following criteria. Randomized controlled clinical trials comparing silk sutures and cyanoacrylate for surgical closure in patients e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article

R-46

undergoing lower third molar surgery. The studies for inclusion in this review represents comparison of silk sutures and cyanoacrylate in reducing postoperative pain and bleeding after third molar surgery. The databases PUBMED CENTRAL and MEDLINE were searched for the related topic till November 2016. The search identified 1 publication. Hand search was done and 3 articles were obtained. Full articles were obtained for 4 studies. Therefore, a total of 4 publications fulfilled all criteria for inclusion.

Conclusion

From this systematic review it can be concluded that, cyanoacrylate is a better alternative for intraoral minor surgical procedures as tissue glue, was found to be haemostatic in nature, was helpful in reduction of pain and patients need not visit again for suture removal. Even this procedure was comfortable for the surgeon.

Acknowledgements

Saveetha Dental College and Hospital, Chennai, India

Reference 1.

Setiya, S., Halli, R., Shah, A., Chhabaria, G., & Singh, T. (2015). Comparative evaluation of efficacy of tissue glue and sutures after surgical removal of impacted mandibular third molars – A prospective controlled clinical study. Journal of

Oral and Maxillofacial Surgery, Medicine, and Pathology, 27(2), 183–188. 2.

Ghoreishian, M., Gheisari, R., & Fayazi, M. (2009). Tissue adhesive and suturing for closure of the surgical wound after removal of impacted mandibular third molars: A comparative study. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics., 108(1).

Gogulanathan, M., Elavenil, P., Gnanam, A., & Raja, V. (2015). Evaluation of fibrin sealant as a wound closure agent in mandibular third molar surgery--a prospective, randomized controlled clinical trial. International journal of oral and maxillofacial surgery., 44(7), 871–5.

Joshi, A. D., Saluja, H., Mahindra, U., & Halli, R. (2011). A comparative study: Efficacy of tissue glue and sutures after impacted Mandibular Third molar removal. , 10(4).

Surindar NB, Joe F. Use of cyanoacrylate adhesives in dentistry. JADA 1968;77:831–7.

Kulkarni S, Dodwad V, Chava V. Healing of periodontal flaps when closed with silk sutures and N-butyl cyanoacrylate: a clinical and histological study. Indian J Dent Res 2007;18:72–7.

Milton DB, Fran AC, Marvin PL, Louis JB. Pulpal response to isobutyl cyanoacrylate in human teeth. JADA 1971;83:140–5.

Fauad AA, Maged ZA. Hemostatic effect of n-butyl2-cyanoacrylate (histoacryl) glue in warfarin treated patients undergoing oral surgery. JOral Maxillofac Surg 2003;61:1405–9.

*Corresponding author: Dr. Divya James, Saveetha Girls Hostel, Saveetha Dental College and Hospital, 162, Poonamallee High Road, Vellapanchavadi, Chennai – 600077, India. Email: divyajames3112@gmail.com Date of Submission : 20.09.2017 Date of Acceptance : 12.10.2017 Date of Publication : 29.10.2017

Financial or other Competing Interests: None.

http://www.pacificejournals.com/aabs

Original Article DOI: 10.21276/AABS.1655

Assessment of Peripheral Blood Smear Preparation Technique in Laboratories with High Sample Load Sumanashree Mallappa*, Sachin Kolte, Nimisha Sharma, Arti Khatri and Indrani Dhawan VMMC & Safdarjung Hospital, New Delhi, India

ABSTRACT Background: Examination of the blood film is an important part of the hematological evaluation. The reliability of the information obtained depends heavily on well-made and well-stained films that are systematically examined. Commonly used method of smear preparation in hematology the wedge method. In wedge method two slides are used one slide is placed on a flat surface with all necessary conditions maintained and the other slide is used as a spreader slide with is used to spread the blood film producing a good tongue shaped smear. In laboratory settings with high case load, same spreader is used to produce blood smears of multiple patients. Methods: The study was designed to know if there is any carry over of cells if the same spreader is used to make blood smears of multiple patients in laboratories having high patient load. Same spreader was used to make multiple smears from pancytopenia sample after using it initially to make smears from samples of leukemia and neutrophilic leucocytosis. Result: Slides were examined and results were tabulated, which showed that there was a definite carry over of cells from one smear to another if the same spreader is used consequetively for multiple patients Conclusion: Caution need to be used in laboratories with high case load where multiple smears need to be prepared and time constraint is a limiting factor. Use of advanced coulters can help in correlation of cells counts and typing instead of only depending on smears for cells counts and reporting. Collecting clinical details of the patient is an important step which is indispensable Keywords: Peripheral Smear, High Case Load, Carry Over Of Cells

Introduction

Examination of the blood film is an important part of the hematological evaluation.1 Initiation of a PBF is often a clinical request by the attending clinician on account of a clinical suspicion or less frequently initiated by the laboratory 2. The reliability of the information obtained depends heavily on well-made and well-stained films that are systematically examined.1 The method of making the films are : The two-slide or wedge method, the cover glass method, and the spinner method. Commonly used method of smear preparation in hematology the wedge method. In wedge method two slides are used one slide is placed on a flat surface with all necessary conditions maintained and the other slide is used as a spreader slide with is used to spread the blood film producing a good tongue shaped smear.1 In laboratory settings with high sample load, same spreader is used to prepare peripheral blood smears of multiple patients. Automation in smear preparation done by few hemoslider also uses the same technique of smearing but the spreader blade is cleaned each time before making the next smear.3

The speed and quality of information have become essential items in the release of laboratory reportsÂ 4. Among the numerous advantages of using automated equipment are the reduce time to release results, high sensitivity, greater accuracy with reduced coefficient of variation, better reproducibility and higher productivity in laboratory testing 5,6. Due to economic constraints faced by developing countries majority of the labs cannot go for automated slide makers and stainers, requiring strict quality controls and rechecks in manual techniques employed in smear preparation and staining. So, the study was planned to know if there is any carry over of cells if the same spreader is used to make blood smears of multiple patients in laboratories having high patient load.

Materials and Methods:

One sample was taken each of â&#x20AC;&#x201C; 1) Pancytopenia, 2) Leukemia and 3) Neutrophilic leucocytosis Study was done to see if the use of same spreader used initially for leukemia and later for pancytopenia leads to any changes in TLC and PS findings of the pancytopenia case.

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

A-151

AABS; 4(4): 2017

Likewise it was also studied whether the use of same spreader initially for leucocytosis and later for pancytopenia led to any changes in TLC and PS findings of the latter case. Study was Divided into two Parts1 A) One smear was prepared with leukemia sample initially and 6 successive smears were prepared with pancytopenia case using the same spreader, and B) One smear was prepared with neutrophilic leucocytosis sample initially and 6 successive smears were prepared with pancytopenia case using the same spreader 2 A) One smear was prepared with leukemia sample initially and one smear from pancytopenia sample using the same spreader and this step was repeated 6 times, and B) One smear was prepared with neutrophilic leucocytosis sample initially and one smear from pancytopenia with the same spreader and this step was repeated 6 times.

Result

One sample was taken each of â&#x20AC;&#x201C; 1) Pancytopenia, 2) Leukemia and 3) Neutrophilic leucocytosis, With values as follows â&#x20AC;&#x201C; (table1) After the study results were tabulated and analysed (Table-2,3,4 and 5).

Discussion:

In the first part of study (Table 2 and 3)1. A)

By the use of same spreader TLC was raised in the smears made from pancytopenia case. TLC was high in smear numbered L1 and L2, L3 smear also showed a raised TLC with respect to the pancytopenia case. And there was not so significant raise of TLC in smears which came later in the order namely L4 and L5. There was a definite carry over of blast cells into successive smears in case of L1 and L2 smears and the blast cells were present with high N/C ratio, scant agranular cytoplasm, indented nuclear membrane and inconscpicuous nucleoli. In smears numbered L3 and L4 there were cells with poorly maintained features which could not be clearly typed as blasts and smear L5 showed no blasts.

Irregular distribution of cells were seen predominantly in the head region of the smear (Table2) B) TLC was also raised when successive smears were prepared from pancytopenia case using the same spreader used for leucoerythroblastic reaction casev(Table 3). TLC was significantly raised in N1 Smear, TLC was also raised in N2 and N3 case with respect to the pancytopenia case and TLC raise was not so significant in N4 and N5 cases. Cells were distributed irregularly mainly in tail end and margins of the smear and predominant population of cells constituted of mature neutrophils, band forms and few neutrophilic precursors. Smear N1 and N2 also showed Nucleated RBCs. 2) Second part of the study includes 5 sets of smears (table 4 and 5). A) Same spreader used for making a leukemia smear was used to make one pancytopenia smear and the steps were repeated 5 times. There was a definite carry over of cells in almost all pancytopenia smears. TLC was significantly raised in almost all cases. Blast cells were positive in all the smears. There was irregular distribution of cells predominantly in head and in margins in few smears (Table 4) B) Same spreader used for making a leucoerythroblastic smear was used to make one pancytopenia smear and the steps were repeated 5 times. There was a definite carry over of cells in almost all pancytopenia smears. TLC was significantly raised in almost all cases. Predominant population of cells constituted of neutrophils, band forms and shift to left cells. There was irregular distribution of cells predominantly in tail end and in margins (Table 5) It was seen from the tabulation that there was clear carry over of the cells from the leukemia case and leucoerythroblastic reaction case to the subsequent smears if the same spreader is used.

Table 1: Case

Pancytopenia

Leukemia

Neutrophilic leucocytosis

RBC

2.01million/mm3

1.99million/mm3

2.66million/mm3

6.1g/dl

5.3g/dl

8.6g/dl

HCT

18.1%

18.0%

26.6%

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

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

Original Article

A-152

Case

Pancytopenia

Leukemia

Neutrophilic leucocytosis

MCV

90fl

91mm3

100/mm3

MCH

30.4pg

26.8pg

32.2pg

MCHC

33.9g/dl

29.6g/dl

32.2g/dl

RDWcv

14.3%

13.8%

13.4%

RDWsd

46fl

45fl

48fl

PCT

11000/mm3

44000/mm3

115000/mm3

TLC

400/mm3

1.5 lakh/mm3

61900/mm3

DLC

P-23.6 L-69.4 M-6.0 E-0.3 B-0.7

95% blasts 5% lymphocytes

P-90% L-08% M-01% E-01%

PS findings

RBC- normocytic normochromic RBC picture.; WBC- leucopenia seen TLC <1000/mm3 PLT-Decreased Impression-Pancytopenia

RBC-normocytic normochromic RBC picture. WBC-Very high leucocytosis. Predominant population of blast cells which show high N/C ratio, scant agranular cytoplasm,indented nuclear membrane and inconscpicuous nucleoli PLT – decreased Impression –acute leukemia

RBC-normocytic normochromic RBCpicture WBC- high leucocytosis Predominant population of neutrophils seen with moderate shift to left. NRBCS + - 10/100wbcs

Table 2: Case

DLC

PERIPHERAL SMEAR REPORTING

1.5 lakh/mm3

95% blasts 5% lymphocytes

Pancytopenia

400/mm3

Pred lymphocytes

RBC- normocytic normochromic RBC picture. WBC- leucopenia seen PLT-Decreased Impression-Pancytopenia

65000/mm3

Blasts +

RBC- normocytic normochromic RBC picture WBC-Irregular distribution of cells predominently in base

8000/mm3

Blasts +

RBC- normocytic normochromic RBC picture WBC- Irregular distribution of cells predominently in base

3000/mm3

Few suspicious cells +

RBC- normocytic normochromic RBC picture WBC- few cells predominently in base

1500/mm3

Few suspicious cells+

RBC- normocytic normochromic RBC picture WBC- Leucopenia with Very few cells in base

<1000/mm3

Pred lymphocytes

RBC- normocytic normochromic RBC picture WBC- leucopenia

Leukemia

TLC

http://www.pacificejournals.com/aabs

A-153

AABS; 4(4): 2017

Table 3: CASE

TLC

DLC

PERIPHERAL SMEAR REPORTING

Neutrophilic leucocytosis

61900/mm3

P-90% L-08% M-01% E-01% Nrbcs-14/100wbcs

RBC-normocytic normochromic RBC picture; WBC- high leucocytosis. Predominant population of neutrophils seen with moderate shift to left. NRBCS + - 10/100wbcs

Pancytopenia

400/mm3

Pred lymphocytes

RBC- normocytic normochromic RBC picture.; WBCleucopenia seen; PLT-Decreased Impression-Pancytopenia

30000/mm3

Pred neutrophils. Mature neutrophils-70% Band forms and shift to left cells- 25% NRBCs-5/100wbcs

RBC- normocytic normochromic RBC picture.; WBC-Irregular distribution of cells pedominently neutrophils and precursors in margins and tail end

5000/mm3

Pred neutrophils. Mature neutrophils-80% Band forms and shift to left cells- 20% NRBCs-2/100wbcs

4000/mm3

Pred neutrophils. Mature neutrophils-90% Band forms and shift to left cells- 10% NRBCs-nil

RBC- normocytic normochromic RBC picture.; WBCfew neutrophils and band forms seen in tail end

<1000/mm3

Few neutrophils + band forms seen

RBC- normocytic normochromic RBC picture. WBC-few neutrophils and band forms seen in margin and tail end

<1000/mm3

Few neutrophils + band forms seen

RBC- normocytic normochromic RBC picture. WBC-leucopenia

RBC- normocytic normochromic RBC picture. WBC-Irregular distribution of cells pedominently neutrophils and precursors in margins and tail end

Table 4: CASE

DLC

PERIPHERAL SMEAR REPORTING

95% BLASTS 5% lymphocytes

RBC-normocytic normochromic RBC picture.; WBC- Very high leucocytosis. Predominant population of blast cells which show high N/C ratio, scant agranular cytoplasm, indented nuclear membrane and inconscpicuous nucleoli; PLT â&#x20AC;&#x201C; decreased. Impression â&#x20AC;&#x201C; acute leukemia

Pancytopenia 400/mm3

Pred lymphocytes

RBC- normocytic normochromic RBC picture.; WBC- leucopenia seen; PLT-Decreased. Impression-Pancytopenia

LA+

90000/mm3

BLASTS+

RBC-normocytic normochromic RBC picture; WBC-Irregular distribution of cells mainly in base and margins. High leucocytosis predominant population of blasts

LB+

1lakh/mm3

BLASTS+

RBC-normocytic normochromic RBC picture; WBC- Irregular distribution of cells mainly in base. High leucocytosis predominant population of blasts

LC+

60000/mm3

BLASTS+

RBC-normocytic normochromic RBC picture; WBC- Irregular distribution of cells mainly in base. High leucocytosis predominant population of blasts

LD+

80000/mm3

BLASTS+

RBC-normocytic normochromic RBC picture; WBC- Irregular distribution of cells mainly in base and margins. High leucocytosis predominant population of blasts

LE+

55000/mm3

BLASTS+

RBC-normocytic normochromic RBC picture; WBC- Irregular distribution of cells mainly in base. High leucocytosis predominant population of blasts

Leukemia

TLC

1.5 lakh/mm3

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

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

Original Article

A-154

Table 5: CASE

TLC

DLC

PERIPHERAL SMEAR REPORTING

Neutrophilic leucocytosis

61900/mm3

P-90%; L-08%; M-01%; E-01%; NRBSs-14/100wbcs

RBC-normocytic normochromic RBC picture; WBC- high leucocytosis. Predominant population of neutrophils seen with moderate shift to left. NRBCS + - 10/100wbcs.

Pancytopenia

400/mm3

Predominantly lymphocytes

RBC- normocytic normochromic RBC picture.; WBCleucopenia seen. ; PLT-Decreased. ImpressionPancytopenia

NA+

30000/mm3

Pred neutrophils. P-92%; L-08%; NRBCs-6/100wbcs

RBC- normocytic normochromic RBC picture. ; WBCLeucocytosis seen with predominant population of neutrophils.

NB+

25000/mm3

Pred neutrophils. P-85%; L-14%; E-1%; NRBC10/100wbcs

RBC- normocytic normochromic RBC picture.; WBCLeucocytosis seen with predominant population of neutrophils

NC+

20000/mm3

Pred neutrophils. P-95%; L-55%; Nrbcs-2/100wbcs

RBC- normocytic normochromic RBC picture.; WBCLeucocytosis seen with predominant population of neutrophils

ND+

32000/mm3

Pred neutrophils. P-90%; L-10%; Nrbcs-nil

RBC- normocytic normochromic RBC picture.; WBCLeucocytosis seen with predominant population of neutrophils

NE+

18000/mm3

Pred neutrophils. P-91%; L-09%; Nrbcs-1/100wbcs

RBC- normocytic normochromic RBC picture. ;WBCLeucocytosis seen with predominant population of neutrophils

Fig. 1 :

http://www.pacificejournals.com/aabs

A-155

AABS; 4(4): 2017

Fig. 2:

Fig. 3 : Common representational image LA and LA + to LF and LF+

Fig. 4 : Common representational image for NA and NA = to NE and NE+

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

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

Original Article

A-156

Conclusion

There was clear carry over of cells to the successive smears by use of same spreader. Carry over was more significant in the successive 3 smears in order. There was irregular distribution of carried cells. In case of malignancy carried over cells were more commonly distributed in head of the smear and in case of neutrophilic leucocytosis carried over cells were more commonly found in tail end and margins of the smear. Caution need to be used in laboratories with high case load where multiple smears need to be prepared and time constraint is a limiting factor. Use of advanced coulters can help in correlation of cells counts and typing instead of only depending on smears for cells counts and reporting. Collecting clinical details of the patient is an important step which is indispensable.

Acknowledgements

Faculty, friends, and family members have helped me to complete this study. I would like to express my gratitude to these individuals for their support and assistance. I would love to express my gratitude to all the staff of central collection center of Safdarjung hospital for their support and help.

Reference 1.

Vajpayee N, Graham SS, Sylva Bem. Basic Examination

of Blood and Bone Marrow. In: McPherson RA,

Pincus MR. Henry′s Clinical Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia: Elsevier Saunders; 2007. 2.

Bain BJ. Diagnosis from the blood Smear. N Engl J Med. 2005;353:498–507

Levine MS Levine DS. Automatic blood film preparation method.1998

Ryan DH. Automated analysis of blood cells. In: Hoffman R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Silberstein LE, editors. Hematology: basic principles and practice. 2nd ed. New York: Churchill Livingstone; 1995. pp. 2223–2235.

Buttarello M, Plebani M. Automated blood cell counts: state of the art. Am J Clin Pathol. 2008;130(1):104–116

6. Barnes PW, McFadden SL, Machin SJ, Simson E, International Consensus Group for Hematology The international consensus group for hematology review: suggested criteria for action following automated CBC and WBC differential analysis. Lab Hematol. 2005;11(2):83–90

*Corresponding author: Sumanashree Mallappa “ Malayashree Nilaya” 2 cross, 1 B main Nagarbhavi village, Bangalore – 560072 India hone: +91 9990476487 Email: simplysumana@yahoo.co.in Date of Submission : 20.08.2017 Date of Acceptance : 01.10.2017 Date of Publication : 14.10.2017

Financial or other Competing Interests: None.

http://www.pacificejournals.com/aabs

Original Article DOI: 10.21276/AABS.1687

Syphilis in Blood Donors in Tertiary Care Teaching Hospital Ravi Jain And Ashok Yadav* Dept. 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. Aims & Objectives: To find out the seroprevalence of Syphilis in blood donors, to find the incidence of spectrum of Transfusion transmitted dieasess in blood bank donation & to find the age distribution of the cases studied. Material & Methods: The present study was undertaken in the Department of Pathology MGM Medical College Indore. This is a retrospective study that was conducted, during the period 2008 –2015. The screening for Syphilis was done by rapid chromatographic assay for detection of antibodies to T. pallidum Results: Out of total 137689 blood donations, majority of donors are voluntary donors 83.02 % as compared to replacement donors 17.05 %. Seroprevalence of Anti TP is 0.26 %. Seroprevalence of Anti TP is higher in the age group 26-35 year . Among Voluntary & replacement/relative donors. Overall seropositivity of TTI’s (HIV, HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.71 % as compared to voluntary donors 1.75 %. 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. Keywords: Treponema Pallidum, Seroprevalence, 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. Thereafter, 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. 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 + spirochetes 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: 1. Human immunodeficiency virus (HIV) 2. Hepatitis B virus 3. Hepatitis C virus 4. Hepatitis A virus 5. Hepatitis G virus 6

Non - A, Non - B Hepatitis

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

Original Article

A-158

7. Epstein Barr Virus 8. Cytomegalo virus (CMV) 9. Human T Lymphocytic virus (HTLV - 1 & HTLV - 2) Syphilis, an ancient disease is caused by Spirochete Treponema pallidum. According to The World Health organization estimates2, there are approximate 12 million new cases diagnosed each year , with more than 90% in developing countries. Syphilis has acquired new potential for morbidity with the advent of HIV & AIDS. Natural History of syphilis3: Syphilis is a chronic disease caused by Treponema pallidum. Treponemes (trepos – to turn & nema - thread)4 are relatively short, slender spirochetes with fine spirals & pointed or round ends. Some of them are pathogenic while others occur as commensals in the mouth, intestines & genitalia. Treponemes causes the following diseases in the humans4. 1. 2. 3. 4.

Venereal Syphilis: Treponema pallidum. Endemic Syphilis: Treponema pallidum (T endemicum) Yaws: Treponema pertenue Pinta: Treponema carateum

They are almost identical in their morphology & antigenic structure though there are differences in clinical features & natural history. Treponema Pallidum: the causative agent of syphilis was discovered by Schaudinn & Hoffmann (1905)4 in the chancres & inguinal lumph nodes of syphilitic patients. The name pallidum refers to its pale staining. Treponema pallidum is a thin, delicate spirochete with tapering ends, about 10 μm long (range 4-14 μm) & 0.1-0.2 μm wide. It has ten regular spirals, which bare sharp, angular at regular intervals. The spirochete is actively motile, rotating around long axis with backward & forward movements. Treponema pallidum cannot be seen under light microscope but can be appreciated under dark ground or phase contrast microscope. Treponema pallidum can be demonstrated in tissue sections using silver impregnation methods4. Antigenic Structure of Treponema Pallidum: Infection induces three types of antibodies4. 1. Reagin Antibody: reacts in Wasswrmann, Kahn & VDRL tests, in which hapten extracted from beef heart (Cardiolipin) is used as Antigen. 2. Group Antigen: found in Treponema pallidum & non-pathogenic treponemes. 3. Species Specific: antibody to this antigen is demonstrated by specific Treponema pallidum tests & which are positive in sera of patients infected with Treponema pallidum.

On the basis of clinical presentation, infectivity & progression Syphilis is divided into 5 clinical stages – primary, secondary, latent & tertiary syphilis. 1. Primary Syphilis: painless genital ulcer (chancre) following exposure to infection. 2. Secondary Syphilisl macular popular rash involving palms & soles with high bacteriemia in blood. 3. Latent Phase: asymptomaticbut positive serological test for syphilis 4. Tertiary Syphilis: cardiovascular/neurological symptoms. Transfusion Transmitted Syphilis: The first case of Transfusion transmitted syphilis was reported in 19153. 138 cases were reported in the literature by 19415. Cases were mostly discovered in donors with primary or secondary stage of disease6. Treponema pallidum may be found in blood, not levels are variable & bacteremia is short lived. Treponemes are senisitve to cold; hence risk of transmission through stored blood at 4-8◦C is very low7-8. In India, most blood donors are first-time donors3. The prevalence of syphilis among blood donors in India was reported to be 0.7%9. The global incidence of syphilis in blood donors is variable ranging from 0.75% in Pakistan10 to 12.7% in Tanzania11. Testing Methodologies3: Three methods are currently used. 1. Direct microscopic examination in early stage. 2. Non-treponemal Serological Tests e.g : RPR & VDRL 3. Indirect Treponemal Tests: FTA-ABS(Fluorescent treponemal antibody absorption test), TPHA (Treponema pallidum Hemagluttination assay), EIA (enzyme immunoassay), Rapid assays (immunechromatographic strips) & western Blotting 4. Molecular Based Methods: PCR (Polymerase chain reaction)

Aims and Objectives

The study is being conducted in the department of pathology, M.G.M.M.C, Indore. 1. To find out the seroprevalence of Syphilis in blood donors. 2. To find the age distribution of the cases studied. 3. To find the incidence of spectrum of Transfusion transmitted diseases in blood bank donation.

Material and Methods

The present study is being undertaken in the Department of Pathology MGM Medical College Indore. This is a

http://www.pacificejournals.com/aabs

A-159

AABS; 4(4): 2017

retrospective study that will be conducted, during the period 2008 –2015. 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 Syphilis was done by rapid chromatographic assay for detection of antibodies to T.pallidum . 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.

Graph 1: Out of total 137689 blood donations, majority of donors are voluntary donors 83.02 % as compared to replacement/relative donors 17.05 %

Results

Graph 4: Age wise distribution of Anti TP in the year 2008-15. Seroprevalence of Anti TP is higher in the age group 26-35 year

The present study was 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 –2015. In the present study, 137689 blood donors are observed in the year 200815 in the M. Y. Blood Bank. The data collected from donor register record book, donors form, master record book, HIV, & HBV positive bag number records. The results and observations studies are presented below:

Graph 2: Out of total 137689 blood donations, majority of donors are male donors 95.59 % as compared to female donors 4.40% Graph 3: Seropositive donors for Anti TP in 2008-15. Seroprevalence of Anti TP is 0.26 %.

Table 1: Seropositivity of transfusion transmitted diseases (HIV, HBV, HCV, Syphilis & Malaria) in 200815. Among Voluntary & replacement/relative donors . Overall seropositivity of TTI’s (HIV, HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.71 % as compared to voluntary donors 1.75 %.

Table 1: Seropositivity of transfusion transmitted diseases (HIV,HBV,HCV,Syphilis & Malaria) in 2008-15. Among Voluntary & replacement/relative donors . Overall seropositivity of TTI’s (HIV,HBV,HCV,Syphilis & Malaria) is higher in replacement donors 3.71 % as compared to voluntary donors 1.75 %. S.No

Total No of Voluntary donors (2008-15)

Total No of Voluntary donors found seropositive for TTI (2008-15)

Total No of Replacement/ relative donors (2008-15)

Total No of Replacement/relative found seropositive for TTI (2008-15)

Number

114246

2007

24093

896

%age

83.02 %

1.75 %

17.05 %

3.71 %

Graph 1: Number of blood units collected during the year 2008-15. Out of total 137689 blood donations, majority of donors are voluntary donors 83.02 % as compared to replacement/relative donors 17.05 %

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

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

Original Article

A-160

Graph 2 : Number of male and female donors during the year 2008-15.Out of total 137689 blood donations, majority of donors are male donors 95.59 % as compared to female donors 4.40%.

Graph 3 : Seropositive donors for Anti TP in 2008-15 Seroprevalence of Anti TP is 0.26 %.

Graph 4: Age wise distribution of Anti TP in the year 2008-15. Seroprevalence is higher in the age group 26-35 year

http://www.pacificejournals.com/aabs

A-161

Discussion

Voluntary or Replacement/Relative Donor -In our study, out of total 137689 blood donations, majority of donors are voluntary donors 83.02 % as compared to replacement/ relative donors 17.05 % (Graph 1) Similarly majority of donors are voluntary in another study by Nagarekha Kulkarni12 , ,, out of 19135 blood donors, 11165 (58%) were voluntary and 7970 (42%) were replacement donors Male or Female Donors: In our study, out of total 137689 blood donations, majority of donors are male donors 95.59 % as compared to female donors 4.40 % (Graph 2) . Similarly another study is comparable for majority of donors are male 96.22 % by Dimple Arora and Bharti Arora et al13 in 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 et al14, conducted in New Delhi. Seroprevalence of Anti TP (Anti Treponema pallidum): In our study, the Seroprevalence of Anti TP is 0.26 %. in total blood donations in the year 2008-15 (Graph 3). Seroprevalence of Syphilis is comparable to another study with seroprevalence of Anti TP was 0.91 % by Sultan S, Murad S Irfan m et al 15conducted in Pakistan. In another study by Elyamany G et al16 seroprevalence was found to be 0.044% . In a study conducted at Mangalore, India by Zulfikar A et al 17 seropositivity of syphilis was found to be 0.07% . Age Wise Distribution: In our study, overall Seroprevalence of Anti TP (2008-15) is higher in the age group 26-35 years for anti TP (0.09 %) (Graph 4 ). In a study conducted at Mangalore, India by Zulfikar A et al 17 incidence of seropositivity was found to be more in donors in the group aged 18-35 years old than in the group 36-55 years old. In a study by Tessema et al18 , seropositivity of syphilis was found to be 0.9% & 1.7% in age groups 17-25 & 26-35 yrs respectively. In another study by Elyamany G et al16 seropositivity was found to be highest in age group 21-30 yrs. Seropositivity in Volunatry/replacement Donors: Among Voluntary & replacement/relative donors, Overall seropositivity of TTI’s (HIV, HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.71 % as compared to voluntary donors 1.75 % (Table 1). In study by Nagarekha Kulkarni12 , the seroprevalence was more in relative/replacement donors as compared to voluntary donors.

Conclusion

The present study was conducted in the Department of Pathology MGM Medical College Indore and M. Y. Annals of Applied Bio-Sciences, Vol. 4; Issue 4: 2017

AABS; 4(4): 2017 Hospital blood bank. This is a retrospective study that was conducted, during the period 2008 –2015. 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 137689 blood donations, majority of donors are voluntary donors 83.02 % as compared to replacement/relative donors 17.05 %. Out of total 137689 blood donations, majority of donors are voluntary donors 83.02 % as compared to replacement donors 17.05 %. Seroprevalence of Anti TP is 0.26 %. Seroprevalence of Anti TP is higher in the age group 26-35 year . Among Voluntary & replacement/relative donors . Overall seropositivity of TTI’s (HIV,HBV,HCV,Syphilis & Malaria) is higher in replacement donors 3.71 % as compared to voluntary donors 1.75 %. HBV and HIV are the most prevalent transfusion-transmissible 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.

Acknowledgements

We are Highly grateful to Dr C.V.Kulkarni, Prof.& Head, Dept. of Pathology for providing us the opportunity & full support.

References 1.

Tang, J. and Kaslow, R. A. (2003). “The impact of host genetics on HIV infection and disease progression in the era of highly active antiretroviral therapy”. AIDS 17 (Suppl 4): S51–S60.

World Health Organization. Global prevalence & incidence of selected curable sexually transmitted infections: overview & estimates (2001). Available at : http://www.who.int/hiv/ pub/sti/en/who_hiv_aids_2014.05.pdf.

Kaur.G, Kaur.P : Syphilis testing in blood donors: an update. Blood Transfus 2015;13:197-204

Ananthnarayan.R & Panicker C.K in Ananthnarayan. & Panicker’s Textbook of Microbiology 8th Edition Chapter 42 pg 371. University Press.

De Schryver A, Meheus A. Syphilis & blood transfusion: a global perspective. Transfusion 1990;30:844-7

Gardella C, Marin AA, Kahn RH et al. Persons with early syphilis identified through blood or plasma donation screening in United states. J Infect Dis 2002; 185:545-9

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

Original Article

A-162

Orton S. Syphilis & blood donors: what we know, what we do not know, & what we need to know. Transfus Med Rev 2001;15:282-91.

Wendel S. Current concepts on transmission of bacteria & parasites by blood components. Vox Sang 1994;67(Suppl3):161-74

Kaur G, Basu S, Kaur R et al. Patterns of infections blood donors in a tertiary care center: A retrospective study. Natl Med J India 2010:23;147-9

10. Bhalti FA, Ullah Z, Salamat N, et al . Anti-Hepatitis B core antigen testing, viral markers & occult Hepatitis b infection in Pakistani blood donors: implication for transfusion practice. Transfusion 2007; 47:74-9 11. Matee MI, Magesa PM, Lyamuya EF. Seroprevalence of Human immunodeficiency virus, Hepatitis B & C virus and syphilis infections among blood donors at Muhimbili National hospital in Dar es Salam, Tanzania. BMC Public health 2006;6:21 12. Kulkarni N. Analysis of the seroprevalence of HIV, HBsAg, HCV and syphilitic infections detected in the pretranfusion blood: A short report. International Journal of Blood Transfusion and Immunohematology 2012;2:1-3.

13. Dimple Arora, Bharti Arora, AnshulKhetarpal“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. Sultan S, Murad S Irfan m et al . Trends of venereal infections among healthy blood donors at Karachi. Arch Iran Med 2016;19(3):192-196 16. Elyamany G et al. Prevalence of syphilis among blood & stem cell donors in Saudi Arabia: an institutional experience.2016;Vol 8 Issue 8: 2747-2751 17. Zulfikar A, Umaru N, Shreesha K. Seroprevalence of Transfusion transmitted Infections among blood donors in Mangalore. Medica Innov. Dec 2012, Vol 1, issue 2; 24-27 18. Belay Tessema et al “Seroprevalence of HIV, HBV, HCV and syphilis infections among blood donors at Gondar University Teaching Hospital, Northwest Ethiopia: declining trends over a period of five years” BMC Infectious Diseases201010:111

*Corresponding author: Dr. Ashok Yadav, Postal Address: Yadav clinic, Newpanchsheel colony, Musakhedi, Indore (M.P )452001 Phone: +91 9893273236 Email: drashokmyh@gmail.com, ravijainpatho@gmail.com Date of Submission : 24.09.2017 Date of Acceptance : 01.10.2017 Date of Publication : 14.10.2017

Financial or other Competing Interests: None.

http://www.pacificejournals.com/aabs

Original Article DOI: 10.21276/AABS.1701

Hematological Profile of Pregnant and non Pregnant females: A comparative Study in a Tertiary Care Hospital in Faridabad, Haryana

Sujata Raychaudhuri1*, Shveta Lukhmana2, Deepsikha Rana1, Mukta Pujani1, Nimisha Sharma1 and Rashmi Ahuja3 2

1 Pathology dept, Employees State Insurance Corporation Medical College and Hospital, Faridabad, India Community Medicine dept, Employees State Insurance Corporation Medical college and Hospital, Faridabad, India 3 Gyaenecology dept, Employees State Insurance Corporation Medical College and Hospital, Faridabad, India

ABSTRACT Background: There are marked variations in the various haematological parameters during the pregnancy and also during the different trimesters of pregnancy. Due to paucity of literature on the haematological profile of pregnant population in Haryana, India the present study aims to fill the gap by drawing a comparison between cases and controls for the different haematological indices among patients attending a tertiary care hospital and also highlight the difference among the pregnant women across the three trimester(s). Methods: A case control study was conducted to study and compare the haematological profile among 119 pregnant and 119 non-pregnant women attending the out-patient department of ESIC medical, college & hospital Faridabad,a tertiary care hospital in Faridabad district of Haryana. Statistical analysis of the data was done using SPSS software 17.0. Unpaired students t-test was applied to compare various haematological parameters between cases and controls. One way Anova was applied and Tukey and Games Howell post hoc test was applied to study between group and within group differences among the three trimester with hematological indices. Result: White blood cells (WBCs), neutrophils, nucleated Red Blood Cells(NRBC), immature granulocytes(IG) and Red cell Distribution Width-Standard deviation (RDW-SD) were found to be higher among cases as compared to controls. Hemoglobin, hematocrit, mean corpuscular volume(MCV),mean corpuscular haemoglobin(MCH), lymphocyte and platelet count were found to be significantly higher among controls as compared to cases. On one-way ANOVA a statistically significant difference in hemoglobin, hematocrit, WBCs, neutrophils, lymphocytes, immature granulocytes and nucleated RBCâ&#x20AC;&#x2122;s was found between the three trimester(s). Conclusion: Haemoglobin and haematocrit and platelets showed a fall while WBCs and neutrophils showed a rise when compared to the nonpregnant state.The findings of the study will be a value addition in comparative analysis of studies between pregnant and non-pregnant females.

Introduction

Keywords: Hematological Indices, Pregnancy, Trimester, Case-Control

Haematological profile of an individual is a reflection of general state of body and is of significance. It is quick, cheap and easy to perform and reliable.1 Pregnancy is a physiological condition with profound effects on various organ systems of the body. It is a state of adaptation to meet the increased requirements of fetoplacental unit. There is a marked variation in the haematological profile during pregnancy. Many studies have shown that haematological profile of an individual is a major determinant affecting the pregnancy and its outcome.2,3,4 Normally in the absence of co-morbidity associated with pregnancy the body can recover, however in the presence of abnormal coagulation states, anemia, hypertension, haemorrhage etc. the parameters remain deranged. Pregnancy is influenced by many factors which includes socioeconomic, enviornmental and cultural conditions which impact the nutritional

state of an individual.5 It is essential for the health care workers to be aware of both normal and abnormal changes during pregnancy and the resulting lab values.6 This study was undertaken to determine the haematological profile of patients attending tertiary care hospital in Faridabad district of Haryana. This present study evaluates various haematological parameters including haemoglobin (Hb), packed cell volume (PCV), red cell indices, white blood cell (WBC) counts, red blood cell (RBC) counts, RDW, nucleated red blood (NRBCs) and platelet count among normal pregnant women and their comparision with normal control and also study the inter-trimester variation among these parameters.

Materials and Methods:

A case control study was conducted in Department of Pathology in collaboration with Department of Gynaecology at ESIC Medical College & Hospital,

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

Original Article

A-164

Faridabad among pregnant women attending the outpatient antenatal clinic of the hospital to compare the haematological indices of pregnant and non-pregnant women and to evaluate the haematological parameters of these pregnant women at different trimesters. The study was conducted over a period of three months.119 pregnant women in first, second and third trimester, irrespective of their gravidae, were selected for the purpose of study using convenient sampling and were classified as ‘cases’. 119 non-pregnant women attending the out-patient department of the ESIC medical college & hospital were selected and classified as ‘controls’. Matching was not done. 4 ml of venous blood was collected in ethylene diamine tetra acetic acid (EDTA) vial. CBC was done using automated sysmex analyser XN 1000 in the central lab of the hospital. Ethical clearance was obtained from the Institutional ethical committee. Any coagulation abnormalities, hypertension, diabetes, respiratory diseases, cardiac, renal and/or haemolytic diseases that alters the test result were excluded from the study. Informed consent was taken from all the participants. Subsequently haematological profile of the patients were studied which is also a part of their routine investigation. The information thus collected was converted into a computer based spreadsheet using Microsoft Excel software. Statistical analysis of the data was done on the Statistical Package for the Social Sciences [SPSS, Chicago, IL] software 17.0. Unpaired student t-test was applied to compare hemoglobin, hematocrit, red cell indices, total WBC count, differential, nucleated RBC’s, immature granulocytes and platelet count between cases and controls. One way Anova was applied to study association of haematological indices between three trimester and Tukey and Games Howell post hoc test was applied to study within group differences among the three trimester with haematological indices. p value of 0.05 was taken to be statistically significant.

Result

The number of patients in first, second and third trimester were 38.6%, 31.9% and 29.4% respectively. The mean of various haematological parameters is reflected in Table 1. On applying t-test, hemoglobin was found to be higher among controls as compared to cases and this was found to be statistically significant (p=0.009). Similarly, hematocrit was found to be higher among controls as compared to cases and this was found to be statistically significant (p=0.001). MCV and MCH were also found to be higher among controls as compared to cases and this was found to be statistically significant (p = 0.00; p=0.00 respectively) (Table 1). WBC’s were found to be higher among cases as compared to controls and this was found to be statistically significant (p=0.044). Neutrophils were

found to be higher among cases as compared to controls and this was found to be statistically significant (p=0.00). Lymphocytes were found to be higher among controls as compared to cases and this was found to be statistically significant (p=0.00). Similarly immature granulocytes were found to be higher among cases as compared to controls and this was found to be statistically significant (p=0.00). Platelets were found to be higher among controls as compared to cases and this was found to be statistically significant (p=0.004) (Table 1). On one way ANOVA, a statistically significant difference in red cell parameters like hemoglobin [F (2,116) = 3.17], p = 0.046., hematocrit [F (2,116) = 3.387], p = 0.037 was found between the three trimester(s). Similarly, a statistically significant difference in white blood cell indices like WBC [F (2,116) = 5.1], p = 0.007, neutrophils [F (2,116) = 11.72], p = 0.000, lymphocytes [F (2,116) = 12.39], p = 0.000 and immature granulocytes [F (2,116) = 8.18], p = 0.00, NRBC [F (2,116) = 3.2], p = 0.042 was found between the three trimester(s) as shown in Table 2. Tukey’s post hoc test was applied to determine the difference between the three trimester. WBC’s were found to be significantly higher among subjects in third trimester as compared to those in first trimester (p=0.006). Subjects in second trimester were found to have a higher hemoglobin as compared to those in third trimester and this was found to be statistically significant (p=0.00). Hematocrit was higher among subjects in first trimester as compared to those in third trimester and this was found to be statistically significant (p=0.025) (Table 3). Assumption of equal variances was violated by some independent variables like neutrophils, lymphocytes, immature granulocytes and nucleated RBC’s as shown by Levene’s test (p = 0.004, p= 0.003, p=0.005, p=0.000 respectively), therefore, Games Howell post hoc test was used to determine differences between the groups in these set of variables. Neutrophils were found to be higher among subjects in second and third trimester as compared to first trimester and this was found to be statistically significant (p= 0.022, p = 0.00 respectively). Lymphocytes were found to be higher among first trimester as compared to second and third trimester and this was found to be statistically significant (p= 0.035, p=0.00 respectively). Immature granulocytes were significantly higher among second (p= 0.041) and third trimester (p=0.000) as compared to first trimester (Table 3).

Discussion

Hematological changes that occur during pregnancy are physiological. Changes in pregnancy are brought about by hormones namely estrogen and progesterone secreted by the placenta.7 This stimulates renin secretion which activates the renin angiotensin system along with mild

http://www.pacificejournals.com/aabs

A-165

AABS; 4(4): 2017

Table 1: Comparison of Hematological Profile Among Pregnant and Non-Pregnant Women Hematological profile

Socio-economic status

Mean ± S.D.

T statistic

Confidence interval

p value

Lower limit

Upper limit

1.125

-0.095

0.349

0.262

-2.165

-0.9871

-0.1389

0.009

-3.224

-3.19

-0.7713

0.001

-3.816

-7.9595

-2.5397

0.00

-3.692

-2.76

-0.8416

0.00

0.082

-0.504

0.548

0.935

-3.566

-6.57

-1.89

0.00

0.529

-0.52

0.90

0.597

Red cell parameters RBC Hemoglobin Hematocrit MCV MCH MCHC RDW-SD RDW-CV

Case

4.22 ± 1.04

Control

4.09 ± 0.65

Case

10.9 ±1.66

Control

11.55 ± 1.65

Case

34.68 ± 4.25

Control

36.66 ± 5.19

Case

84.33 ± 8.67

Control

89.58 ±12.2

Case

26.7 ± 3.5

Control

28.5 ± 3.97

Case

31.5 ± 1.6

Control

31.5 ± 2.4

Case

47.06 ± 7.8

Control

15.43 ± 2.5

Case

15.62 ± 3.05

Control

15.43 ± 2.5 White cell parameter

WBC Neutrophil Lymphocyte Monocyte Eosinophil Basophil Immature granulcyte NRBC

Case

10.32 ± 3.01

Control

9.48 ± 3.44

Case

69.35 ± 8.5

Control

64. ± 11.4

Case

21.13 ± 7.3

Control

25.4 ± 9.33

Case

7.3 ± 5.77

Control

8.16 ± 7.4

Case

2.5 ± 2.7

Control

3.11 ± 3.03

Case

0.266 ±0.59

Control

0.240 ± 0.195

Case

0.802 ± 0.811

Control

0.36 ± 0.45

Case

0.093 ± 0.326

Control

0.017 ± 0.045

2.021

0.021

1.67

0.044

4.074

2.76

7.9

0.00

-3.927

-6.42

-2.13

0.00

-0.958

-2.52

-.871

0.39

-1.463

-1.29

0.1917

0.145

0.437

-0.0885

0.139

0.663

5.181

0.2739

0.6101

0.000

2.53

0.0167

0.1362

0.013

-2.895

-0.561

-0.106

0.004

Platelet count Platelet count

Case

2.3 ± 0.74

Control

2.6 ± 1.01

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

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

Original Article

A-166

Table 2: Comparison of Hematological Parameters Between The Trimester(S) Hematological parameters

Trimester

Mean ± S.D.

F statistic

P value

2.5

0.084

3.17

0.046

3.387

0.037

0.430

0.652

0.583

0.56

1.67

0.19

0.196

0.822

0.257

0.774

5.1

0.007

11.72

0.000

12.396

0.00

1.688

0.189

0.139

Red cell parameters RBC

Hemoglobin

Hematocrit

MCV

MCH

MCHC

RDW-SD

RDW-CV

First

4.49 ± 1.51

Second

4.08 ± 0.58

Third

4 ± 0.44

First

11.45 ± 1.5

Second

10.73 ± 1.78

Third

10.63 ± 1.6

First

35.9 ± 4

Second

33.78 ± 4.4

Third

34 ± 4.1

First

85 ± 9.67

Second

83.2 ± 8.4

Third

84.57 ± 7.6

First

27.14 ± 3.9

Second

26.43 ± 3.5

Third

26.4 ± 3.11

First

31.8 ± 1.55

Second

31.65 ± 1.69

Third

31.19 ± 1.54

First

47.54 ± 6.4

Second

46.46 ± 6.6

Third

47 ± 10.46

First

15.37 ± 3.2

Second

15.18 ± 2.24

Third

15.75 ± 3.6

White cell parameters WBC

NEUT

LYMPH

MONO

First

9.32 ± 2.95

Second

10.59 ± 2.99

Third

11.36 ± 2.77

First

65.2 ± 8.7

Second

70.5 ± 9

Third

73.54 ± 4.7

First

24.66 ± 7.55

Second

20.445 ± 7.6

Third

17.23 ± 4

First

8.4 ± 9

Second

6.13 ± 1.05

Third

7.22 ± 1.6

First

2.8 ± 2.8

Second

2.9 ± 3.3

Third

1.7 ± 1.89

http://www.pacificejournals.com/aabs

A-167 Hematological parameters BASO

NRBC

AABS; 4(4): 2017 Trimester

Mean ± S.D.

F statistic

P value

First

0.22 ± 0.13

0.357

Second

0.379 ± 1

Third

0.191 ± 0.11

First

0.45 ± 0.37

8.18

0.00

Second

0.93 ± 1.13

Third

1.11 ± 0.64

First

0.037 ± 0.06

3.2

0.042

Second

0.055 ± 0.122

Third

0.209 ± 0.57 1.05

0.351

Platelet count Platelet count

First

2.41 ± 0.77

Second

2.43 ± 0.73

Third

2.2 ± 0.7

Table 3: Group-Wise Comparison of Hematological Parameters within The Three Trimester(S). Hematological parameter

Trimester

Mean difference

p value

Red cell parameter RBC

Hemoglobin

Hematocrit

MCV

MCH

MCHC

RDW-SD

RDW-CV

First

Second

0.41

0.167

First

Third

0.45

0.119

Second

Third

0.04

0.978

First

Second

0.72

0.112

First

Third

0.82

0.068

Second

Third

0.963

0.000

First

Second

2.13

0.054

First

Third

1.91

0.025

Second

Third

-0.219

0.97

First

Second

1.73

0.63

First

Third

0.44

0.972

Second

Third

-1.29

0.80

First

Second

0.71

0.634

First

Third

0.73

0.627

Second

Third

0.025

0.999

First

Second

0.179

0.866

First

Third

0.645

0.174

Second

Third

0.4665

0.429

First

Second

1.08

0.806

First

Third

0.45

0.965

Second

Third

-0.62

0.938

First

Second

-0.44

0.79

First

Third

-0.37

0847

Second

Third

0.06

0.99

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

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

Original Article Hematological parameter

A-168 Trimester

Trimester

Mean difference

p value

White cell parameter WBC

Neutrophil

Lymphocyte

Monocyte

Eosinophil

Basophil

Immature granulocyte

NRBC

First

Second

-1.27

0.12

First

Third

-2

0.006

Second

Third

-0.77

0.498

First

Second

-5.30

0.022

First

Third

-8.33

0.000

Second

Third

-3.0

0.174

First

Second

4.22

0.035

First

Third

7.4

0.000

Second

Third

3.2

0.067

First

Second

2.3

0.164

First

Third

1.21

0.615

Second

Third

-1.09

0.696

First

Second

-0.11

0.979

First

Third

1.05

0.209

Second

Third

1.17

0.170

First

Second

-0.15

0.486

First

Third

0.03

0.959

Second

Third

0.187

0.377

First

Second

-0.48

0.041

First

Third

-0.65

0.00

Second

Third

-0.1746

0.694

First

Second

-0.018

0.68

First

Third

-0.17

0.197

Third

-0.153

0.28

First

Second

-0.021

0.99

First

Third

0.20

0.432

Second

Third

0.227

0.392

Second Platelet count Platelet count

decrease in atrial natriuretic peptide causing sodium and water retention. There is increase the blood volume during pregnancy upto 1.5 litres mainly to meet the increased demands during pregnancy as vascular channels are formed for the fetus and to compensate for the blood loss during delivery.8 By 6 to 8 weeks of gestation there is 10 to 15% rise in plasma volume.9,10 Maternal erythropoietin production which rises due to renin secretion causes increase in red cell mass which is less compared to the increased plasma volume. This leads to reduced haemoglobin and dilutional anemia. The plasma volume increases during the course of pregnancy reaching its peak in the second trimester and hence the haemoglobin and the PCV drops in this trimester.

By the third trimester the reduction in blood volume is less and reduction of haemoglobin is less with mild increase in the PCV. The present study is also consistent with this finding as the Hb level and PCV drops in the second trimester and then stabilises by third trimesrter. This finding is consistent with study from other researchers5,11,12 and other internatonal studies. According to the standard laid down by WHO, anemia in pregnancy is present when the haemoglobin concentration in the peripheral blood is 11 gm/100ml or less.13,14 Anaemia contributes to intrauterine growth restriction, preterm labour, abortions and it is also a primary cause of low immunity of both the mother and the baby, which makes them prone for several life

http://www.pacificejournals.com/aabs

A-169 threatening infections.15 In the present study the red cell indices MCH and MCHC shows a steady decline by third trimester but the MCV is showing a decline in the second trimester but again rising back in the third trimester. The MCH and MCV are statistically significant which matches with the mentioned previous studies. The decrease in MCV during second trimester can be attributed to the reduction in hemoglobin which is more pronounced in the second trimester which would have resulted in microcytosis. By the third trimester the RBC production is increased to meet the increased demand and more immature RBCs are released into the circulation resulting in macrocytosis and increased MCV. This finding is not in concordance with other previous studies. Our study shows increase in total leucocyte count and neutrophil count in the course of pregnancy. Both these parameters are statistically significant when compared with control group. However no definite trend was noted for eosinophils, monocytes and basophils in our study and also all the three parameters were not statiscally significant. The lymphocyte count during pregnancy declined in our study and is statistically significant. This is in concordance with previous studies16,17 although study in Lagos, Nigeria7 shows increase in the lymphocyte count. The stresss during pregnancy results in increase in WBC count reaching maximum during third trimester.Neutrophils are major leukocytes in the differential count as this is due to reduced apoptosis of neutrophils in pregnancy18 and also there is reduced chemotactic and phagocytic activity of neutrophils19 due to the inhibitory substances present in the plasma of the of the pregnant women. Toxic granules in the neutrophils also increased. There is absolute monocytosis in the first trimester in the present study. Monocytes induce decidual cells to secrete PGE2 to prevent fetal allograft rejection.20 Due to different geographical locations and physiological conditions the changes in different WBC cell types causing leucocytosis cannot be clearly defined7. The present study shows reduction in the platelet count with the progress of pregnancy reaching a minimum in the third trimester. This correlates with other similar previous Indian studies5,11,12.This reduction is known as gestational thrombocytopenia. This is due to hemodilution, accelerated platelet clearance and platelet activation21. Platelet count is lower when compared with control but not significant. As in the present study most pregnant females have normal platelet count. If the platelet count in the beginning of pregnancy is towards the lower side of normal range then only it will fall below normal. This is due to two phenomenon that is increased blood volume and damage Annals of Applied Bio-Sciences, Vol. 4; Issue 4: 2017

AABS; 4(4): 2017 to the platelets as it flows through the rough and scarred surface of the placenta22. Most of the thrombocytopenia is mild. Lower limit of platelet count in pregnancy is 1.15 lakh/mm3. 23 Also the present study shows increase in the nucleated red blood cells and immature granulocytes with the progress of pregnancy both reaching peak in the third trimester and both these parameters are statistically significant. The immature granulocytes correlates with leucocytosis and neutrophilia which is also maximum in the third trimester. Immature granulocytes may be found in the peripheral blood of the pregnant women and is not pathological.24

Conclusion

Hemoglobin was significantly higher among second trimester and haematocrit was significantly higher in first trimesrer as compared to third trimester in both the cases. WBC was significantly higher among third trimester as compared to first. Neutrophils and immature granulocytes were found to be significant higher among second and third trimester as compared to first trimester. Lymphocytes were significantly higher among first trimester as compared to second and third. The findings of the study shall be a value addition in comparative analysis between pregnant and non pregnant females.

Reference 1.

Shen C, Jiang YM, Shi H, et al. A prospective, sequential and longitudinal study of haematological profile during normal pregnancy in Chinese women. J Obstet Gynaecol. 2010;30(4):357â&#x20AC;&#x201C;361.

Allen LH: Anemia and Iron Deficiency:effects on pregnancy outcomes. Am J clin nutr,2000;71(5):1280-84.

Meng LZ, Goldenberg RL, Cliver S, Cutter G, Blankson M; The relationship between maternal hematocrit and pregnancy outcome. Obstet Gynecol, 1991; 77: 190-94.

Osonuga IO, OsonugaOA, Onadeko AA et al;Hematological profile of pregnant women in south west of Nigeria. Asian Pac J Trop Dis, 2011;3(1):232-234.

Purohit G, Shah T, Harshoda JM. Hematological profile of normal pregnant women in western India. Sch. J. App. Med. Sci.,2015;3(6A):2195-2199.

Harrison KA. Blood Volume Changes in normal pregnant Nigerian women. The Journal of obstretics and gynecology of the British Commonwealth.1996 ;73(5):717-723.

Akinsegun A Akinbami et al. Hematological Profile of normal pregnant women in Lagos, Nigeria. Int J Womens Health. 2013;5:227-232.

Margaret R. Normal hematological changes during pregnancy and the puerperium. In:Pavord S, Hunt B (eds). The obstetric hematology manual. Cambridge University Press, Cambridge, 2010.

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

Original Article 9.

A-170

Bernstein IM, Ziegler W, Badger GJ. Plasma volume expansion in early pregnancy. J Obstet Gynecol 2001;97:669.

10. Bjorksten B, Soderstrom T, Damber M-G et al. Polymorphonuclear leucocyte function during pregnancy. Scand J Immunol. 1978;8(3):257–262.

18. Gatti L, Tinconi PM, Guarneri D et al. Hemostatic parameters and platelet activation by ﬂow-cytometry in normal pregnancy: a longitudinal study. Internat J Clin Lab Res. 1994;24(4):217–219

11. Chandra S., Tripathi AK, Mishra S et al. Physiological Changes in Hematological Parameters during Pregnancy. Indian J of Hematol Blood Transfus. 2012;28(3):144-146.

19. Jessica M, Badger F, Hseih CC et al. Plasma volume expansion in pregnancy: implications for biomarkers in population studies. Cancer Epidemiol Biomarkers. 2007;16:1720

12. Das S, Char D, Sarkar S et al. Study of Hematological Parameters In Pregnancy. Journal of Dental and Medical Sciences. 2013;12(1):42-44.

20. Kline AJ, Williams GW, Hernandez-Nino J. D-Dimer concentration in normal pregnancy: new diagnostic thresholds are needed. Clin Chem. 2005;51(5):825–829.

13. Svanberg B. Absorption of iron in pregnancy. Acta Obstet Gynecol Scand Suppl. 1975;48:1–108.

21. Shehlata N, Burrows RF, Kelton JG Gestational thrombocytopenia. Clin Obstet Gynecol. 1999;42:327–334.

14. Foulkes J, Goldie DJ. The use of ferritin to assess the need for iron supplements in pregnancy. J Obstet Gynaecol. 1982;3(1):11–16.

22. Fay RA, Hughes AO, Farron NT. Platelets in pregnancy: hyperdestruction in pregnancy. Obstet Gynecol. 1983;61(2):238–240.

15. Imam TS and Yahaya A. Packed cell volume of pregnant women attending Dawakin Kudu General Hospital,kano State Nigeria. Int for P App Scs. 2008;2(2):46 -50.

23. Sultana GS, Haque SA, Sultana T et al. Role of red cell distribution width (RDW) in the detection of iron deficiency anaemia in pregnancy within the first 20 weeks of gestation. Bangladesh Med Res Counc Bull. 2011; 37(3): 102-105.

16. Pitkin RM, Witte DL. Platelet and leucocyte count in pregnancy, JAMA 1979;242(24): 2696-2698. 17. Luppi P. How immune mechanisms are affected by pregnancy. Vaccine 2003; 21(24): 3352-3357.

24. Karalis L, Nadan S, Yemen EA. Platelet activation in pregnancy induced hypertension. Thromb Res. 2005;116(5):377–383.

*Corresponding author: Dr. Sujata Raychaudhuri, D 1101, Central Park 1, Golf Course Road, Sector 42, Gurgaon. 122009 India Phone: +91 8527393445 Email: Sujatabulu@yahoo.co.in Date of Submission : 07.10.2017 Date of Acceptance : 19.10.2017 Date of Publication : 11.11.2017

Financial or other Competing Interests: None.

http://www.pacificejournals.com/aabs

Original Article DOI: 10.21276/AABS.1733

“Laboratory Parameters in Clinically Suspected Dengue Cases in Tertiary Care Teaching Hospital in North Maharashtra” Madhuri Magan Suryawanshi*, Shubhngi C. Dange, M. N. Dravid, Sunil P. Lilani, Pooja Shah Dept. of Microbiology, Shri Bhausaheb Hire Govt. Medical College Dhule Maharashtra India

ABSTRACT Background: Dengue an arbovirus infection, has significantly increased in past decade causing increased mortality & morbidity in temperate countries including India. Earlier diagnosis helps in prompt treatment resulting in decrease in mortality & prevention of complications. In present study we have evaluated laboratory parameters for diagnosis along with different demographic profile for prevention of disease. Methods: All clinically suspected patients tested for NS1Ag & IgM by ELISA along with platelet count & Peripheral blood smear. Individual results were used in comparative analysis according to demographic (gender, age) and laboratory (platelet counts, NS1 Ag & IgM) profiles Result: Total serologically confirmed cases were 583( 21.12%) . Lower platelet counts was the most important factors in predicting dengue infection. Furthermore, all demographic and laboratory profiles presented a conservative temporal pattern throughout this long-lasting outbreak. Conclusion: As consistency throughout the epidemic facilitated defining the conservation pattern throughout the early stages, this was useful for improving management during the remaining period. Also NS1 Ag is early & reliable test.

Introduction

Keywords: Demographic Profile, Dengue Fever, NS1 Ag, IgM ELISA

Dengue an important emerging disease of tropical & sub tropical region is a Vector born disease is caused by Dengue Virus belonging to the family Flaviviridae & genus Flavivirus. Globally dengue is most important mosquito born viral disease presenting with varied clinical symptoms ranging from flue like fever to deadlier DHF & Shock. (1). Epidemiologically, globally 50 million people are infected with dengue virus every year while 2 & half billions are at risk of infection2. Similarly in India with population of more than 1 billion, it is estimated that India has largest number of dengue cases, with about 33 millions apparent & another 100 million asymptomatic infections occurring annually.3 The first epidemic of clinical dengue-like illness was recorded in Madras (now Chennai) in 1780 and the first virologically proved epidemic of DF in India occurred in Calcutta and Eastern Coast of India in 1963-1964. 4/5/6. The first major wide spread epidemics of DHF/DSS occurred in India in 1996 involving areas around Delhi 7 and Lucknow 8and then it spread to all over the country9 India has documented infections from all four serotypes. According to WHO , the incidence of dengue globally has shot up 30 folds in last 50 yrs. The cumulative dengue diseases burden has attained an unprecedented proportion in recent times with a sharp increase in the size of

population at risk. Dengue disease presents highly complex pathophysiological, economic & ecological problem10 Currently neither effective antiviral agents to treat dengue infection nor an effective vaccine are available. So main approach for prevention, control & management of dengue lays with vector control & early diagnosis of disease. In current study, we have studied different parameters like seasonal variations, gender, age along with laboratory parameters in relation with clinical symptoms which will help us to determine burden of disease in this region & preventive measures to be taken.

Materials and Methods

Study was conducted at SBH GMC, Dhule after the Ethical committee approval. Study caters to both urban population and referred rural cases during the period Jan 2016-Dec 2016 .Blood samples were collected in the plain & EDTA bulb from patients clinically diagnosed as dengue fever .The serum was separated. Cold chain was maintained during transportation of the serum samples whenever required. Data were extracted from patient’s notes collect to socio-demographic factors, clinical characteristics of presenting illness, probable diagnosis, management & outcome of disease. In laboratory investigations Platelet count, Peripheral Blood Smear for Malarial Parasite was done for all cases .Investigations like NS1 antigen ELISA, IgM antibody ELISA were done on all serum samples.

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

Original Article

A-172

Cases with Positive NS1 antigen , IgM Dengue Antibody or both were recognized as Dengue fever.( NS1 antigen ELISA(Dengue NS1Ag MICROLISA)&IgM antibody ELISA kits(NIV DEN IgM Capture ELISA Kit) were standardized and supplied by NIV Pune. Ig G antibody is not reliable marker as it is a cross reacting antibody to other flaviviruses also hence not considered in this study. 11

was diagnosed at age of 4 months with total 13 cases were detected in less than 1 yr age group. During Study period highest number of cases were noted in the month of September 210 (36%) followed by October 171(29.34%) & November 93(15.95) respectively. Sporadic cases were noted throughout year.( Figure 1) Out of 2755 serum samples tested, 583 (21.16%)were positive for either one or both serological marker. Of these 583 sample 378( 64.8%) were exclusively positive for NS1 antigen while 167( 28.6%) sample showed IgM antibody titer positivity. 38 serum samples were positive for both serological marker i.e. NS1 antigen & IgM antibody (Table 2)

Result

Clinically suspected dengue patients from Jan. 2016 to Dec 2016 were included in study. Total 2755 samples from patients were collected & tested as per protocol. Patients tested positive for malaria were excluded from study. Of these 583 were confirmed dengue cases & remaining 2172 were considered as dengue negative & appropriate control. Among the dengue positive cases,67.93% ( 396/583) were male & 32.07% (187/583) were female. (Table 1)

Among 583 sero-positive cases thrombocytopenia (Platelets <1 Lac) was evident in 299 (51.29%) while out of 2172 seronegative cases thrombocytopenia was seen in 159(7.3%) Out of the 378 NS1 positive cases, 224( 59.26%) had platelet count less than 100,000/ml while out of 167 IgM positive samples, 83(49.7%) had thrombocytopenia. (Table 3 & 4)

A substantial number of patients belonged to the age group of 16-30 yrs i.e. 325(55.75%) followed by age group 1 month -15 yrs. i.e. 156 (26.7%). Youngest case Table:1 Age, Sex & Month wise distribution of cases. Characteristic 1 month-15 yrs 16-30 yrs 31-40 yrs 41-50 yrs 51-60 yrs 61-75yrs

No. of patients Age Group 156 325 58 22 16 6 Sex

Male Female

396 187

Percentage 26.7 55.75 10 3.7 2.7 1 67.93 32.07

Month August September October November Other

50 210 171 93 59

8.57 36 29.34 15.95 10.13

Table 2: Serological marker wise distribution of cases. Dengue specific marker NS 1antigen only IgM Only NS1+ IgM

Total number of positive serum sample 378 167 38

Percentage 64.8 28.6 6.6

Table: 3 Comparison of Dengue marker & Platelet count. Dengue specific marker NS 1 Antigen only IgM Only NS1+ IgM Total

Total positive serum sample 378 1867 38 583

Platelet counts (<1,00,000) 224 83 12 299

http://www.pacificejournals.com/aabs

Percentage (%) 59.26 49.7 31.5 54.7

A-173

AABS; 4(4): 2017

Table: 4 Study of dengue cases with platelet count (n=583). Platelet range

Dengue positive cases

Dengue negative cases

Platelet counts less than <1,00,000/cmm

299

159

Platelet counts more than >1,00,000/cmm

284

2013

Total

583

2172

Fig. Line Diagram showing month wise break down of cases.

Discussion

Dengue is an important emerging disease of the tropical & sub tropical regions today. India is one of the seven countries in the South East Asia, endemic for DF & Dengue Hemorrhagic Fever & may soon become a major niche for dengue infection. 12 In present study, maximum numbers of patients suffering from dengue were in age group of16-30 yrs. This finding is in accordance with study done by Doke et al in Maharashtra13 Mohamed Murtuza Kauser et al 14 from Central Karnataka, Kumar et al from Udupi Karnataka12Hemant Kumar et al from Surat 15. Though maximum positive cases belonged to young adult group who are exposed to more outdoor activities, extreme age variations were observed. Youngest patient in present study was 4 months old while oldest was 72 yrs in age. The youngest patient reported by Jonathan G. Lim et al in a study done in 2000-04 was 4 months old 16. M.J. Kulkarni et al reported 6 cases of newborn admitted for dengue 17 evidently showing all ages are more or less susceptible for dengue infection.

from Malayasia16.Male to female ratio in a study by Mohan Kashikanti et al in 2013 found a ratio of 1.2:119 Majority of patients (92.03%) were found to be males and females constituted7.96% by a study done by Hemant Kumar et al15 A. Abrol et al reported male to female ratio of 1:1 from a study in Chandigarh20 The present study shows that outbreak occurred from August to December. Dengue outbreak in India have generally occurred between August and November21 M.J. Kulkarni et al in their study of Dengue cases in a tertiary care center in Jaipur reported cases from September to November17 Mohan D.K. reported most of the cases during the month of June to September19 A study of 766 patients of Dengue in 2011-2013 in A.J. Institute of Medical Sciences and Research Center, Mangalore, Karnataka, reported cases throughout the year with peak admission in April followed by May and June. 15

Further majority of patients were male (396) while female constitute rest (187) thus male to female ratio is 2.1: 1. M.J. Kulkarni in 2010 found two third patients to be males17 In contrast to this, a study reported by CV Prathyusa et al from Andhra Pradesh state showed almost equal distribution of male and female ratio in 201218. Equal sex distributions was reported by study of Jonathan G. Lin et al

Before the advent of NS1 Ag testing, demonstration of dengue specific IgM /Ig G antibodies was mainstay for diagnosis of dengue infection since viral culture or detection of viral genome by molecular method is not feasible at grass root level. As antibodies begin to appear on 5th day of fever in primary & 3rd day in secondary infection, diagnosis is delayed due to lag period in this test. In contrast NS1 Ag is detectable in blood from day 1 of fever & can be seen up to 9 day even when viral RNA detection is negative by RT PCR. 22

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

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

Original Article

A-174

Out of total 583 sero-positive cases, 378(64.8%) were positive for NS1 Ag, 167(28.6%) for IgM Antibody & 38(6.6%) for both. Similar studies by Santosh Tathe et al 23 shows NS1 Positivity in 88.23 % , IgM 68.75% & both NS1+ IgM 100% respectively. Also Sindhanai V et al documented 86.5% NS 1 positivity with 44.4.% positivity for IgM. 24 While most studies shows more NS1 positivity compared to IgM, study by Mohite et al 25 This study also tried to find the association of thrombocytopenia with dengue parameter. Among 583 seropositive dengue cases thrombocytopenia was detected in 299(51.3%) while only 159(7.3%) cases of 2172 were thrombocytopenic, signaling importance of thrombocytopenia as precious clue for diagnosis. Out of 378 NS1 positive cases, 224(59.26%) showed thrombocytopenia while 83(49.7%) of exclusively IgM positive cases were thrombocytopenic. This difference was statistically significant using Z test. This is in accordance with study by Tanvi Panwala et al 26, Kulkarni et al17. It also signifies importance of doing NS1 Ag detection test in all febrile patients with thrombocytopenia.

Conclusion

The present study shows that dengue infection is more common in young adult age group favoring males as this group more involved in outdoor activities, which exposes them to mosquitoes. Since monsoon in our region in not heavy & has intermittent showers, peak of case is seen during mid & end of monsoon. For all febrile patients with thrombocytopenia, Dengue NS1 Ag, IgM antibodies detection should be done as low platelet is important but not exclusive test for this infectious disease.

Acknowledgements

We are thankful to Mrs. Suhasini Gavit for her technical support. We also extend thanks to authors /editors/ publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

Reference 1.

WHO Classification of Dengue cases D Gupta-sapir & B. Schimmer, “ Dengue fever: New paradigms for changing epidemiology”. Emerging Themes in Epidemiology, Vol 2, article 1, 2005.

Bhatt S, Gething PW, Brady OJ, et al. The global distribution & burden of dengue . Nature 2013; 496: 504-507

SarkarJK, Chatterjee SN, Chakravarty SK. Haemorrhagic Fever in Calcutta: some epidemiological observations. Indian J Med Res 1964; 52 : 651-9.

Chatterjee SN, Chakravarti SK, Mitra AC, Sarkar JK. Virological investigation of cases with neurological

complications during the outbreak of haemorrhagic fever in Calcutta. J Indian Med Assoc 1965; 45 : 314-6. 5.

Carey D7. E, Myers RM, Reuben R, Rodrigues FM. Studies on dengue in Vellore, South India. Am J Trop Med Hyg1966; 15 : 580-7.

DarL, Broor S, Sengupta S, Xess I, Seth P. The first major outbreak of dengue hemorrhagic fever in Delhi, India. Emerg Infect Dis 1999; 5 : 589-90.

Agarwal R, Kapoor S, Nagar R, Misra A, Tandon R, Mathur A, et al. A clinical study of the patients with dengue hemorrhagic fever during the epidemic of 1996 at Lucknow, India. Southeast Asian J Trop Med Public Health 1999; 30 : 735-40.

Shah I, Deshpande GC,TardejaPN. Outbreak of dengue in Mumbai and predictive markers for dengue shock syndrome. J Trop Pediatr2004; 50 : 301-5.

Gubler DJ. The economic burden of dengue . Am. J. Trop. Med. Hyg. 2012; 86; 743-744

10. Shrivatavaa, Dash PK, Tripathi NK, Sahni AK et al” Evaluation of a commercial dengue NS1enzyme linked immune-sorbent assay for early diagnosis of dengue infection. Indian J. Med. Microbiology 2011; 29:51-5)\ 11. Kumar A, Rao CR, Pandit V, Shetty S, Bammigatti C, Samarasinghe CM. Clinical Manifestations & Trend of dengue cases admitted in a Tertiary Care Hospital Udupi District, Karnataka. Indian Journal of Community Medicine: Official Publication of Indian Association of Preventive & Social Medicine. 2010;35(3):386-90). 12. Prakash Doke, Satish Pawar: Profile of dengue fever outbreak in Maharashtra; Indian Journal of Community Medicine 2000; Vol 25, No 4 (2000-10-2000-12) 13. Mohamed Murtuza Kauser, Kalavathi GP, Mehul Radadiya, Karthik M, Asfiya Afreen, Kumarswamy R. C. : A study of clinical & LaboratoryProfile of Dengue fever in Tertiary Care Hospital in Central Karnataka , India. Global Journal of Medical Research(B) volume XIV Issue V Version I Yr 2014 Page 7-12 14. Hemant Kumar, Saba Mohmmed Mansoor : A study of Clinico- Demographic profile of Dengue cases in A Teaching Hospital. National Journal of Community Medicine 2015/13557:2038) 15. Jonathan G. Lim, Salvacion R, Gatchalian Ma, Rosario Z. Capeding. Profile of Pediatric patients with DF/ DHF over a Five Year Period( 2000-04). Pediatric Infectious Disease Society of Philippines Journal (2010) 11 (1) 26-34) 16. Manjunath J. Kulkarni, Vijayasarathi, Vikas Bhalla, Deepak Shivpuri, Usha Archarya. Clinico-Epidemiological profile of Children Hospitalized with Dengue. Indian Journal of Pediatr(2010) 77: 1103-1107. 17. C.V.Prathyusha, M.SrinivasaRao, P.Sudarsini and K.UmamaheswaraRao. Clinico-haematological profile and outcome of dengue fever in children. Int.J.Curr. Microbiol. App.Sci 2013; 2(10): 338-346

http://www.pacificejournals.com/aabs

A-175 18. Mohan D Kashinkunti, Shiddappa, Dhananjaya M. A Study of Clinical Profile of Dengue Fever in a Tertiary Care Teaching Hospital. Sch. J. App. Med. Sci., 2013; 1(4):280-282. 19. A Abrol, ADewan, N Agarwal, A Galhotra, N Goel, H Swami. A Clinico-Epidemiological Profile of Dengue Fever Cases in a Peri-Urban Area of Chandigarh. The Internet Journal of Epidemiology. 2006 Volume 5 Number 1) 20. V K Singh, J M Haria, S K Jain. Hospital Based Study of Dengue Hemorrhagic Fever in Western Uttar Pradesh Region.International Journal of Scientific Study. (2014) 1 (5): 32-34. 21. Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M. Enzymeâ&#x20AC;&#x2018;linked immunosorbent assay specific to dengue virus type 1nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol 2002;40:376â&#x20AC;&#x2018;81. 22. Santosh Shivajirao Tathe, Chincholkar VV, Kulkarni DM, Nilekar SL, Ovhal RS & Halgarkar CS: A study of NS1

AABS; 4(4): 2017 antigen & Platelet count for early diagnosis of dengue infection. Int. J. Curr. Microbiology & Applied science. Vol 2 Number 12 (2013) PP40-44 23. Sindhanai V, Sageera Bano, Rajkumar N, Suresh Chander VC: Evaluation of correlation between dengue serological markers & Platelet count. Scholars journal of applied medical sciences,2016; 4{2D}: 618-622 24. Mohite Ravindra, Karamje C Nisha, Shubhangi A Gadgil, Inamdar Dhanashree, Jadhav Pradnya: Association of IgG, IgM antibodied , N1 antigen & Platelet count in the diagnosis of Dengue Virus Infection in patients attending Bharati Vidyapeeth Deemed university Medical College & Hospital , sangali. International Journal of contemporary Medical research. Vol 3, issue 10 oct 2016 page no.2942-43. 25. Tanvi H Panawala, Summaiya A Mulla: Evaluation of two diagnostic methods for dengue virus infection & its correlation with thrombocytopenia. International Journal of Health & Allied Sciences Vol 5 Issue 2 Apr-Jun2016 page no88-91.

*Corresponding author: Dr. Madhuri M. Suryawanshi, 23, Sadguru Colony, Nr. Dollys House Deopur Dhule, Maharashtra India. Phone: +91 9422755260, 8087482679 Email: dr.madhurijk@gmail.com

Financial or other Competing Interests: None.

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

Date of Submission : 01.11.2017 Date of Acceptance : 12.11.2017 Date of Publication : 17.11.2017

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

Original Article DOI: 10.21276/AABS.1736

An Integrated Approach to Detection of Mycobacterium Tuberculosis Using BD MGIT 320 System and other Diagnostic Modalities Gargi Choudhury*, Partha Pratim Das, Lahari Saikia Department of Microbiology, Assam Medical College, Dibrugarh, India

ABSTRACT Background: Application of automated liquid culture system like Becton, Dickinson and Company (BD) MGIT 320 can shorten the recovery time of Mycobacteria in clinical diagnostic laboratories as compared to Lowenstein Jensen (LJ) medium. The present study aimed to compare the results of BD MGIT 320 culture system with that of LJ medium and also to evaluate the usefulness of molecular methods as an adjunct. Methods: Specimen from clinically suspected cases of tuberculosis were processed as per standard guidelines for smear microscopy, solid culture in LJ media, liquid culture in BD MGIT 320 system and Line probe assay. Polymerase chain reaction was performed on extracted DNA from MGIT positive specimen to detect IS6110 and MPB64 gene targets. Result: Out of total 83 specimens, a statistically significant higher isolation rate (25.3%, n=21) was shown by MGIT 320 system in comparison to LJ medium (12.04%, n=10). Line Probe Assay was positive in 37.3% (n=31) and direct microscopy was positive in only 13.25% (n=11) specimen. The average duration of detection was found to be more rapid by MGIT320 than LJ culture (18 days vs 24.1 days). Among the 21 MGIT positive specimens 7 were found positive for both IS6110 and MPB64 gene while 7 specimens were found positive only for IS6110. Conclusion: The automated liquid culture systems like BD MGIT 320 have a higher isolation rate and are more rapid as compared to solid media. But adoption of Line probe assay combined with MGIT culture will definitely yield rapid and efficient detection of Mycobacteria. Keywords: Liquid Culture, Multi Drug Resistant Tuberculosis, Extrapulmonary Tuberculosis, Line Probe Assay.

Introduction

The increasing incidence of tuberculosis (TB) and emergence of multidrug resistant tuberculosis (MDRTB) has made it essential for health care laboratories to detect Mycobacteria rapidly along with drug susceptibility pattern. In 2015, the world had an estimated 10.4 million new TB cases; India recorded an incidence of 2.84 million with 0.13 million drug resistant TB cases.[1] As conventional culture methods are cumbersome and need longer incubation, adoption of automated culture system like BD (Becton, Dickinson and Company) MGIT (Mycobacterium Growth Indicator Tube) 320 liquid culture system can shorten the recovery time of Mycobacteria.[2] The MGIT TBc identification test based on detection of MPT64, a protein secreted by Mycobacterium tuberculosis (MTb) complex can detect MTb complex in 15 minutes from positive MGIT cultures. Liquid culture shortens the time of detection in comparison to solid culture but it is still not a rapid test as labor intensive biochemical methods are required for species identification and it takes longer incubation for smear-negative specimens.[3,4,5] In recent times, laboratories are adopting nucleic acid amplification (NAA) diagnostic technologies owing to their rapidity

and sensitivity. Methods like Line probe assay (LPA) or Polymerase Chain Reaction (PCR) assay as adjunct to detect MTb complex from clinical specimen can overcome the limitations of liquid culture. The present study aimed to compare the results of BD MGIT 320 culture system with that of Lowenstein-Jensen (LJ) medium for recovery rates and the mean time required to detect Mycobacteria from clinical specimens. Our study also aimed to evaluate the usefulness of molecular methods as an adjunct for rapid detection.

Materials and Methods

The study involves specimens from clinically suspected cases of tuberculosis (both pulmonary and extrapulmonary) received in Mycobacteriology laboratory for detection of Mycobacteria during a period of 18 months from September, 2015 to February, 2017. Specimen Collection and Processing: A total number of 83 clinical specimens were collected after obtaining prior consent from the patients and processed in bio-safety level (BSL)-3 laboratories in the Department of Microbiology. Specimens were digested and decontaminated with an equal volume of N-acetyl- L-cysteine (NALC)â&#x20AC;&#x201C;NaOH (final

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

A-177 concentration 2% NaOH and 0.5% NALC) for 15 minutes at room temperature and then neutralized with sterile 0.067 M phosphate buffer (pH 6.8).[6] After centrifugation at 3000g for 15 minutes, the sediment was re-suspended in 2.0 mL of phosphate buffer. An aliquot of the suspension was used for Ziehl Neelsen (ZN) stain to detect acid-fast bacilli (AFB); 0.5 mL of the suspension was inoculated into a BD MGIT 320 tube, and another 0.2 mL was inoculated onto one Lowenstein-Jensen (LJ) slant (Himedia lab, Mumbai). Another aliquot of the suspension was used for molecular identification by HAINS MTBDRplus Line Probe Assay as well as conventional PCR assay for IS6110 and MPB64 gene targets. LJ Culture: After inoculation into liquid media, 0.2 ml of each processed specimen was also inoculated onto the surface of an LJ slant procured from Himedia Laboratory Pvt. Ltd, Mumbai and the cultures were incubated at 37 degree for 8 weeks. The LJ medium was examined on alternate days from day 2 to day 7 and once a week from day 8 to day 56 according to established laboratory procedures.[7] BD MGIT 320 Culture: [8] Principle: MGIT tube contains Middlebrook 7H9 liquid media and an oxygen quenched fluorochrome embedded in silicon at the bottom of the tube. During bacterial growth, the free oxygen is utilized and replaced with carbon-dioxide. With the depletion of free oxygen the fluorochrome is no longer inhibited resulting in fluorescence within the MGIT tube when visualized under UV light. Instrument declares a tube negative if it remains negative for six weeks (42 days). Inoculation of MGIT medium: After labeling MGIT tube with specimen number, 0.8 mL of Growth Supplement/ MGIT PANTA (polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) antibiotic mixture was added just prior to inoculation of 0.5 mL of the suspension into a BD MGIT 320 tube. After proper mixing, tubes were entered into the instrument and allowed for automatic testing for the duration of the recommended 42 days. Positive MGIT tubes were processed for the MGIT TBc identification test; positive result of which confirmed growth of Mtb complex. Line Probe Assay (LPA): The GenoType MTBDRplus VER 2.0 (Hain Lifesciences GmBH, Nehren, Germany) is a commercially available line probe assay based on multiplex PCR combined with reverse hybridization on nitrocellulose strips, targeting common mutations and was performed according to the manufacturer’s instructions [9]. This assay identifies Rifampicin (RIF) and Isoniazid (INH) resistance by detecting the most common mutations of the rpoB gene and the katG and inhA genes, respectively, and Annals of Applied Bio-Sciences, Vol. 4; Issue 4: 2017

AABS; 4(4): 2017 can be used on direct specimens as well as culture isolates. Mycobacterial DNA was extracted in BSL-3 laboratory according to manufacturer’s instructions.[9] Conventional PCR : Multiplex PCR assay was performed for identification of M. tuberculosis using specific pair of primers designed to amplify IS6110 and MPB64 gene in the Mtb complex and the expected band size was 123bp for IS6110 and 240 bp for MPB64.[10] The sequences of primer used were IS6110F: 5’-CCTGCGAGCGTAGGCGT-3’, IS6110R: 5’-CTCGTCCAGCGCCGCTTCGG-3’, MPB64F: 5’-TCCGCTGCCAGTCGTCTTCC-3’, MPB64R: 5’- GTCCTCGCGAGTCTAGGCCA-3’ .[10] Briefly, following components were added to a sterile eppendorf tube (for 20 µL reaction volume)- 10 µL universal PCR mix 2X (Promega), 2 µL of each of the forward and reverse primers (10pm/ µL), 1 µL of template and rest nuclease free water. The positive control included was the DNA of M. tuberculosis H37Rv strain and negative control was the PCR grade water. DNA amplification was performed for 40 cycles following an initial denaturation step at 95o C for 5 minutes, denaturation at 94 o C for 1 minute, annealing at 65 o C for 1.5 minutes, extension at 72 o C for 1.5 minutes and final extension at 72 o C for 10 minutes followed by storage at 4o Celsius until detection. The amplified products were then run on 1.5% agarose gel electrophoresis stained with ethidium bromide using 100bp ladder followed by detection of target band using gel documentation system (BIORAD) (Fig 1). Quality control of all the methods was performed using M. tuberculosis, H37Rv strain.

Result

Out of 83 clinical specimens (pulmonary =38, extrapulmonary= 45) processed, a total of 21 (25.3%) were found positive for MTb complex by the BD MGIT 320 culture systems, confirmed by MGIT TBc identification test (pulmonary =8, extra-pulmonary= 13) (Table 1). Conventional culture in LJ medium showed growth of Mycobacteria in 10 (12.04%) number of specimen (pulmonary =3, extra-pulmonary= 7). Initially HAINS Genotype MTBDRplus (LPA) was positive in 31 (37.3%) number of specimen while direct microscopy (ZN Stain) for acid fast bacilli was positive only in 11 (13.25%) numbers. Among the MGIT positive specimens, sputum and pus shared the top of the list (5 nos. each) followed by 4 nos. of tissue specimen (Table 1). All MGIT positive specimens (except one pleural fluid) were initially found positive for MTb complex by LPA, of which only 8 specimens were positive on direct microscopy. The average duration of MGIT positivity was e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-178

found to be 18 days while LJ media showed an average duration of 24.1 days for positive growth (Table 2). In case of pulmonary specimens, the mean recovery time was 17.3 days whereas in extra-pulmonary specimens it was found to be 18.9 days by the BD MGIT 320 TB system in the present study. Multiplex PCR of all MGIT positive specimens showed positive result for both IS6110 and MPB64 gene in 7

numbers, negative for both the targets in another 7, while 7 specimens were found positive only for IS6110 (Table 2). Drug Susceptibility: Results of LPA positive specimens showed that 12 were susceptible for both Rifampicin (RIF) and Isoniazid (INH), 3 were indeterminate and 3 were resistant to both the drugs while one specimen was susceptible to Rifampicin and indeterminate to Isoniazid; yet another was Isoniazid monoresistant.

Table 1: Distribution of clinical specimen and result of different assay. No. of sample processed (n=83) 18 17 10 10 5 7 5 10 1

Sample type Sputum Pus BAL Pleural fluid Ascitic fluid Urine CSF Tissue Bone marrow

ZN stain result (n=11) 2 5 0 0 0 3 0 1 0

MTBDR plus (LPA) Positive (n=31) 7 8 4 2 1 3 2 4 0

MGIT culture Positive (n=21) 5 5 2 1 0 2 2 4 0

LJ Culture Positive (n=10) 2 3 0 1 0 0 0 4 0

Table 2: Results of other diagnostic methods in MGIT positive cases. Patient ID Age Gender 106 107 111 145 157 158 167

54 54 20 30 25 25 18

Female Female Male Female Female Female Female

178

Male

185 192 219 224 226 227 245 320 339 357 367 482 531

28 10 35 23 65 25 28 28 11 24 24 7 11

Female Female Female Male Male Male Male Male Female Male Male Male Male

Direct Line Probe MGIT Time to LJ Time PCR result Type of Microscopy Assay culture MGIT Culture to LJ Specimen (AFB) result result result Positivity Result Positivity IS6110 MPB64 Pus Neg Pos Pos 12 days Neg Pos Pos Tissue Neg Pos Pos 15 days Pos 21 days Neg Neg Sputum Neg Pos Pos 13 days Neg Neg Neg Pus Pos Pos Pos 14 days Neg Pos Pos Sputum Pos Pos Pos 13 days Neg Pos Neg Sputum Neg Pos Pos 21 days Neg Pos Neg Pus Pos Pos Pos 14 days Pos 23 days Pos Neg Pleural Neg Neg Pos 15 days Pos 18 days Pos Neg Fluid Urine Pos Pos Pos 16 days Neg Pos Neg Pus Pos Pos Pos 34 days Pos 15 days Neg Neg Urine Pos Pos Pos 18 days Neg Pos Pos Tissue Pos Pos Pos 17 days Pos 24 days Neg Neg BAL Neg Pos Pos 22 days Neg Neg Neg BAL Neg Pos Pos 22 days Neg Neg Neg Sputum Neg Pos Pos 18 days Pos 24 days Pos Pos Sputum Pos Pos Pos 15 days Pos 27 days Pos Pos Pus Neg Pos Pos 30 days Pos 18 days Neg Neg CSF Neg Pos Pos 20 days Neg Pos Neg Tissue Neg Pos Pos 27 days Pos 31 days Pos Pos Tissue Neg Pos Pos 14 days Pos 40 days Pos Pos CSF Neg Pos Pos 15 days Neg Pos Neg

Foot Note: Pos- Positive; Neg- Negative http://www.pacificejournals.com/aabs

A-179

AABS; 4(4): 2017 statistically significant (p value= <0.05 using Chi square test). The obtained results are in agreement with those reported by Hanna et al, Tortoli et al which showed that the MGIT system had a recovery rate higher than in solid media.[3,12] Dongsi and Dunne also found that the MGIT culture system consistently gave better isolation rates of all Mycobacterium species from a variety of clinical specimens than the traditional L.J. slants.[13] Rishi S et al reported that isolation rate in MGIT system was higher as compared to solid media (50.6% vs 33.6%) so as Rodrigues et al (41% vs 24%).[14,15] In addition, Bunger R et al also reported that the highest mycobacterial recovery rate was by MGIT (91.6%) as compared to LJ media (58.3%).[16]

Fig. 1: Multiplex PCR for IS6110 and MPB64 gene; Starting from left: Lane 1- 100 bp ladder; Lane 2,4,5 - PCR negative specimen; Lane 3- specimen positive for IS6110 (123 bp) and MPB64 (240 bp); Lane 6- positive control (H37Rv strain), Lane 7- Negative control.

Discussion

The increased incidence of tuberculosis worldwide has made rapid and efficient culture strategies very essential which can easily be adopted in a clinical Mycobacteriology laboratory. Solid egg based medium like LJ medium is the current “gold standard” culture method for detection of Mycobacteria [6,11]. One of the culture methods, MGIT liquid culture system is easy to handle, non-radiometric, and at present does not need costly instrumentation. The BD MGIT 320 culture system contains only one drawer, holds 320 MGIT tubes and can be placed on a bench top. It is designed for laboratories with limited space and workload. MGIT 320 can be used by a laboratory that cultures up to 40 Mycobacteriology specimens per week, with an incubation period of 6 weeks, and performs up to five drug susceptibility tests per week. [2]

The present study revealed that the mean time to detection of M. tuberculosis complex were 18 days by BD MGIT system and 24.1 days by solid media which is in agreement with an earlier study by Hanna et al (14.4 days by MGIT vs. 24.1 days for solid media).[3] The average time to detection of mycobacterial growth according to the smear positivity was noted to be shorter by MGIT culture in our study (15.3 days for AFB positive and 18.7 days for AFB negative specimen) compared to solid media (22.2 days for AFB positive and 25.3 days for AFB negative specimen). This is further supported by Rishi et al, Bunger et al and Chihota et al.[14,16,17] One smear positive pus specimen took 34 days to detection by MGIT system while another smear negative pus specimen flagged positive on day 30. Both the isolates were found to be MDRTB by LPA. Thus, in this study MGIT 320 was found to be more rapid and efficient method than the conventional LJ media. Though culture contamination is often encountered in most of the laboratories, meticulous adherence to the manufacturer’s manual and protocols, and rapid transport and/or refrigeration of samples could overcome any such problem in our study.

Our study compared BD MGIT 320 system with LJ culture method for Mycobacteria and evaluated following important parameters - the rate of recovery and mean time to detection. Combined use of liquid and solid media is essential in good laboratory practice for successful isolation of mycobacterium which we followed in our study. The present study showed higher isolation rate in BD MGIT 320 system than LJ culture (25.3% vs 12.04%) and was found

NAA based diagnostic techniques have the potential to increase the sensitivity as well as to considerably reduce the detection time.[18] Most studies have used IS6110 as a target for PCR-based diagnosis of tuberculosis with variable degrees of success.[19] However, this insertion element is absent in a proportion of Mtb isolates which limits its use as a sole target for the detection of tuberculosis in India. [20,21] Use of two or more gene targets for amplification has been demonstrated to increase the diagnostic yield of MTB infection in some earlier studies.[22,23] We evaluated the role of PCR using two different targets IS6110 and MPB64 specific for MTb complex in MGIT positive cases and found presence of IS6110 in 66.6% isolates while both IS6110 and MPB64 in 33.3% and none of the targets amplified in the rest.

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

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

Original Article

A-180

Limitation of The Study Though we detected the susceptibility to Rifampicin and Isoniazid using LPA in all cases, we performed MGIT DST only in those isolates which were found resistant/ indeterminate by LPA. Comparison of DST result by both the methods would have been more useful which we intend to follow in future studies. Also due to limited number of cases in the present study it is too early to comment on the usefulness of PCR assay using multiple targets.

Conclusion

Hence, we conclude that the automated culture systems like BD MGIT 320 have a higher isolation rate as compared to solid media (LJ). But adoption of Line probe assay combined with MGIT culture will definitely yield rapid and efficient detection of Mycobacterium as well as multidrug resistant TB cases.

Acknowledgements

We thank Mr Lakhi Gogoi, Mr. Dipankar Borah, Department of Microbiology, Assam Medical College, Dibrugarh and Mr. Manash P Borah, Multidisciplinary Research Unit, Assam Medical College, Dibrugarh for their technical assistance. We are grateful to the Department of Biotechnology, Govt. of India for providing us the research platform.

Reference 1.

SEARO WHO, New Delhi, India; 2017 [cited 2017 Sep 25]. Available from: https://www.searo.who.int/topics/ tuberculosis

Duque A, Lin SY, Desmond E, Rienthong S, Rienthong D, Boonin C.Evaluation of the BD Bactec MGIT 320 for detection of Mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis. J. Clin. Microbiol 2013;51:3403– 3405. http://dx.doi.org/10.1128/JCM.01357-13

Hanna B A, Ebrahimzadeh A, Elliott L B, Morgan M A, Novac S M, Gerdes S R et al. Multicenter evaluation of the BACTEC MGIT 960 system for recovery of mycobacteria. J Clin Microbiol 1999; 37: 748–752. https://www.ncbi.nlm. nih.gov/pmc/articles/PMC84542

Somoskovi A, Kodmon C, Lantos A, Bartfai Z, Tamasi L, Fuzy J et al. Comparison of recoveries of Mycobacterium tuberculosisusing the automated BACTEC MGIT 960 system, the BACTEC 460 TB system, and LowensteinJensen medium [Internet]. J Clin Microbiol. 2000 Jun;38(6):2395-2397 [cited 2017 Sep 26] https://www.ncbi. nlm.nih.gov/pmc/articles/PMC86818 Dowdy D, Lourenço M, Cavalcante S, Saraceni V, King B, Golub J et al. Impact and Cost-Effectiveness of Culture for Diagnosis of Tuberculosis in HIV-Infected Brazilian Adults. PLoS ONE [Internet]. 2008; 3(12):e4057 [cited 26 September 2017] https://doi.org/10.1371/journal.pone.0004057

Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA: U.S. Dept. of Health and Human Services, Public Health Service, Centers for Disease Control; 1988.

7. Revised National TB Control Programme. Manual of standard operating procedures for culture of Mycobacterium tuberculosis and drug susceptibility testing on solid medium [Internet], Version No. 01.01, 2009. [cited 16 September 2017] http://tbcindia.nic.in/ WriteReadData/l892s/6995271860Training manual M tuberculosis C DST.pdf 8.

Siddiqi, S. H., & Rüsch-Gerdes, S. For BACTEC™ MGIT 960™ TB System [Internet] 2006. [cited 16 September 2017] https://www.finddx.org/wp-content/uploads/2016/02/ mgit_manual_nov2006.pdf

Hain Lifescience, GmbH, Nehren. Germany. Genotype MTBDRplus TM, version 2.0. Instruction manual [Internet] [cited 2017Sep26] http://www.hainlifescience.com/pdf

10. Sharma K, Sinha S, Sharma A, Prasad K, Rana S, Sharma M, et al. Multiplex PCR for rapid diagnosis of gastrointestinal tuberculosis. [Internet] J Glob Infect Dis 2013;5(2):49-53 [cited 2017 Sep 27] https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC3703210 11. Nolte, F. S., B. Metchock. Mycobacterium. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken, ed. by. Manual of clinical microbiology. 6th ed. ASM Press, Washington, D.C.; 1995. p. 400–437. 12. Tortoli E, Cichero P, Piersimoni C, Simonetti T, Gesu G, Nistta D. Use of BACTEC MGIT for recovery of mycobacteria from clinical specimens: multicentric study. J Clin Microbiol. 1999;37: 3578- 3582. http://jcm.asm.org/ content/37/11/3578.full.pdf 13. Dongsi L, Bobby H, Dunne W M. Comparison of the automated mycobacteria growth indicator tube system (BACTEC MGIT 960) with Lowenstein Jensen medium for recovery of mycobacteria from clinical specimens. Microbiol Infect Dis. 2002; 118:542-545. https://academic.oup.com/ ajcp/article-pdf/118/4/542/4984895/ajcpath118-0542.pdf 14. Rishi S, Sinha P, Malhotra B, Pal N. A Comparative study for the detection of mycobacteria by BACTEC MGIT 960, Lowenstein Jensen media and direct AFB smear examination. Ind J Med Microbiol. 2007 Oct; 25(4):383386. https://www.ncbi.nlm.nih.gov/pubmed/18087090 15. Rodrigues C, Shenai S, Sadani M, Sukhadia N, Jani M, Ajbani K et al. Evaluation of the bactec MGIT 960 TB system for recovery and identification of Mycobacterium tuberculosis complex in a high volume tertiary care centre. Indian J Med Microbiol [serial online] 2009; 27:217-221. [cited 2017 Aug 28] http://www.ijmm.org/text.asp?2009/27/3/217/53203 16. Bunger R, Singh VA, Avneet, Mehta S, Pathania D. Evaluation of BACTEC Micro MGIT with Lowenstein Jensen media for detection of Mycobacteria in clinically suspected patients of extra pulmonary tuberculosis in a tertiary care hospital at Mullana (Ambala). J Med Microb

http://www.pacificejournals.com/aabs

A-181 Diagn. 2013,2:2. https://www.omicsonline.org/evaluationof-bactec-micro-mgit-with-lowenstein-jensen-media-fordetection-of-mycobacteria-in-clinically-suspected-patientsof-extra-pulmonary-tuberculosis-2161-0703.1000123. php?aid=12037 17. Chihota VN, Grant AD, Fielding K, Ndibongo B, Zyl AV, Muirhead D et al. Liquid versus solid culture for tuberculosis: performance and cost in a resource-constrained setting. Int J Tuberc Lung Dis. 2010;14(8):1024-1031. https://www.ncbi. nlm.nih.gov/pubmed/20626948 18. Horner PJ, Moss FM: Tuberculosis in HIV infection. Int J STD AIDS 1991, 2(3):162-167. https://www.ncbi.nlm.nih. gov/pubmed/1863645. 19. Jatana S. K., Nair M. N., Lahiri K. K., Sarin N. P. Polymerase chain reaction in the diagnosis of tuberculosis. Indian Pediatr. 2000; 37, 375–382. http://indianpediatrics. net/april2000/april-375-382.htm 20. Narayanan S, Parandaman V, Narayanan P, Venkatesan P, Girish C, Mahadevan S, Rajajee S. Evaluation of PCR

AABS; 4(4): 2017 using TRC4 and IS6110 primers in detection of tuberculous meningitis. J Clin Microbiol. 2001; 39, 2006–2008. https:// www.researchgate.net/publication/12010310 21. Radhakrishnan I, Manju Y K, Kumar R A & Mundayoor S. Implications of low frequency of IS6110 in fingerprinting field isolates of Mycobacterium tuberculosis from Kerala, India. J ClinMicrobiol. 2001 ; 39, 1683. pubmedcentralcanada.ca/ pmcc/articles/PMC88004/pdf/jm001683.pdf 22. Bhigjee AI, Padayachee R, Paruk H, Hallwirth-Pillay KD, Marais S, Connoly C. Diagnosis of tuberculous meningitis: Clinical and laboratory parameters. Int J Infect Dis. 2007;11(4):348–354. http://www.ijidonline.com/ article/S1201-9712(06)00190-1/fulltext 23. Rafi W, Venkataswamy MM, Ravi V, Chandramuki A. Rapid diagnosis of tuberculous meningitis: A comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen. J Neurol Sci. 2007;252:163–168. http://www. jns-journal.com/article/S0022-510X(06)00529-6/fulltext

*Corresponding author: Dr. Gargi Choudhury, Assistant Professor, Department of Microbiology, Assam Medical College, Dibrugarh, Assam, India, Pin-786002 Phone: +91 9435284089 Email: gargicb@gmail.com Date of Submission : 03.11.2017 Date of Acceptance : 12.11.2017 Financial or other Competing Interests: None. Date of Publication : 17.11.2017

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

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

Original Article DOI: 10.21276/AABS.1770

Transfusion Transmitted Diseases Among Blood Donors in Tertiary Care Teaching Hospital of Central India Ashok Yadav and Ravi Jain* Dept. 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. Among all infections HIV and hepatitis are the most dreadful. Aims & Objectives: To find out the Seroprevalence of transfusion-transmissible infections in blood donors, to find the incidence of spectrum of diseases in blood bank donation, to find the age 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 was conducted, during the period 2001 –2016. Tests are routinely done on every blood unit to exclude HIV, HBV, HCV, syphilis and malaria. The screening for HIV was done by ELISA using kits. HBS Ag was detected by ELISA. Anti-HCV test was done by ELISA& Syphilis by VDRL. Results; In the present study, 241571 blood donors were observed in the year 2001-16, majority of donors are voluntary donors 68.13 % as compared to replacement/relative donors 31.86 %. Majority of donors are male donors 96.25 % as compared to female donors 3.74%. Seroprevalence of HBV,HCV, HIV& SYPHILIS are 1.80 %, 0.098%, 0.20% and 0.26 % respectively. Seroprevalence is higher in the age group 26-35 years. Overall seropositivity of TTI’s (HIV,HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.12 % as compared to voluntary donors2.01 %. Over all Seroprevalence of transfusion transmitted disease in all donations in the year 2001-16 is 2.36 %. Conclusion -Voluntary blood donation should be encouraged for prevention of transfusion-transmissible diseases. Keywords: Hepatitis B, Hepatitis C, Transfusion Transmitted Diseases, Voluntary Donors, Syphilis, 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. By this study, we intend to find out the seroprevalence of Transfusion transmitted diseases amongst blood donors & age distribution of Transfusion transmitted diseases amongst blood donors. Infectious Agents1 There are four main groups of microorganisms 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. Viruses Viruses are the simplest forms of life. Following are some of the viruses which are known to be transmitted through blood2,3:

1. Human immunodeficiency virus (HIV) 2. Hepatitis B virus 3. Hepatitis C virus 4. Hepatitis A virus 5. Hepatitis G virus 6. Non - A, Non - B Hepatitis 7. Epstein Barr Virus 8. Cytomegalo virus (CMV) 9. Human T Lymphocytic virus (HTLV - 1 & HTLV - 2) SyphilisSyphilis, an ancient diseaseis caused by Spirochete Treponema pallidum. According toThe World Health organization estimates4, there are approximate 12 million new cases diagnosed each year. Syphilis has acquired new potential for morbidity with the advent of HIV & AIDS. Syphilis is a chronic disease caused by Treponema pallidum. Treponemes (trepos – to turn & nema - thread )6 are relatively short, slender spirochetes with fine spirals & pointed or round ends. Transfusion transmitted Syphilis – The first case of Transfusion transmitted syphilis was reported in 19155. 138 cases were reported in the literature by 19417. Cases were mostly discovered in donors with

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

A-183

AABS; 4(4): 2017

primary or secondary stage of disease8. Treponemes are senisitve to cold; hence risk of transmission through stored blood at 4-8◦C is very low9-10. In India, most blood donors are first-time donors5. The prevalence of syphilis among blood donors in India was reported to be 0.7%11. The global incidence of syphilis in blood donors is variable ranging from 0.75% in Pakistan12 to 12.7% in Tanzania13. Aims and Objectives: The study was conducted in the department of pathology, M.G.M.M.C, Indore. 1. To find out the seroprevalence of Transfusion transmitted diseases (HBV , HCV ,HIV & syphilis) in blood donors. 2. To find the incidence of spectrum of diseases in blood bank donation. 3. To find the age distribution of the cases studied.

Material and Methods

Graph 1: Number of blood units collected during the year 2001-16. Out of total 241571blood donations, majority of donors are voluntary donors 68.13 % as compared to replacement/ relative donors 31.86 % Graph 2: Number of male and female donors during the year 2001-16.Out of total 241571 blood donations, majority of donors are male donors 96.25 % as compared to female donors 3.74% Graph 3: Seropositive donors for HBV , HCV ,HIV & SYPHILIS in 2001-16 Seroprevalence of HBV , HCV , HIV & SYPHILIS are 1.80 %, 0.098%, 0.20% and 0.26 % respectively. Graph 4: Age wise distribution of HBV , HCV ,HIV & SYPHILIS in 2001-16 Seroprevalence is higher in the age group 26-35 years

The present study is being undertaken in the Department of Pathology MGM Medical College Indore. This is a retrospective study that was conducted, during the period 2001 –2016. 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.

Table 1: Seropositivity of transfusion transmitted diseases (HIV, HBV, HCV, Syphilis & Malaria) in 200116. Among Voluntary & replacement/relative donors. Overall seropositivity of TTI’s (HIV, HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.12 % as compared to voluntary donors2.01 %. Over all Seroprevalence of transfusion transmitted disease in all donations in the year 2001-16 is 2.36 %.

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.

Discussion

Results and Observation

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 2001 –2016. In the present study, 241571 blood donors are observed in the year 2001-16 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:

Voluntary or Replacement/Relative Donor - In our study, Out of total 241571 blood donations, majority of donors are voluntary donors 68.13 % as compared to replacement/ relative donors 31.86 % 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 Kulkarni14Associate Professor, Department of Pathology, Vijayanagara Institute of Medical Sciences, Bellary - 583104, Karnataka, India. Male or Female Donor: In our study, .Out of total 241571 blood donations, majority of donors are male donors 96.25 % as compared to female donors 3.74%. Similarly another study is comparable for majority of donors are male 96.22

Table 1: Seropositivity of transfusion transmitted diseases. Total No of Voluntary donors (2001-16)

Total No of Voluntary donors found seropositive for TTI (2001-16)

Total No of Replacement/relative donors (2001-16)

Total No of Replacement/ relative found seropositive for TTI (2001-16)

Number

164586

3319

76985

2406

%age

68.33 %

2.01 %

31.85 %

3.12 %

S.No

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

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

Original Article

A-184

Graph 2: Number of male and female donors during the year 2001-16.Out of total 241571 blood donations, majority of donors are male donors 96.25 % as compared to female donors 3.74%.

Graph 3: Seropositive donors for HBV , HCV ,HIV & SYPHILIS in 2001-16 Seroprevalence of HBV , HCV , HIV & SYPHILIS are 1.80 %, 0.098%, 0.20% and 0.26 % respectively.

http://www.pacificejournals.com/aabs

A-185

AABS; 4(4): 2017

Graph 4: Age wise distribution of HBV , HCV ,HIV & SYPHILIS in 2001-16 Seroprevalence is higher in the age group 26-35 years.

% by Arora Det al15 in Haryana. In the another study, the percentage of male patients was 73% (860/1178) as compared with 27% (318/1178) for female patients by Jain et al16 conducted in New Delhi. Seroprevalence of HBV: In our study, the seroprevalence of HBV is 1.80 %in total blood donations in the year 200116. Seroprevalence of HBV is comparable to another study with seroprevalence of HBS Ag was 1.7 % by AroraD et al15 conducted in Haryana. The Seroprevalence of hepatitis B surface antigen was 0.87% noted in hospital-based population by Sood et al17conducted 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 0.098%in total blood donations in the year 200116. This seroprevalence is much lower than the 0.4-5.2% seroprevalence reported in an earlier study conducted in various European countries by Hahneet al19. Another study by Viet Le at al20 found HCV prevalence to be 0.17%.

HIV was 2.21% by Nagaloet al21 conducted in Koudougou. Seroprevalence of HIV is low as in another study seroprevalence of HIV was 0.91% Nagarekha Kulkarni14 in Karnataka.In our study Seroprevalence is low as compared to overall Seroprevalence of HIV (3.8%) by Tessema et al22conducted in University of Gondar, Ethiopia Seroprevalence of Syphilis; Anti TP (Anti Treponema pallidum) - In our study, the Seroprevalence of Anti TP is0.26 %. in total blood donations in the year 2008-15 (Graph 3).Seroprevalence of Syphilis is comparable to another study with Seroprevalence of Anti TP was 0.91 % by Sultan S, Irfanm et al23conducted in Pakistan.In another study by Elyamany G et al24Seroprevalence was found to be 0.044%. In a study conducted at Mangalore, India by Zulfikar A et al 25seropositivity of syphilis was found to be 0.07%.

Seroprevalence of HIV: In our study, the Seroprevalence of HIV is 0.20 % in total blood donations in the year 200116. Seroprevalence of HIV is low as compared to another study 0.3% in total donors by AroraD et al15conducted in Haryana. In another study, the Seroprevalence of antibodies to HIV in hospital population was 0.35% by Sood et al17conducted in Rajasthan. This is in accordance with the 2006 estimates of NACO (National AIDS Control Organization), NIHWF (National Institute of Health and Family Welfare), and NMS (National Medical Statistics) which suggest that the national adult HIV prevalence in India is 0.36%. Our Seroprevalence of HIV is very low as compared with another study, the overall Seroprevalence of

Age Wise Distribution: In our study, Seroprevalence is higher in the age group 26-35 years. . The seroprevalence of HBV was significantly higher donors in the group aged 20-29 years old than in the group 30-40 years old by Nagalo and Sanou et al21 conducted in Koudougou. The highest seroprevalence for anti-HIV was found in the age group 31-40 years by Sood et al19conducted in Rajasthan. Seroprevalence of Anti TP (2008-15) is higher in the age group 26-35 years for anti TP (0.09 %) .In a study conducted at Mangalore, India by Zulfikar A et al 25 incidence of seropositivity was found to be more in donors in the group aged 18-35 years old than in the group 36-55 years old. In a study by Tessema et al22, seropositivity of syphilis was found to be 0.9% & 1.7% in age groups 17-25 & 26-35 yrs respectively. In another study by Elyamany G et al24seropositivity was found to be highest in age group 21-30 yrs.

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

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

Original Article

A-186

Seropositivity in Voluntary/replacement Donors: Among Voluntary & replacement/relative donors, overall seropositivity of TTI’s (HIV, HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.12 % as compared to voluntary donors2.01 %. Over all Seroprevalence of transfusion transmitted disease in all donations in the year 2001-16 is 2.36 %. (Table 1). In study by Nagarekha Kulkarni14 , the seroprevalence was more in relative/replacement donors as compared to voluntary donors.

& estimates (2001). Available at : http://www.who.int/hiv/ pub/sti/en/who_hiv_aids_2014.05.pdf. 5.

Kaur.G, Kaur.P : Syphilis testing in blood donors: an update. Blood Transfus 2015;13:197-204

Ananthnarayan.R&Panicker C.K in Ananthnarayan. &Panicker’s Textbook of Microbiology 8th Edition Chapter 42 pg 371. University Press.

De Schryver A, Meheus A. Syphilis & blood transfusion: a global perspective. Transfusion 1990;30:844-7

Gardella C, Marin AA, Kahn RH et al. Persons with early syphilis identified through blood or plasma donation screening in United states. J Infect Dis 2002; 185:545-9

Orton S. Syphilis & blood donors: what we know, what we do not know, & what we need to know. Transfus Med Rev 2001;15:282-91.

Conclusion

The present study was 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 2001 –2016. Seroprevalence of HBV, HCV, HIV& SYPHILIS are 1.80 %, 0.098%, 0.20% and 0.26 % respectively. Seroprevalence is higher in the age group 26-35 years. Overall seropositivity of TTI’s (HIV, HBV, HCV, Syphilis & Malaria) is higher in replacement donors 3.12 % as compared to voluntary donors 2.01 %. Over all Seroprevalence of transfusion transmitted disease in all donations in the year 2001-16 is 2.36 %. HBV, Syphilis & HIV are the most prevalent transfusion-transmissible 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.

Acknowledgements

We are Highly grateful to Dr C.V. Kulkarni, Prof.& Head, Dept.of Pathologyfor providing the opportunity & full support

References 1.

Tang, J. and Kaslow, R. A. (2003). “The impact of host genetics on HIV infection and disease progression in the era of highly active antiretroviral therapy”. AIDS 17 (Suppl 4): S51–S60.

10. WendelNeto, Silvano. (1995). Current concepts on the transmission of bacteria and parasites by blood components. Sao Paulo Medical Journal, 113(6), 1036-1052. 11. Kaur G, Basu S, Kaur R et al. Patterns of infections blood donors in a tertiary care center: A retrospective study. Natl Med J India 2010:23;147-9 12. Bhalti FA, Ullah Z, Salamat N, et al . Anti-Hepatitis B core antigen testing, viral markers & occult Hepatitis b infection in Pakistani blood donors: implication for transfusion practice. Transfusion 2007; 47:74-9 13. Matee MI, Magesa PM, Lyamuya EF. Seroprevalence of Human immunodeficiency virus, Hepatitis B & C virus and syphilis infections among blood donors at Muhimbili National hospital in Dar es Salam, Tanzania. BMC Public health 2006;6:21 14. Kulkarni N. Analysis of the seroprevalence of HIV, HBsAg, HCV and syphilitic infections detected in the pretranfusion blood: A short report. International Journal of Blood Transfusion and Immunohematology 2012;2:1-3. 15. Arora D, Arora B, Khetarpal A. Seroprevalence of HIV, HBV, HCV and syphilis in blood donors in Southern Haryana. Indian J PatholMicrobiol 2010;53:308-9 16. Jain M, Chakravarti A, Verma V, Bhalla P. Seroprevalence of hepatitis viruses in patients infected with the human immunodeficiency virus. Indian J PatholMicrobiol 2009;52:17-9 17. Sood S, Malvankar S. Seroprevalence of Hepatitis B Surface Antigen, Antibodies to the Hepatitis C Virus, and Human Immunodeficiency Virus in a Hospital-Based Population in Jaipur, Rajasthan. Indian Journal of Community Medicine :2010;35(1):165-169.

Ronald E. Engle et al “Transfusion-associated hepatitis before the screening of blood for hepatitis risk factors”. Transfusion. 2014 Nov; 54(11): 2833–2841

Hariri S, McKenna MT. Epidemiology of Human Immunodeficiency Virus in the United States. Clinical Microbiology Reviews. 2007;20(3):478-488.

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.

World Health Organization. Global prevalence & incidence of selected curable sexually transmitted infections: overview

19. Hahné SJ, Veldhuijzen IK, Wiessing L, Lim T-A, Salminen M, Laar M van de. Infection with hepatitis B and C virus

http://www.pacificejournals.com/aabs

A-187

AABS; 4(4): 2017

in Europe: a systematic review of prevalence and costeffectiveness of screening. BMC Infectious Diseases. 2013;13:181.

University Teaching Hospital, Northwest Ethiopia: declining trends over a period of five years”BMC Infectious Diseases201010:111

20. 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.

23. Sultan S, Murad S Irfan m et al . Trends of venereal infections among healthy blood donors at Karachi. Arch Iran Med 2016;19(3):192-196 24. Elyamany G,Al amro Mohamed, Pereira WC,Alsuhaibani O. Prevalence of Syphilis among Blood and Stem Cell Donors in Saudi Arabia: An Institutional Experience. Electronic Physician. 2016;8(8):2747-2751.

21. Nagalo MB, Sanou M, Bisseye C, et al. Seroprevalence of human immunodeficiency virus, hepatitis B and C viruses and syphilis among blood donors in Koudougou (Burkina Faso) in 2009. Blood Transfusion. 2011;9(4):419-424. 22. Belay Tessema et al “Seroprevalence of HIV, HBV, HCV and syphilis infections among blood donors at Gondar

25. Zulfikar A, Umaru N, Shreesha K. Seroprevalence of Transfusion transmitted Infections among blood donors in Mangalore. MedicaInnov. Dec 2012, Vol 1, issue 2; 24-27

*Corresponding author: Dr Ravi Jain, 373,Goyal Avenue, Nipania Indore 452010 INDIA Phone: +91 09981494144 Email: ravijainpatho@gmail.com, ravij939@gmail.com

Financial or other Competing Interests: None.

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

Date of Submission : 22.11.2017 Date of Acceptance : 15.12.2017 Date of Publication : 20.12.2017

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

Original Article DOI: 10.21276/AABS.1775

Histomorphology of Upper GI Endoscpic Biopsies: A Study In Urban Care Centre Puvitha. R. Duraisamy1, Lalitha chithambram2, Shifa Seyed Ibrahim3*, Kavitha Madasamy4, Lavanya Krishnagiri Bala4 and Pavithra Thandavarayan4 1 Department of Pathology,Dharmapuri Medical College. India Department of Pathology, Coimbatore medical college, Coimbatore, India 3 Department of Pathology, Madurai Medical College, Madurai, India 4 Department of Pathology, Coimbatore medical college, Coimbatore, India 2

ABSTRACT Background: Upper gastro intestinal [GI] disorders are one of the most commonly encountered problems in clinical practice. A variety of disorders can affect upper GI mucosa, ranging from dysphagia, GI bleed, dyspepsia to altered bowel habits. For investigating symptoms related to upper gastro intestinal tract, endoscopic surveillance followed by biopsies from the grossly abnormal areas are the standard protocol followed. Upper GI Endoscopy is a simple safe and well tolerated OPD procedure. Differentiating benign and malignant lesions needs histopathological aid. The definitive diagnosis of gastric disorders relies on the histopathological confirmation and is one of the bases for planning proper treatment. The objective of our study was to find the prevalence of various disease entities in upper GI lesions in our area Methods: This is a cross-sectional study includes 196 specimens from oesophagus, stomach and duodenum for a period of one year 2016-2017. H&E slides were reviewed by panel of pathologists, the datas were compiled and analysed. Result: Among 196 specimens, male predominance was noted among malignant lesions. In malignancies, squamous cell carcinoma moderately differentiated grade was highly predominant. Out of the seven biopsies from the oesophagogastric [OG] junction, 57% of the cases were non neoplastic. Two cases each of squamous cell carcinoma and adenocarcinoma was diagnosed from the OG junction during our study period. The incidence of duodenal pathology was comparatively less, adenocarcinoma was very less [3.2%] and inflammatory pathologies were more prevalent with female predominance was noted. A case of carcinoid was diagnosed in the duodenum during our study period. Conclusion: We had encountered wide variations of histopathology in the received biopsies and the incidence seen in our study matched those seen in the literatures.

Introduction

Keywords: Oesopahgus, Stomach, Duodenum, Non meoplastic, Malignancy

For investigating symptoms related to upper gastro intestinal tract, endoscopic surveillance followed by biopsies from the grossly abnormal areas are the standard protocol followed. Differentiating benign and malignant lesions needs histopathological aid. This being the gold standard in the diagnosis in upper GI symptoms, the incidence of various neoplastic entities that were diagnosed were taken up for our study in a hope to find the prevalence of various tumors in our area .196 specimens were received during our study period which included oesophagus, stomach and duodenum. According to national cancer registry 2012-14, in Chennai stomach is the second leading cancer site in male and comes fifth among females [1]. This incidence and incidence of carcinomas of the upper GIT of our study was compared along with those found in other literatures.

Materials and Methods

It is a retrospective study carried on for six months. 196 biopsy specimens were included in our study that was sent as a part of investigations for upper GI symptoms. Both non-malignant and malignant lesions that were diagnosed during that period were included in our study. Slides were reviewed by a panel of pathologists and sorted out into malignant and non-malignant lesions. Among them malignant lesions were tabulated, graded and compared with those available in the literatures.

Result

Out of 196 cases, 132 were male and the rest were female. In that, 122 cases were malignant and the rest were non neoplastic except two specimens, which were inadequate for processing[Table 1]. Inflammation was the most common non neoplastic entity seen during the study period

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

A-189 and stomach was the organ commonly involved in the inflammatory pathology in our study Male: female ratio among those with malignancy was 2:1 in our study. The age range was from 23 years to 90 years with a mean of 57 years. In those who were less than 40 years of age, female predominance was noted among the oesophageal squamous cell carcinoma. As the age advances male predominance were noted. In case of oesophageal adenocarcinoma male predominance were noted [Table 2]. Totally 131 biopsies were received from the oesophagus and oesophagogastric junction. 2.3% of the cases out of it were diagnosed as Barrettâ&#x20AC;&#x2122;s oesophagus [Fig 1]. Out of the 122 malignancies reported from the endoscopic biopsies, most cases were oesophageal carcinoma which constitutes about 65.57%. Squamous cell carcinoma was the commonest carcinoma seen in the oesophagus [Fig 2] and next comes the stomach in which adenocarcinoma was seen in a higher proportion [Fig 3]. In the oesophagus moderately differentiated squamous cell carcinoma was highly predominant [Table 3]. Out of 68 gastric biopsies we had received 48.5% of the cases were malignant. Almost all the cases were adenocarcinoma except one case of diffuse large B cell

AABS; 4(4): 2017 Lymphoma [DLBC] [Fig 4]. The age incidence varied from fourth to seventh decade with a mean of 45 years. Poorly differentiated adenocarcinoma was seen among older individuals [seventh decade] .Male: Female ratio was 1.5:1 in our study. Stomach adenocarcinoma also showed mixed patterns when age and sex were correlated. In the lower age group female predominance were noted and as the age progress there were male predominance and later there were equal sex incidence. Most of the cases were of moderately differentiated grade [Table 3]. . In our study we had included biopsies from the oesophagogastric [OG] junction as well. Out of the seven cases that were received, 57% of the cases were non neoplastic. Two cases each of squamous cell carcinoma and adenocarcinoma was diagnosed from the OG junction during our study period [Table 3]. Comparatively the incidence of duodenal adenocarcinoma was very less [3.2%] and inflammatory pathologies were more prevalent. Female predominance was seen in the fourth decade and in the sixth decade there were equal sex incidence. A case of carcinoid was also diagnosed in the duodenum during our study period. A single biopsy from the periampullary region was received and it showed inflammatory pathology [Table 3].

Table 1: Specimens received during our study period with site wise stratification.

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

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

Original Article

A-190

Table 2: Age and sex distribution of various grades of carcinomas 21-30 Yrs 31-40 Yrs 41-50 Yrs 51-60 Yrs 61-70 Yrs 71-80 Yrs 81-90 Yrs M

SITE

F DUODENUM ESOPHAGUS STOMACH

SQAUMOUS CELL CARCINOMA MODERATELY DIFFERNTIATED GRADE

OG JN PERI AMPULLARY DUODENUM 1

ESOPHAGUS STOMACH OG JN

SQUAMOUS CELL CARCINOMA POORLY DIFFERENTIATED GRADE

PERIAMPULLARY DUODENUM ESOPHAGUS 1

STOMACH OG JN

ADENOCARCINOMA WELL DIFFERENTIATED GRADE

PERI AMPULLARY DUODENUM 3 1

ESOPHAGUS 4

STOMACH

OG JN

ADENOCARCINOMA MODERATELY DIFFERENTIATED GRADE

PERI AMPULLARY 1

DUODENUM

ESOPHAGUS 3

STOMACH OG JN

ADENOCARCIOMA POORLY DIFFERENTIATED GRADE

PERI AMPULLARY DUODENUM 1 1

ESOPHAGUS 1

STOMACH

ADENOSQUAMOUS CARCINOMA

OG JN PERI AMPULLARY 1

DUODENUM 1

ESOPHAGUS STOMACH

CARCINOID

OG JN PERI AMPULLARY 1

DUODENUM ESOPHAGUS

STOMACH OG JN PERI AMPULLARY

http://www.pacificejournals.com/aabs

DLBCL

A-191

AABS; 4(4): 2017 Table 3: Site wise stratification of various carcinomas.

Fig. 1: Shows Squamous epithelium of the esophagus replaced by mucin secreting columnar epithelium – Barrett’s esophagus [40x H&E].

Fig. 2: Shows sheets of malignant squamous cell – Squamous cell carcinoma - Moderately differentiated grade [100x H&E].

Fig. 3: Shows malignant epithelial cells arranged in a glandular pattern- Adenocarcinoma[100x H&E].

Fig. 4: Shows sheets of lymphoblast admixed with lymphocytes - DLBCL [ 40x H&E].

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

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

Original Article

A-192

Discussion

Direct visualisation of the upper GIT even though is valuable in the diagnosis of a symptomatic patients, histopathology is the gold standard. Endoscopies are done to evaluate gastritis, gastric ulcer or in cases of suspected malignancies of the upper GIT 2.According to the study done by Shennak MM, et al and Paymaster JC, et al the investigations for upper GI symptoms were higher in males when compared to females3, 4. This is in concordance with our study. There are regional variations in the incidence of various carcinomas of the upper GIT. This study was done to find out the incidence of various carcinomas seen in the endoscopic specimens received as a part of investigations for upper GI symptoms and to compare it with the incidence of various other studies in the literature. The number of new cases of oesophageal cancer was 4.2 per 100,000 men and women per year according to SEER registry. In Asia, squamous cell carcinoma of oesophagus is more prevalent compared to adenocarcinoma in contrast to the western world5, This is in correlation with our study where we saw more number of oesophageal biopsies [124 cases] and out of that 80 cases were squamous cell carcinoma and five were adenocarcinoma. 40% of the cases were from fifth to sixth decade which was comparable to Qureshi NA ,et al and Bazaz -Malik G,et al’ studies6, 7. Male predominance was noted in the oesophageal squamous cell carcinoma in our study as seen in Bukhari U, et al’study8. Female predominance was noted in younger age groups, although female predominance was seen in B Ziaian, et al’s study, they did not see in it in younger age groups as seen in our study9. This increase in the incidence among younger females may be due to the risk factors they are exposed and when intervened we will be able to prevent it. Smoking and alcohol are the two major risk factors associated with squamous cell carcinoma of the oseophagus10. Deficiency of vitamins and celiacsprue is also associated with oesophageal squamous cell carcinoma. Squamous dysplasia is the precursor lesion to squamous cell carcinoma.30% of the cases with dysplasia progress to carcinoma and 15- 30% of the squamous cell carcinoma are multiofcal11.Dyplasia has prognostic significance as well , as the presence of dysplasia at the margins of squamous cell carcinoma has inverse relation to the depth of invasion of the tumor12. In our study the incidence of dysplasia of the oesophagus was 0.8% and it was 4.1% in Memon F, et al’s study group13. Dysplasia of the oesophagus either progress from low grade to high grade or regress.Waf1p21 reactivity decreases in the dysplastic epithelium whereas immuno reactivity for PCNA and

P53 increase 14. In our study 65.57% of the cases had squamous cell carcinoma which was in consistent with Memon F, et al’s study [78.6%] 13. Barrett’s oesophagus is defined as metaplastic replacement of squamous epithelium by columnar epithelium as a response to prolonged gastro oesophageal reflux. Barrett’s oesophagus may or may not be associated with atypia/ neoplasm. Dysplasia may be low grade, high grade, crypt dysplasia, foveolar dysplasia, submucosal invasion, intra mucosal carcinoma. All of our cases presented without dysplasia. The incidence of Barrett’s oesophagus seen in our study correlated with Jawalkar S, et al’s study 15.AMACR and P53 are used to assess the disease progression from dysplasia to carcinoma16, 17. Adenocarcinoma arises from Barrett’s oesophagus in 95% of the cases and the rest from heterotrophic glands or sub mucosal glands. Incidence of adenocarcinoma of the oesophagus was 24.4% in B Ziaian, et al’s study which was in contrast with our study[4%]and in correlation with Mustafa SA, et al’ study 9,18. The incidence of adenocarcinoma of the western world was comparatively higher than Asian population and this may be the reason for the contrast observed between our studies19. Reflux, obesity, diet alcohol and smoking are the risk factors for adenocarcinoma of the oesophagus. Prognosis of this tumor as such is poor and it is worst in mucinous and signet ring cell variants of adenocarcinoma20. In Memon F, et al’s study group 1.35% of the cases showed intestinal metaplasia with atypia in correlation with our study group13. Our case presented without dysplasia, in contrast their cases had dysplasia. Among gastric carcinomas, the incidence of non cardia gastric adenocarcinoma is decreasing worldwide21. Risk factor for cardia adenocarcinoma is H.Pylori and for non cardia it is obesity and reflux. Decrease in the incidence of cardia adenocarcinoma is due to various treatment methods available for H.Pylori. Rashmi K, et al in their study had detected27.94% malignancy among the received gastric biopsies which is in correlation with our study22ShreeshaKhandige in his study had mentioned that inflammatory gastric lesions predominated in his study, but in our study both inflammatory and malignancy showed equal prevelance23.According to Kabir MA, et al’s study, mean age was 51.05±14.98years which was in correlation with our study24. Male predominance is seen in our study and it was in correlation with Kabir MA, et al’ s study24.. In Kato Y, et al’s study the incidence of well differentiated adenocarcinoma had decreased which correlated with our study25. Jawalkar S, et al in their study had mentioned that

http://www.pacificejournals.com/aabs

A-193 squamous cell carcinoma was the predominating diagnosis in contrast to our study group where inflammatory condition predominated in the OG junction biopsies15. Because the duodenum has actively dividing epithelial lining which is susceptible to injury, inflammatory pathologies are common13.Likewise, inflammatory pathologies predominated in our study which correlated with Shepherd NA, et al’s and Memon F, et al’s studies 26, 13 .Kimchi NA, et al in their study had observed 15% of non-neoplastic lesions in the periampullary region27. In our study we had a single non neoplastic lesion in that site.

Conclusion

Endoscopy of the gastro intestinal tract though is diagnostic; histopathology is the gold standard in the diagnostic arena. We had encountered wide variations of histopathology in the received biopsies. The incidences we had seen were in correlation with those seen in the literatures. And it provided an excellent study tool to work up future programmes targeted on our population.

Reference 1.

National Cancer Registry Programme. Three-Year Report ofPopulation Based Cancer Registries 2012-2014Indian Council ofMedical Research. Bangalore, India. 2016.

Mustapha SK, Bolori MT, Ajayi NA, Nggada HA, Pindiga UH, Gashau W, Khalil MIA. Endoscopic Findings and The Frequency

of Helicobacter Pylori Among Dyspeptic Patients in NorthEastern Nigeria. Highland Medical Research Journal. 2007; 5: 78-81

Shennak MM, Tarawneh MS, Al-Sheik; Uppergastrointestinal diseases in symptomaticJordanians: A prospective endoscopic study.Annals of Saudi Medicine, 1997; 17(4): 471-474.

Paymaster JC, Sanghvi LD, Ganghadaran P; Cancer of gastrointestinal tract in western India. Cancer, 1968; 21: 279-287.

Zhang HZ, Jin GF, Shen HB. Epidemiologic diﬀerences in esophageal cancerbetween Asian and Western populations. Chin J Cancer 2012;31:281-6

Qureshi NA, Hallissey M T, John W;Fielding Outcome of index upper gastrointestinalendoscopy in patients presenting with dysphagia ina tertiary care hospital – A 10 years review. BMC Gastroenterology, 2007; 7: 43.

Bazaz -Malik G, Lal N; Malignant tumors of the digestive tract. A 25 year study. Indian Journal of Pathology and Microbiology, 1989; 32(3): 179-185.

Bukhari U, Siyal R, Ahmed F, Memon JH. Oesophageal carcinoma. A reveiw of endoscopic biopsies, Pak J Med Sciences 2009; 25 (5); 845-848.

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

AABS; 4(4): 2017 10. B Ziaian, V Montazeri, R Khazaiee, S Amini,M Karimi, D Mehrabani. Esophageal cancer occurrence in southeastern Iran. J Res Med Sci ,2010 sept-oct:15 (5);290-291 11. Boffeta P, Hashibe M. Alcohol and cancer. Lancet Oncol.2006;7:149–156 12. Kuwano H, Ohno S, Matsuda H, Mori M, Sugimachi K. Serialhistologic evaluation of multiple primary squamous cell carcinomas of the esophagus. Cancer 1988;61:1635 13. Kuwano H, Matsuda H, Matsuoka H, Kai H, Okudaira Y, Sugimachi K. Intra-epithelial carcinoma concomitant with esophageal squamous cell carcinoma. Cancer 1987;59:783 14. Memon F, Baloch K, Memon AA. Upper gastrointestinal endoscopic biopsy; morphological spectrum of lesions. Professional Med J 2015; 22(12): 1574-1579. 15. Wang LD, Yang WC, Zhou Q, Xing Y, Jia YY, Zhao X. Changes in p53 and Waf1p21 expression and cell proliferation in esophageal carcinogenesis. World J Gastroenterol 1997 June 15; 3(2): 87-89. 16. Jawalkar S, and Arakeri U S. Role of Endoscopic Biopsy in Upper Gastrointestinal Diseases. RJPBCS. 2015;6(4):977-983. 17. Kaye PV, Haider SA, Ilyas M, James PD, Soomro I, Faisal W, et al. Barrett’s dysplasia and the Vienna classification: reproducibility, prediction of progression and impact of consensus reporting and p53 immunohistochemistry. Histopathology2009;54:699-712. 18. Kastelein F, Biermann K, Steyerberg EW, Verheij J, Kalisvaart M, Looijenga HJ L, et al. Value of alphamethylacyl-CoAracemase immunochemistry for predicting neoplastic progression in Barrett’s oesophagus. Histopathology 2013;63:630-639. 19. Mustafa SA, Banday SZ, Bhat MA, Patigaroo AR, Mir AW, Bhau KS. Clinico-Epidemiological Profile of Esophageal Cancer in Kashmir. Int J Sci Stud 2016; 3(11):197-202 20. Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2014, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2014/, based on November 2016 SEER data submission, posted to the SEER web site, April 2017. 21. Chirieac LR, Swisher SG, Correa AM, Ajani JA, Komaki RR, Rashid A, Hamilton SR, Wu TT. Signet-ring cell or mucinous histology after preoperative chemoradiation and survival in patients with esophageal or esophagogastric junction adenocarcinoma. Clin Cancer Res 2005; 11:2229. 22. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol. 2006; 24:2137–50. 23. Rashmi K, Horakerappa MS, Karar A, Mangala G. A Study on Histopathological Spectrum of Upper Gastrointestinal

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

Original Article

A-194

Tract Endoscopic Biopsies. Int J Med Res Health Sci. 2013;2 (3):418-424 24. Khandige S; The Conceding of Upper Gastrointestinal Lesion Endoscopic Biopsy: A Bare Minimum For Diagnosis. International Journal of Scientific Research, 2015; 4(2): 264 â&#x20AC;&#x201C; 266 25. Kabir MA, Barua R, Masud H, Ahmed DS, Islam MMSU, Karim E et al.Clinical Presentation, Histological Findings and Prevalence of Helicobacter pylori in Patients of Gastric Carcinoma. Faridpur Med. Coll. J. 2011;6(2):78-81.

26. Kato Y, Kitagawa T, Nakamura K. Changes in the histologic types of gastric carcinoma in Japan. Cancer 1981; 48:2084-87 27. Shepherd NA, Valori RM The effective use of gastrointestinal histopathology: guidance for endoscopic biopsy in the gastrointestinal tract Frontline Gastroenterology 2014;5:84-87. 28. Kimchi NA, Mindrul V, Broide E, Scapa E. The contribution of endoscopy and biopsy to the diagnosis of periampullary tumors. Endoscopy. 1998; 30(6):538-43.

*Corresponding author: Shifa Seyed Ibrahim, 82, J.N. Nagar, Old Natham Road, Madurai-17 India Phone: +91 09486669274 Email: shifafrin@gmail.com Date of Submission : 27.11.2017 Date of Acceptance : 16.12.2017 Date of Publication : 20.12.2017

Financial or other Competing Interests: None.

http://www.pacificejournals.com/aabs

Original Article DOI: 10.21276/AABS.1784

Tender Coconut (Cocos nucifera L.) Husk Anaerobic Leachate as A Potent Antifungal and Antibacterial Agent Praveen Krishnakumar* and Treesamol Antony Immunology & Toxicology Research Lab, Dept. of Zoology, Christ College, Irinjalakuda. Kerala, India.

ABSTRACT Background: Leaching of tender coconut husk in anaerobic condition may leads to the production of a wide range of secondary metabolites including, alkaloids, terpenoids and phenolic compounds. In this study we have evaluated the antibacterial and antifungal potential of tender coconut husk leachate against Escherichia coli, Lactobacillus plantarum, Staphylococcus aureus, Klebsiella pneumoniae, Penicillium sps, Aspergillus niger and Mucor indicus. Methods: Leachate was treated separately as unaltered raw leachate and lyophilized leachate.Qualitative phytochemical analysis of the tender coconut husk leachate was carried out using standard procedures. There are differences in the constituents present with raw and lyophilized leachate. Antibacterial activity were determined using disc diffusion method and also by calculating MIC and MBC. Antifungal activity was determined using poison plate method. Result: Anaerobic leaching leads to the extraction of potent secondary metabolites which possess antibacterial and antifungal activities. Both the lyophilized and air dried raw leachate was effective in preventing the growth of gram negative bacteria (E.coli and K.pneumoniae) whereas the gram positive bacteria ( S.aureus and L.plantarum) were least affected.In higher concentrations leachate exhibited similar inhibitory activity as that of positive control used. We could also observe a concentration dependent growth inhibition in majority of the treatments. Conclusion: From this study we could conclude that the anaerobic leachate of tender coconut husk exhibits antimicrobial activity against different pathogenic organisms. In all the trials the raw and lyophilized leachate showed very prominent control over both the bacteria and fungus studied. Further studies like specific characterization of potent molecules from the leachate and their action on multidrug resistant microorganisms are necessary todevelop effective and safe antimicrobial products. Keywords: Phenolic Compounds, Antibacterial Activity, Antifungal Activity, Lyophilized Leachate, Raw Leachate.

Introduction

The wide varieties of plant products having ethnopharmacological importance have been used against many infectious and non-infectious diseases by traditional medical practitioners for thousands of years with or without proper scientific validation. Even though antibiotics are undisputedly considered as one of the important therapeutic discoveries of 20thcentury, only one third of the infectious diseases known have been treated from this synthetic products[1].This implies the considerable increase in the rate of resistant pathogens against antibiotics. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents.The resistance developed by many microbes against synthetic drugs was the major reason for switching over the search from synthetic chemicals to highly potent plant derived molecules which acts against a wide spectrum of microbes. Fungi are among the important biotic agents which play a significant role in deteriorating aesthetic and nutritive value of the stored food commodity[2-3]. They are

considered as significant destroyers of stored foodstuffs and grains, rendering them unfit for human consumption by retarding their nutritive value and often by producing mycotoxins[4-6]. A significant portion of the agricultural product in the country and the world over become unfit for human consumption due to mycotoxins contamination of grains, especially those produced by species of Aspergillus, Mucor and Penicillium. Even though the precise mechanism of action of many plant extracts against bacteria is not well studied, but it has been proved that the stress and overload experienced by bacterial cell wall is the major reason behind bacteriostatic or bactericide activity[7]. This suggests that the action of same compound on gram positive and gram negative bacteria may vary considerably. Despite extensive progress in the past few years, the morbidity and mortality of invasive bacterial and fungal infections are still unacceptably high. It would therefore be novel to evaluate and identify antimicrobial drugs with new mechanisms of action having broad spectrum of activity, less toxicity, flexible route of administration. Current trends in drug development

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

Original Article

A-196

process are focused on natural sources, especially sources of plant origin due to some proven correlation between the folkloric medicinal uses of some of these plants to biological activity [8]. Coconut (Cocos nucifera L.) which comes under the family Arecaceae is commonly considered as an important fruit crop in tropical and subtropical countries. The coconut fruit comprises an outer epicarp, a mesocarp, and an inner endocarp. The mesocarp of coconut, commonly known as coconut “husk” is the major source of the coir fibre which is extracted from the husk by a process called as retting [9]. Retting of coconut husks encompasses the biodegradation of mainly polyphenols and pectins which play a major role in binding the fibre in the husk. Resorcinol, pyrogallolic acid and catechol were found to be major phenolic compounds leached out during retting of coconut husks.Cocos nucifera is a widely dispersed plant that has important pharmacological effects with low toxicity. Different constituents of endocarp and coconut water exhibit antioxidant activity whereas the fibre showed antibacterial, antiparasitic, and anti-inflammatory activities [10]. After scrutiny of published literature showing its medicinal importance as antioxidant, anthelminthic, antithrombotic, antimicrobial, antiatherosclerotic, immunostimulatory, antidiabetic, hepatoprotective andanticholecystitic effects the present study has been outlined regarding the antifungal and antibacterial activity of tender coconut husk leachate. Thus the aims of this study were 1.Anaerobic leaching of tender coconut husk, 2.Lyophilizing half of the extract obtained after leaching, 3.Qualitative phytochemical screening of anaerobic leachate, 4.To evaluate the antibacterial and antifungal activity of unaltered raw leachate and lyophilized leachate separately.

Materials and Methods

Collection and Extraction of Plant Material: Tender coconut husk was selected as experimental material. Tender coconuts of moderate weight were collected from local market of Irinjalakuda, Thrissur district and its total weight was taken. Outer husk of the fruit was decorticated, washed and cut into small pieces.Extraction was done in different air tight containers. Approximately1kg of husk was weighed from different tender coconuts, and it was immersed in 5litres of de-aerated water. The husk was hammered well before immersing so as to make the extraction process easier. The extraction process was continued in anaerobic condition for about 30 days. Lyophilization and Phytochemical Screening : Lyophilization or freeze drying is defined as a stabilizing process in which the sample is frozen followed by a reduction

of the water content by sublimation and then by desorption to standards that will no longer allow biological growth or chemical reactions[11].After 30 days of anaerobic leaching, leachate was filtered using Syringe-driven filters (0.22µ) and transferred to amber coloured bottles ensuring less air contact. Leachate was then separated into two halves. One half of the leachate was dried using a rotary evaporator and stored in air tight vials. Other half was centrifuged in 5000rpm for 10minutes. The supernatant was filtered and lyophilized in a freeze dryer (Operon Freeze Dryer) and at 4oC. Qualitative phytochemical analysis of the tender coconut husk leachate was carried out using standard procedures to assess the different types of phytochemical constituents present in the dried leachate. Screenings were done for polyphenols, saponins, flavonoids, alkaloids, tannins and terpenoids and carbohydrates[12-14]. Antimicrobial Susceptibility Test Microbial Strains: The antimicrobial activities of the raw and lyophilized leachate were tested againstEscherichia coli(MTCC 1652),Lactobacillus plantarum(MTCC 1407), Staphylococcus aureus(MTCC 3160), Klebsiella pneumonia(MTCC 2403),Penicillium sps(MTCC 1995), Aspergillus niger(MTCC 872) and Mucor indicus(MTCC 3318). Disc Diffusion Assay: Disc diffusion method was used to screen the anti-bacterial activity of tender coconut husk leachate. The plates were prepared by pouring 15 ml of molten sterile nutrient agar media into sterile petri plates. The plates wereallowed to solidify for 5 min. The test microorganisms 10µl (106 cells/ml) from overnight broth cultures of bacteria in nutrient broth were seeded into respective plates with medium by spread plate method. Inoculated cultures were allowed to dry. Concentrations of leachate were taken as 1, 2, 5 and 10 mg/ml. Sterilized paper discs of 6 mm diameter were taken for assay. Prepared sterile paper discs were saturated with tender coconut husk leachate of different concentrations and dried. Saturated discs were then placed on the surface of agar medium of petri plate and allowed to diffuse for 3 minutes. Pre-pared plates were kept for incubation at 37 0 C for 24 hrs. Streptomycin (20µg/ml) discs were used as positive control. DMSO (100µg/ml)discs were used as negative control. Post-incubation inhibition zones around the extract disc were measured with a transparent ruler in mm[15]. Inhibition value is obtained using the formula, Inhibition value = Inhibition diameter in mm – Disk diameter (6mm) / 2 The mean and standard deviation of triplicates of various concentrations of plant extracts were calculated and compared with Streptomycin.

http://www.pacificejournals.com/aabs

A-197 Minimum inhibitory concentration bactericidal concentration assays

AABS; 4(4): 2017 and

Minimum

Minimum Inhibitory Concentration was determined according to Murray’s method [16]with slight modifications. Different dilutions of the leachatein increasing concentrationsviz1, 2, 5 and 10 mg/ml were prepared by dissolving it in DMSO.Standardized suspensions of the test organisms (Escherichia coli, Lactobacillus plantarum, Staphylococcus aureus, and Klebsiella pneumoniae) were inoculated into a series of 96 well microtiter plate including one positive and one negative control. All tubes were incubated at 37°C for 24 hours and then examined for growth, by observing the turbidity.The microtiter plate showing the minimum turbidity was noted for MIC. Ten microliters of bacterial culture from the MIC tubes, which did not show any growth was pipetted, and sub cultured onto Nutrient agar, and incubated at 37oC for 24 hours. After incubation, the concentration at which there was not a single colony of bacteria was taken as the minimum bactericidal concentration (MBC). Determination of Antifungal Activity Poison Plate Assay: Antifungal activity of tender coconut husk leachate was determined by food-poisoned technique[17] with minor modifications. The plates were prepared by pouring 1ml of raw/lyophilized leachate in different concentrations (viz.1, 2, 5 and 10 mg/ml) in to respective plates. To this 15 ml of molten sterile PDA were poured and allowed to solidify for 5 minutes. After solidification fungus were inoculated using sterile wire loop. The inoculated plates were incubated at 370C for 48hrs. Fluconazole(20µg/ml) and DMSO (100µg/ ml) were used as positive control and negative control respectively.

Antimicrobial Susceptibility Test Disc Diffusion Assay: For the disc diffusion assay we have used two gram positive bacteria (MTCC 3160 & MTCC 1407) and two gram negative bacteria (MTCC 1652 & (MTCC 2403). In all the experimental trials with raw and lyophilized leachate, the zone of inhibition increased from low to high concentrations (Table 2).It was also observed that both the lyophilized and raw leachate werecomparatively effective in preventing the colonization of gram positive bacteria than gram negative bacteria. At higher concentrations the diameter of zone of inhibition of raw leachate against two gram positive bacteria S.aureus and L.plantarum were found to be 16 and 16.6 millimetres respectively. In the case of gram negative bacteria E.coli and K.pneumoniae the diameter of zone of inhibition decreased to 11.1 and 11.2 millimetres respectively at higher concentrations of raw leachate. The disc diffusion assay also envisage that in all the four concentrations the lyophilized leachate possess a comparative dominance over the raw leachate in preventing both gram positive and negative bacteria. According to the antimicrobial study on coconut husk extract[20], they observed that the antimicrobial activity of husk extract increased with increasing concentration and was found to be more effective against gram-negative than gram- positive organism which was somewhat similar with our results on both lyophilized as well as raw anaerobic husk leachate. Anaerobic leaching may have enhanced the formation of recalcitrant compounds in higher proportions and this may thought to be effective in preventing the colonization of both gram positive and gram negative bacteria tested.

Phytochemical Screening; The qualitative phytochemical screening test reveals the presence of alkaloids, terpenoids, phenols, tannins and carbohydrates with lyophilized husk leachate. It is noteworthy that the raw samples did not show a positive reaction for alkaloids and terpenoids indicating that these compounds present in the leachate could be heat liable. Carbohydrates were absent in both freeze dried and air dried leachate (Table 1).Phytochemical screening ofC. nucifera conducted by Alvianoet al. has reported that thisplant material is rich in polyphenolic molecules catechin, andepicatechin together with condensed tannins, which conferson its potent antimicrobial properties [18]. Tannins present in the coconut plant extracts possess astringent effect on the mucous membrane. They also form a layer over enamel, thus providing protection against dental caries[19].

Minimum inhibitory concentration and Minimum bactericidal concentration assays: The average values of Minimum inhibitory concentration and Minimum bactericidal concentration assays are plotted in figure 1. The results show that the minimum inhibitory concentration and minimum bactericidal concentration of gram positive bacteria exhibited much lower values than that of gram negative bacteria. On treatment with raw leachate theaverage MIC of gram positive bacteria Lactobacillus was about 2mg/ml followed by S.aureus (5mg/ml). In the case of gram negative bacteria E.coli and K.pneumoniae, the average MIC value was about 10 mg/ml each for unaltered raw leachate. Compared to raw leachate the MIC values of L.plantarum and K.pneumoniae were found to be decreased to 1mg/ml and 5mg/ml respectively when treated with lyophilized leachate. On the other hand the MIC values of S.aureus and E.coli when treated with lyophilized leachate were found to be more or less similar with raw leachate treatment. The average MBC values on treatment with raw leachate were found to be 10mg/ml

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

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

Result and Discussion

Original Article

A-198

each for S.aureus and L.plantarum. In the case of E.coli and K.pneumoniae the average MBC values were15mg/ ml and 20mg/ml respectively. The average MBC value of lyophilized leachate show marked difference from raw leachate with an average value of 1mg/ml against L.plantarum and 10mg/ml against K.pneumoniae. The precise mechanism through which the secondary metabolites from plant materials donate to anti-bacterial activity is not clear, though one of the mechanisms suggests that it is the hydrophobicity which helps partition the cell membrane and rendering them more permeable and leaky[21]

of positive control.The antifungal activity of lyophilized leachate after 24hrs of incubation against Penicillium sps, Aspergillus niger and Mucor indicus were evaluated using various concentration. In all the three fungal strain tested, there was a concentration dependent growth inhibition. At the highest concentration (10mg/ml), leachate was very effective in limiting the growth of Aspergillus niger to a smaller diameter than that of the positive control (flucanazole). The Penicillium sps shows almost similar results that of positive control. TheMucor indicus seems to be less affected even at higher concentrations of lyophilized leachate. In all the trials the fungal growth was proportionately high in negative control plates.

Antifungal Activity Poison Plate Assay: The anti-fungal activity of raw leachate and lyophilized leachate after 24 and 48hrs of incubation against Penicillium sps, Aspergillus niger and Mucor indicus was observed. In the case of raw leachate treatment, when the concentration of leachate increases from 1 mg/ml to 10 mg/ml, we could observe ameasured decrease in the diameter of the fungal colonies in the PDA plate. The maximum inhibition was seen at the concentration 10mg/ml and it was almost equal to the extent of20µg/ml of fluconazole (positive control). The Penicillium sps,Aspergillus niger and Mucor indicusexhibited almost similar growth inhibition at highest concentrations. The diameter of growth of Aspergillus niger at the highest concentration (10mg/ml) was less than the diameter of positive control but in Mucor the diameter of growth at higher concentration was double the value

After 48 hrs of incubation the fungus show similar kind of growth inraw and lyophilized leachate containing PDA plates. In the case ofPenicillium sps and Aspergillus nigergrown in raw leachate containing plates the diameter of growth of the colony at highest concentration (10mg/ ml) were less than that of fluconazole. At the highest tested concentration (10mg/ml) of lyophilized leachate, Aspergillus nigerand Penicillium sps shows almost similar diameter of growth as that of fluconazole.In the case ofMucor indicus, even at higher concentration (10mg/ml) of raw and lyophilized leachate, the diameter of growth was double that of fluconazole showing they are less affected by any of the leachate. After 48hrs Penicillium sps, Aspergillus nigerand Mucor indicus were grown to the full plate in DMSO (negative) control.

Table 1: Preliminary phytochemical screening of Coconut husk anaerobic leachate. SI.No

Phytochemicals

Raw Leachate

Lyophilized leachate

Alkaloids

Terpenoids

Tannins

Phenols

Flavanoids

Carbohydrates

(Key: + presence, - absence)

Table 2: Zone of inhibition of tender coconut husk raw and lyophilized leachate against different bacterial strains. Test organisms E.coli K.pneumoniae

Diameter of zone of inhibition (mm) lyophilized leachate

Diameter of zone of inhibition (mm)Raw leachate 1 mg/ml 2mg/ml

5mg/ml

8.6 ±0.5 11.1±0.2 13.7 ± 0.5 9.3 ±1

11±1.7 14.7 ± 1.1

10mg/ml 16 ± 1 16.6 ±2

Strepto1 mg/ml mycin 18 ± 2

2mg/ml

5mg/ml

10mg/ml

8.7 ± 0.5 11.9 ± 0.7 15.3 ± 0.7 20.1 ± 1

18.7 ±0.5 9.5 ± 0.5 12.5 ± 0.5

16 ± 1

Streptomycin 18.8 ± 0.7

20.6 ± 0.7

19.5 ± 0.5

L.plantarum

6.6 ±0.4 8.5 ±0.5

9.1 ±0.2

11.1 ±0.1 18.5 ±0.5 7.5 ± 0.5 9.4 ±0.7 10.7 ± 0.2 12.1 ±0.4

18.6 ±0.4

S.aureus

6.4 ±0.5 7.3 ±0.2

8.1 ±0.7

11.2 ±0.2

18.2 ±0.7

18.1 ±1

7 ±0.92

8.2 ±0.3

9.5 ±1

12.2 ±0.4

Note: The control disc used for solvent had no zone of inhibition, so there data was omitted from the above data. Data are represented in the form of mean of three tests ± SD of the standard group

http://www.pacificejournals.com/aabs

A-199

AABS; 4(4): 2017

Table 3: Antifungal activity of raw and lyophilized leachate observed after 24hrs of incubation. Diameter of zone of inhibition (mm) Raw leachate

Test organisms

Diameter of zone of inhibition (mm) lyophilized leachate

1 mg/ ml

2mg/ ml

5mg/ ml

10mg/ ml

Flucana- DMSO 1 mg/ zolel ml

2mg/ ml

5mg/ ml

10mg/ ml

Flucanazole

DMSO

Penicillium sps

13.7 ±0.4

12.3 ±0.3

10.1 ±1.1

6.5 ±0.5

6.3 ±0.5

69 ±1.6

12 ±0.5

11 ±0.5

8.9 ±0.2

7.6 ±0.3

7.7 ±0.6

63.6 ±1.3

A.niger

14.1 ±0.4

11.9 ±0.1

9.3 ±0.6

6.6 ±0.4

8.1 ±1

69.9 ±1

18.5 ±0.4

15.6 ±1

9.1 ±0.7

6.7 ±0.5

7.9 ±0.3

66.3 ±1

M.indicus

33.6 ±1.5

25 ±1.4

20.2 ±0.7

18.4 ±0.6

9.2 ±1

70.1 ±1.2

32.6 ±0.5

24.6 ±1.1

19.9 ±0.7

17.8 ±0.2

9.5 ±0.8

73.4 ±1

Note: Data are represented in the form of mean of three tests ± SD of the standard group

Table 4: Antifungal activity of raw and lyophilized leachate observed after 48hrs of incubation. Testorganisms

Diameter of zone of inhibition (mm) Raw leachate 1 mg/ 2mg/ ml ml

5mg/ ml

Diameter of zone of inhibition (mm) lyophilized leachate

10mg/ Flucana10mg/ FlucanaDMSO 1 mg/ml 2mg/ml 5mg/ml DMSO ml zole ml zole

Penicillium sps

18 ±0.9

15.4 ±0.5

12.8 ±0.2

10.3 ±0.5

11.3 ±1.1

18 ±1

17.5 ±1.3

12.7 ±0.3

10.7 ±0.6

11.8 ±0.8

A.niger

18.5 ± 0.4

15.6 ±1

12.2 ±1

9.9 ±1

10.5 ±0.9

18.2 ±0.6

17.1 ±1

11.3 ±0.5

10.8 ±0.7

10.5 ±0.7

M.indicus

50.7 ± 0.6

45 ± 1.1

35.3 ±1.3

28 ±0.4

11.13 ±1

62.2 ±0.9

46.6 ±1.1

35.6 ±0.9

29.2 ±0.8

11.5 ±0.6

Note: Data are represented in the form of mean of three tests ± SD of the standard group. FG: Fully grown.

Fig. 1: Showing average MIC and MBC values of raw and lyophilized leachate.

Conclusion

From this study we could conclude that the anaerobic leachate of tender coconut huskexhibits antimicrobial activity against different pathogenic organisms studied. In all the trials the raw and lyophilized leachate showed very prominent control on microbial growth compared to DMSO (negative) control. The phytochemical analysis revealed

the presence of different secondary metabolites in the raw leachate and lyophilized leachate varies. Among these identified metabolites the level of phenolic compounds was reasonably high and this is supposed to be a major factor in limiting the growth of microbes in culture plates. These results help to enhance the possibilities of future studies to isolate potent molecules from coconut husk leachate and

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

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

Original Article

A-200

test for their activity against broad spectrum of multi drug resistant microbes and this may leads to the commercial production of effective and safe antimicrobial products.

Acknowledgement

The authors are thankful to the Principal, Christ College, Irinjalakuda for the facilities provided for this work. We are thankful to Dr. Leyon Varghese, Assistant Professor Christ College Irinjalakuda, for his constant support and guidance throughout the project.We also acknowledge Dr. Pius K Jacob, HOD, department of zoology for his endless support throughout the project.

10. Lima EBC, Sousa CNS, Meneses LN, Ximenes NC, Santos. Junior MA, Vasconcelos GS, Lima NBC, Patrocínio MCA, Macedo D &Vasconcelos SMM, Cocos nucifera (L.) (Arecaceae): A phytochemical and pharmacological review Brazilian Journal of Medical and Biological Research. 2015;48(11): 953-964. 11. Tushar R. Jadhav and Moon RS, Review on lyophilization technique. World journal of pharmacy and pharmaceutical sciences. 2015;4(5) :1906-1928. 12. Trease GE & Evans WC, Pharmacology (BailliereTindall Ltd., London).1989;60-75.

11thed,

Reference 1.

Sharma A, Antibacterial activity of ethanolic extracts of some arid zone plants. Int. J. of Pharm.Tech. Res. 2011;3(1):283-286.

13. Sofowora A, Medicinal Plants and Traditional Medicines in Africa (Chichester John Wiley & Sons New York). 1993; 97- 145.

Christensen CM, Kaufmann HH, Deterioration of Stored grains by fungi, Annu. Rev. Phytopathol. 1965;3:69-84.

14. Kokate CK, Purohit AP &Gokhale SB, Practical Pharmacognosy, 4th ed. (VallabhPrakashan Publishers, New Delhi). 2006; 107-108

Neergard P. In: Seed Pathology, (TheMcMillan Press Ltd. London and Basingatoke) Vol. I. 1977:118-269.

Park JW, Kim EK & Kim YB, Estimation of the daily exposure of Koreans to aflatoxin B1 through food consumption. Food additives and contaminants. 2004; 2(1):70-75.

Koirala, P, Kumar S, Yadar BK &Premarajan KC, Occurrence of Aflatoxin in some of the food and feed in Nepal. Indian Journal of Medical Sciences. 2005; 59: 331-336.

Domijan A, Feraica M, Jurjevic Z, Ivil D &Cvjetkovic B, Fumonisin B1,fumonisin B2, Zearalenone and ochratoxin a contamination of maize in Croatia. Food additives and contaminants.2005;22: 677-680.

Calvo MA, Arosemena EL, Shiva C&Adelantado C,Antimicrobial activity of plant natural extracts and essential oils, Science against microbial pathogens: communicating current research and technological advances. 2011;1:1179-1185.

ShirishaRao, BibechanaTimsina&Varalakshmi KN, Antimicrobial effects of medicinal plants and their comparative cytotoxic effects on HEPG2 cell line. International Journal of Pharmacy and Pharmaceutical Sciences. 2014;6(1): 101-105. Ravindranath AD, Bhosle S, Development of bacterial consortium for coir retting. Journal of Scientific & Industrial Research. 2000; 59: 140-143.

15. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility by standardized single disk method. American Journal of Clinical Pathology. 1966; 45(4): 493-496. 16. Murray PR, Baron EJ, Pfaller MA, Tenover FC &Yolken, RH, Manual of Clinical Microbiology, 6th edition. (American Society of Microbiology Press, Washington DC). 1995; 1482 p. 17. Schmitz H, Food poisoned technique. Industrial Engineering Chemical Analyst Education. 1930; 361-363. 18. Alviano WS, Alviano DS, Diniz CG, Antoniolli AR, Alviano CS, Farias LM, et al. In vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on Brazilian folk medicine. Arch Oral Biol. 2008;53:545-52. 19. Prashanth GM, Chandu GN. The effect of mango and neem extract on four organisms causing dental caries: Streptococcus mutans, Streptococcus salivarius, Streptococcus mitis, Streptococcussanguis: An in vitro study. Indian J Dent Res. 2007;18:148-51. 20. Shettigar R, Lala R, Nandvikar NY. Evaluation of antimicrobial activity of coconut husk extract. Annals of Applied Bio-sciences. 2014;1:A23-27. 21. Sikkema J, De Bont AM, Poolman BM. Interaction of cyclichydrocarbons with biological membranes. J BiolChem1994;269:8022-8.

*Corresponding author: Praveen Krishnakumar, Immunology & Toxicology Research Lab, Dept. of Zoology, Christ College, Irinjalakuda, Thrissur, Kerala, India. 680125 Phone: +91 09447370509 Email: pvnpraveen1990@gmail.com Date of Submission : 29.11.2017 Date of Acceptance : 16.12.2017 Financial or other Competing Interests: None. Date of Publication : 20.12.2017

http://www.pacificejournals.com/aabs

Case Report DOI: 10.21276/AABS.1801

Paramedian Forehead Flap in Post Radiotherapy Fistula Manish Munjal, Japneet Kaur*, Venus Tilavat, Amanjot Kaur and Shubham Munjal Department of ENT, Dayanand Medical College and Research Institute, Ludhiana, Punjab, India

ABSTRACT The medial canthal fistula gains notoriety for its unsuccessful “ repair “ utilising the varied flaps from its vicinity . Ultimately the conventional forehead flap is lone “ saviour “ in this malady . Keywords: Paramedian Forehead Flap, Reconstruction, Axial

Introduction

Maxillofacial fistulae are a surgeon’s enigma . Particularly so when the primary malignancy is under control as per clinical and positron emission imaging , evaluation . The acute infraorbital - nasolabial junction , component of the established Weber Ferguson incision is likely to “ gape “ due to counter forces of traction . A loss of underlying bony support due to removal of the maxilla and the radiation fibrosis effects the facial aesthetics . Primary suturing using advancement flaps , Imeres ‘ cheek flap or bilobed transposition flaps usually fail to seal the defect .. A median or paramedian flap based on the supra orbital and supra trochlear vessels is the most reliable with the maximum rate of success . Though necessitating a bi staged procedure , the bulk is adequate to obliterate the medial canthal defect . The second stage reposits the redundant flap with trimming if need be , within 4-6 weeks as the case be . The scar at the forehead and the medial canthus is imperceptible at 3-6 months ,subject to individual skin texture . The forehead flap is one of the oldest recorded surgical techniques for nasal reconstruction. As the gold standard for nasal soft tissue reconstruction, the forehead flap provides a reconstructive surgeon with a robust pedicle and large amount of tissue to reconstruct almost any defect. Modifications provided by masters like Burget and Menick have only increased the utility of this exceptional flap 1

Case Report

A 60 yearr old elderly male who had completed a course of fractionated radiotherapy following maxillectomy a year back was taken up for repair of the medial canthal fistula of the left side ( Picture 1) . A right side paramedian forehead flap 7.5 cm in length , 3.5 cm at base and 1.5 cm at apex was marked out .

Pedicle planning 1. The supratrochlear artery was identified at the supraorbital rim using landmarks such as the medial brow border, a point 2cm lateral to the midline. (Picture 2) 2. The pedicle was centered over the artery, with a width of 1.5cm. Smaller pedicles risk damage to the supratrochlear artery. Larger pedicles restrict pivotal movement of the flap. 3. A small amount of extra length was included to allow for flap thickness and swelling. This was done by removing superior standing cutaneous deformity of scalp skin with the distal end of the flap and trimming off during inset.(Picture 3&4) Raise the flap 1. Local anesthetic was injected circumferentially along the pedicle, inject along the borders only to avoid disrupting the supratrochlear artery. 2. Maked borders of the flap were incised. 3. The flap was raised from distal to proximal (ie. superior to inferior)(picture 3) 4. Layers of the flap-The defect portion of the flap was raised in a subcutaneous plane, leaving the galea/ frontalis down.  The pedicle was raised in a subgaleal plane, leaving periosteum down, until 1 cm superior to the level of the eyebrow. It was then transitioned down to a subperiostial plane to keep the supratrochlear artery protected.  When the proximal end of the flap was extended across the orbital rim an extra 1.5 cm of length was obtained. The artery was sandwiched between the corrugator and frontalis muscles in this area

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

C-35 Close the donor defect 1. Undermining is done in a subgaleal plane and deep sutures should be placed through galeal layer to ensure adequate strength.(Picture 5) 2. Suture: interrupted 3-0 PDS, 3-0 vicryl, or 3-0 monocryl for subcutaneous tissues, 5-0 black nylon vertical mattress for skin.(Picture 6) 3. The backside of the flap may be covered with thin Alloderm or a split thickness skin graft as a biological dressing and to decrease oozing. Flap inset 1. The flap was pivoted clockwise to reach the defect. Rotation was done so that the flap skin is facing the ipsilateral eye can decrease ooze from the backend

AABS; 4(4): 2017 of the flap from reaching the eye. Undermining of adjacent defect skin was done to decrease trap door defect.(picture 7) 2. Suture: 5-0 or 6-0 nylon/prolene for skin only. No deep sutures. A minimum of sutures are used so as to minimize risk for flap necrosis(Picture 8 &9) On seventh postoperative day, the sutures were removed. The patient was scheduled for flap detachment approximately 3 weeks from the date of initial surgery. Pedicle separation was done with the patient under local anesthesia. The pedicle was separated sharply, base of the pedicle is returned to the glabellar region to achieve a normal intereyebrow distance.

Picture 1A

Picture 1B

Picture 3

Picture 4

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

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

Case Report

C-36

Picture 5

Picture 6

Picture 7

Picture 8

Picture 9

Picture 10

http://www.pacificejournals.com/aabs

C-37

Discussion

The tint of forehead skin so exactly matches that of the face and nose that it must be first choice. Is not the forehead the crowning feature of the face and important in expression? Why then should we jeopardize its beauty to make a nose? First, because in many instances, the forehead makes far and away the best nose. Second, with some plastic juggling, the forehead defect can be camouflaged effectively.” —Sir Harold Gillies and D. Ralph Millard2 The forehead is acknowledged as the preferred donor site for resurfacing the nose. Small defects less than 1.5 cm can be with local flaps. Superficial defects consisting of skin and minimal subcutaneous fat can be repaired with skin grafts. Limited alar defects can be successfully repaired with a nasolabial flap. The forehead flap, however, remains the premier donor site for a nasal reconstruction because of its size, vascularity, and excellent color, texture, and skin thinness. A paramedian forehead flap allows the transfer of forehead tissue in an efficient and reliable manner with minimal donor deformity and creates the most aesthetically pleasing reconstruction, both to the recipient nose and the donor forehead. Although there has been a shift away from midline forehead tissues toward more extensive or less satisfactory methods such as the scalping flap, skin expansion, or the use of distant or free flaps in nasal reconstruction, the paramedian forehead flap remains the best choice3 The paramedian forehead flap is based on the supratrochlear artery .The supratrochlear artery was precisely located 1.7-2.2 cm from the midline, corresponding to the medial border of the eyebrow. The notch of the supratrochlear vessels is palpable and that is where the skin pedicle was centered.(Picture 2).The supratrochlear artery and the supraorbital artery, which lie laterally, are terminal branches of the ophthalmic artery (branch of the internal carotid artery), and supplies the anterior pericranium and galea.The supratrochlear artery divides into superficial and deep branches. The superficial branch enters the frontalis muscle and runs on the surface of the galea until entering the subcutaneous tissue approximately 3.5 cm above the orbital rim. The deep branch runs within the subgaleal fascia (this layer may be considered a component of the

AABS; 4(4): 2017 pericranium). The deep branches pursue an axial course for approximately 1.5 to 4 cm above the supraorbital rim. There are many penetrating vessels that connect the superficial and deep branches. These vessels are divided if the galea is separated from the underlying pericranium. The pedicle base is perfused by a terminal branch of the angular artery that is actually a major contributor to the flap’s arterial supply. One disadvantage of the paramedian forehead flap is the vertical forehead scar. The wound edges must be carefully approximated to minimize wound tension. For large secondary defects, we achieve this by widely undermining the forehead skin, to the temporalis muscle bilaterally. Additional length can be achieved by performing galeatomies on either side of the incision. Inability to achieve primary closure is not a contraindication to performing a forehead flap. Allowing the donor site to heal in part by secondary intention usually results in an acceptable cosmetic result. Another disadvantage of the paramedian forehead flap is its limited length in patients with low hairlines. Several modifications are possible, such as the oblique forehead flap, tissue expansion, or extension of the flap into hair-bearing scalp.4

Conclusion

A paramedian forehead flap allows the transfer of forehead tissue in an efficient and reliable manner with minimal donor deformity and creates the most aesthetically pleasing reconstruction, both to the recipient defect and the donor forehead. The surgeon should clearly plan the flap and defect dimensions in order to obtain good results.

Reference 1.

Correa BJ, Weathers WM, Wolfswinkel EM, Thornton JF. The Forehead Flap: The Gold Standard of Nasal Soft Tissue Reconstruction. Seminars in Plastic Surgery. 2013;27(2):96103. doi:10.1055/s-0033-1351231.

Gillies, H., and Millard, R. The Principles and Art of Plastic Surgery. Boston: Little, Brown, 1957.

Menick FJ. A ten-year experience in nasal reconstruction with the three-stage forehead flap. Plast. Reconstr. Surg. 109: 1839, 2002

Hoffman HT, Baker SR. Nasal reconstruction with the rapidly expanded forehead flap. Laryngoscope. 1989;99;1096- 1098

*Corresponding author: Dr Japneet Kaur, Department of ENT, Dayanand Medical College and Research Institute, Ludhiana, Punjab, India Phone: +91 09902549237 Email: drjapneetkaur@gmail.com Date of Submission : 04.12.2017 Date of Acceptance : 15.12.2017 Financial or other Competing Interests: None. Date of Publication : 24.12.2017

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

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

Letter to Editor DOI: 10.21276/AABS.2017.1711

Metanephric Adenoma Aneeta Singh Malhotra*, Urvashi Andotra, Harminder kour Rai, Arvind Khajuria, Kuldeep Chander goswami Dept. of Pathology, Acharya Shri Chander College Of Medical Sciences And Hospital, Jammu, India

Dear sir

Metanephric adenoma / renal epithelial tumor are a cortical epithelial tumor with rare incidence of 0.2% of all epithelial neoplasms1. Metanephric adenoma tends to occur more commonly in females with a female:male ratio of 2:12. Tumor is benign3and are typically composed of solid, rare cystic components or calcifications with a poorly derived border4. Less than 200 cases have been reported (till 2015)4. Here we report a case of 20 year old female presented with pain right lumber region. A 20 year old female presented with pain (severe and sudden) right lumber region. On USG and CCET, a tumor involving superior pole of kidney measuring 5x4.7 cm with central necrosis was identified. Right nephrectomy was done and specimen was sent for histopathologic examination. Grossly the size of the kidney was 11x6x3cm, a well circumscribed growth measuring 6x5cm was identified on the upper pole extending upto the middle.

tissue appeared normal. All these features were suggestive of metanephric adenoma Histologically, metanephric adenoma have either sparse fibrous capsule or no capsule at all1. Metanephric adenoma overlapse in morphology with epithelial-predominant nephroblastoma in children younger than 12 years and show morphology similar to papillary renal cell carcinoma in adults5. Immunohistochemistry plays important role in diagnosis. Metanephric adenoma stains for CD57 and focally for CK71. Though it is benign, few cases of metastatic disease have been reported6,7.

On microscopic examination, a well circumscribed tumor with small, uniform, closely packed tubules accompanied by scant stroma. Cells were cuboidal with minimal cytoplasm, bland nuclei and uniform chromatin. Focal papillary architecture and glomeruloid bodies were also seen. Psammoma bodies were also seen . rest of the renal Fig. 2: microscopy, metanephric adenoma with a Psammoma body in the centre.

Fig. 1: a well circumscribed growth on the upper pole.

Fig. 3: Focal papillary architecture.

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

Letter to Editor

L-2

Reference 1.

Khoo CCK, Khetrapal P, Roux J, Bates AW, Mumtaz F (2014) Metanephric Adenoma: A case report with a Discussion of pathology and follow-up Duration. J Urol Res 1(3):1012.

Case report. Department of medical imaging, Subei peopleâ&#x20AC;&#x2122;s Hospital, Medical school of Yangzhou, Jiangsu 225001, P.R. china. Onchology letters 10: 1816-1818,2015 5.

Modern pathology(2015)28,1236-1248;doi:10.1038/ modpathol.2015.81

Jinous Saremian, MD, Melanie J Kubik, MD, Shahla Masood, MD.Cytologic features of metanephric adenoma of kidney : Case report and review of literature. Lab Med Spring 2015;46:153-158.

Pins MR, Jones EC, Martul EV, et al. Metanephric adenomalike tumors of kidney:report of three malignancies with emphasis on discrimating features. Arch Pathol LabMed. 1999;123:415-420.

Brisigotti M, Cozzutto C. Fabbretti G, Sergi C and Callea F: Metanephric adenoma Histol.

Jingtao WU,Qingqiang ZHU, Wenrong ZHU and Hongying Zhang: Metanephric adenoma with diffuse calcifications: A

Nakagawa T, Kanai Y, Fujimotott, et al. Malignant mixed epithelial and stromal tumors of the kidney:a report of the first two cases with a fatal clinical outcome. Histopathology.2004;44:302-4.

*Corresponding author: Dr. Aneeta Singh Malhotra, Acharya Shri Chander College Of Medical Sciences And Hospital, Jammu, India Phone: +91 9419319454 Email: aneetasinghmalhotra@gmail.com Date of Submission : 13.10.2017 Date of Acceptance : 14.11.2017 Date of Publication : 24.11.2017

Financial or other Competing Interests: None.

http://www.pacificejournals.com/aabs

Annals of Pathology and Laboratory Medicine Annals of Pathology and Laboratory Medicine (APALM) is an international, Double-blind peer-reviewed, indexed, open access, online and print journal with an Impact Factor (IFJ): 2.8952 and IC Value (ICV 2014): 67.16

Annals of Woman and Child Health

Cover Design: Dr Prashant Goyal

Annals of Woman and Child Health (AWCH) is an International, Fast Double-blind peer-reviewed, Indexed, Open access, online multidisciplinary journal, related to woman and child health

Pacific Group of eJournals ISO 9001:2008 Certified academic publishing house

www.pacificejournals.com Printed at: Allied Traders, New Delhi