Volume 30, Number 1 April 2018

Department of Medical Research Ministry of Health and Sports No. 5, Ziwaka Road, Dagon Township, Yangon 11191 Republic of the Union of Myanmar

AIMS OF THE JOURNAL

To serve as an important medium for the publication of original research

works in the field of medical science and health research, thus filling gaps

in health knowledge for effective utilization of research findings

To disseminate recent basic, applied and social research findings among

health personnel of different strata for enhancing nation-wide health

development in Myanmar

To offer current medical knowledge and updated scientific information

obtained from research to health professionals for better and appropriate

health care management

MYANMAR HEALTH SCIENCES RESEARCH JOURNAL MEMBERS OF EDITORIAL BOARD

Editor-in-Chief: Dr. Kyaw Zin Thant, HGP, MBBS, DTM, Ph.D, FACTM, Dip. in R & D, FRCP

Department of Medical Research Editors:

Dr. Win Aung, MBBS, MMedSc, FACTM, FRCP Department of Medical Research Dr. Hlaing Myat Thu, MBBS, MMedSc, MACTM, Ph.D, FRCP

Department of Medical Research Dr. Khin Saw Aye, MBBS, MMedSc, Ph.D, FRCP

Department of Medical Research Associate Editor:

Dr. Myat Htut Nyunt, MBBS, MMedSc, DAP & E, Ph.D Department of Medical Research

Dr. Ni Thet Oo, BVS, HGP, Dip ELTM Department of Medical Research

Ms Cho Mar Oo, BA, Dip. LibSc Department of Medical Research

International Editorial Board Members: Prof. Shigeru Okada, M.D, Ph.D Okayama University Hospital, Japan Dr. Sun Dae Song, M.D, M.P.H, Ph.D, D.S, M.S International Tuberculosis Research Center, Republic of Korea Prof. Christopher V. Plowe, M.D, M.P.H, FASTMH Duke Global Health Institute, USA Prof. Anthony David Harries, MA.MB.BChir. MD. FRCP. FFPH, DTM & H International Union Against Tuberculosis & Lung Disease, France London School of Hygiene & Tropical Medicine, UK Prof. David Alan Warrell, MA DM DSc FRCP FRCPE, FMedSci University of Oxford, UK Prof. Sir Roy Anderson, PhD, FRS, FMedSci Imperial College, London, UK

Editorial Board Members: Prof. Zaw Wai Soe, MBBS, MMedSc, FRCS, Dr. MedSc, Dip. Med Edu

University of Medicine (1), Yangon Prof. San San Nwet, MBBS, MMedSc, Ph.D, Dip. Med Edu

University of Pharmacy, Yangon Prof. Myat Thandar, MBBS, Ph.D, Dip. Med Edu

University of Nursing, Yangon Dr. Kyaw Oo, MBBS, MMedSc, M.Sc

Department of Human Resources for Health Prof. Theingi Myint, MBBS, Ph.D, Dip. Med Edu

University of Medicine (1), Yangon

Prof. Chit Soe, MBBS, MMedSc, MRCP, FRCP, Dr. MedSc, Dip. Med Edu University of Medicine (1), Yangon

Prof. Yan Lynn Myint, MBBS, MMedSc, MRCP, FRCP University of Medicine, Mandalay

Prof. Aye Mon, MBBS, MMedSc, FRCS, FICS, Dip. Med Edu University of Medicine (1), Yangon

Prof. San San Myint, MBBS, MMedSc, Dip. Med Edu, MRCOG, Dr. MedSc, FRCOG University of Medicine (1), Yangon

Prof. Khin Nyo Thein, MBBS, MMedSc, Dr. MedSc, FRCP, FRCPCH University of Medicine (2), Yangon

Prof. Wah Win Htike, MBBS, MMedSc, Ph.D, Dip. Med Edu University of Medicine (1), Yangon

Prof. Myint Myint Nyein, MBBS, MMedSc, Ph.D, Dip. Med Edu University of Medicine (1), Yangon

Prof. Hla Hla Win, MBBS, MMedSc, Ph.D, Dip. Med Edu University of Medicine (1), Yangon

Dr. Theingi Thwin, MBBS, MMedSc, Ph.D Department of Medical Research

Dr. Khin Thet Wai, MBBS, MMedSc, MA Department of Medical Research

Dr. Khin Phyu Phyu, BSc(Hons), MSc, Ph.D Department of Medical Research

Editorial Manager: Dr. Moh Moh Htun, MBBS, MMedSc, Ph.D, Post-Graduate Diploma in English

Department of Medical Research Editorial Assistant:

Ms Nilar Soe, BA, Dip. Japanese Language, Dip. LibSc Department of Medical Research

Ms Cho Cho Lwin, BA, Dip. Global English, Dip. IT Department of Medical Research

--------------------------------------------------------------------------------------------------------------- Publisher:

Dr. Zaw Myint, MBBS, Ph.D (01263) Department of Medical Research

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Myanmar Health Sciences Research Journal

Journal description

The Myanmar Health Sciences Research (MHSR) Journal is published by the Department of Medical Research, Ministry of Health and Sports, Republic of the Union of Myanmar. It publishes original articles, review articles, short reports and correspondences in the field of biomedical and health sciences. It has been published since April 1989 with ISSN 1015-0781. The submitted manuscript is double blind peer-reviewed by two referees with expertise in the field of research work related to the paper. The Journal is published every 4th month (i.e. April, August and December) in a year. The annual meeting of the MHSR Editorial Board is usually held in September of the year. The distribution of journal is limited and free of charge around the country and to some international institutions and libraries.

Guidelines to Authors

All manuscripts must be submitted On-line at the website of MHSR Journal (www. myanmarhsrj.com) where "Instruction to Authors for Submission" can be obtained in detail. When submitting, the author must create the cover letter addressing to the Editor, the MHSR Journal, Department of Medical Research. It should contain; description of the novelty and importance of the work briefly, the agreement from all co-authors for submitting the manuscript to MHSR Journal, mentioning that the manuscript and its content have not previously been published elsewhere, and describing that all authors accept responsibility for releasing this material on behalf of any and all co-authors. Also, ethical consideration needs to be mentioned in the manuscript. The manuscript submitted to the MHSR Journal must be prepared as follows:

Original Articles

A manuscript text file consisting of title, names of author and co-authors (not more than 9, email addresses, telephones, fax numbers and the full postal address of the corresponding author), Designation, Department/Institution, keywords (maximum 6 words), Abstract (not more than 350 words), Introduction, Materials and Methods, Results, Discussion, Acknowledgement, References. No figures or tables should be included in this file.

The manuscript must be written in English clearly and concisely. The manuscript should be prepared in single column and double-spacing with sufficient margins (2.5 cm) in

A4 paper. Times New Roman font with size 12 pt is preferable and line numbers should be showed continuously throughout the pages numbered consecutively.

Number of figures and tables should not exceed 4. Tables and Figures should each be numbered consecutively. Line drawings should be of high resolution and high contrast. Black-and-white or color photographs may be

accepted provided that they are of high quality. They should be provided as computer graphic files after the manuscript is accepted for publication in the final form. Details of results presented in this way should not be repeated in the text.

The manuscript should be separated into 3 files: Text, Table and Figure. These files should be created using Microsoft Word (.doc, .docx files)

The manuscript including the combined number of tables and figures should not be more than 3500 words.

Review Articles

These should largely cover the current review concerning the medical and health research publications pertaining to Myanmar. They should not be more than 5000 words.

The manuscript should be prepared in single column and double-spacing with sufficient margins (2.5 cm) in A4 paper. Times New Roman font with size 12 pt is preferable. Line numbers should be shown continuously throughout the pages numbered consecutively.

The file should be created using Microsoft Word (.doc, .docx files)

ISSN 1015-0781

General Information

Short Reports These should be similarly headed, but do not need an abstract nor divisions into sections. They should not

be longer than 1000 words, including references (not more than 5) and one illustration or figure if necessary. The figure should be sent separately.

The manuscript should be prepared in single column and double-spacing with sufficient margins (2.5 cm) in A4 paper. Times New Roman font with size 12 pt is preferable, line numbers should be showed continuously throughout the pages numbered consecutively.

The text and figure files should be created using Microsoft Word (.doc, .docx files). Correspondences Letters to the Editor on general topics of interest related to health, comments on papers published in this

Journal, and, if appropriate, replies to comments are welcome. Letters should generally not exceed 350 words; tables and figures are not accepted. References (not more than 2) may be given only if essential.

References Reference should be cited as outlined by International Committee of Medical Journal Editors. The number

of references should be kept to a minimum; only those that are indispensable to substantiate a statement or as sources of further information should be included. Citation of published literature in the text should be numbered consecutively in the order in which they are first mentioned in the text.

The following form may be used: - Dengue is a major public health problem in tropical and subtropical countries.1, 2 - Tun Lin, et al.3 revealed that An. minimus was mostly collected between 10 and 11 pm in indoor in

Thabwewa Village, Oktwin Township, Bago Region.

References to articles should contain the following: name of author(s), title of the article, name of the journal, year and month of issue, volume (no.), first and last pages. Examples:-- 1. One author: Than Than Htwe. In vitro nitric oxide (NO) production of murine cell line and human

monocyte-derived macrophages after cytokine stimulation and activation. Myanmar Health Sciences Research Journal 1997; 9(2): 85-88.

2. Three to six authors: Kyaw Zin Thant, Morita K & Igarashi A. Sequences of E/NS1 gene junction from four dengue-2 viruses of Northeastern Thailand and their evolutionary relationships with other dengue-2 viruses. Microbiology and Immunology 1995; 39(8): 581-590.

3. More than 6 authors: Khin May Oo, Yi Yi Kyaw, Ohmar Lwin, Aye Aye Yee, San San Oo, Khine Win, et al. Hepatitis B surface antigen sero-prevalence in two border towns near Thailand. Myanmar Health Sciences Research Journal 2009; 21(2): 83-87.

4. Book: Thaw Zin. Role of Analytical Toxicology Laboratory in the prevention, control and management of poisoning: Principle and guidelines. In: Guidelines on Poison Prevention, Control and Management. Department of Medical Research (Lower Myanmar), Ministry of Health, 2003; 76-98.

5. Conference: Moe Kyaw Myint, Khin Lin, Mya Moe, Phyu Phyu Win, Win Htay Hlaing, Thaung Hlaing, et al. Epidemiological assessment of climate change and Malaria Trend. Programme and Abstracts of the 43rd Myanmar Health Research Congress; 2015 Jan 5-9; Yangon, Myanmar. p. 57.

6. Thesis: Win Aung. A study on uirnary N-Acetyl-β-D-Glaucosaminidase excretion in russell's viper bite patients with systemic envenomation. [MMedSc thesis]. Institute of Medicine 2: Yangon; 1993.

7. Internet source: Atherton, J. Behaviour modification [Internet]. 2010 [updated 2010 Feb 10; cited 2010 Apr 10]. Available from: http://www.learningand teaching.info/learning/behaviour_mod.htm

After you have completed Manuscript preparation, please log in to the MHSR Journal On-line Submission and to get successful submission, please follow step-by-step of the submission process. You may suspend a submission at any stage as draft, and save it to submit later. After completed submission, you can also log in any time to check the status of your manuscript.

Myanmar Health Sciences Research Journal has no page charges.

------------------------------------------------------

All inquiries should be directed to:

The Editor, The Myanmar Health Sciences Research Journal, Department of Medical Research, No. 5, Ziwaka Road, Dagon Township, Yangon 11191, Myanmar

Tel: 951-375447, 951-375457, 951-375459 Fax: 951-251514 email: MHSRJadmin@myanmarhsrj.com, http://www.myanmarhsrj.com.

MMYYAANNMMAARR

HHEEAALLTTHH SSCCIIEENNCCEESS RREESSEEAARRCCHH JJOOUURRNNAALL

Department of Medical Research

No. 5, Ziwaka Road

Dagon Township

Yangon, 11191

Republic of the Union of Myanmar

ISSN 1015-0781

Volume 30, Number 1 Published since 1989 April 2018

CONTENTS

Editorial………………………………………………………………………………...... i

Original Articles:

Evaluation of Pharmacokinetics and Pharmacodynamics of Levofloxacin in Patients with Acute Exacerbation of Chronic Obstructive Pulmonary Disease……………….. Khin Hnin Pwint, Mar Mar Kyi, Khin Chit, Yamin Ko Ko, Moe Moe Aye, Phyu Phyu Aye, Phyo Wai Aung, Mya Mya Moe & Thizar Myo

1

Repellency Effect of Artemisia vulgaris Essential Oil against Aedes aegypti Mosquitoes in Laboratory Condition……………………………………………………………..... Maung Maung Mya, Nwe Nwe Oo, Aye Win Oo, Sein Thaung & Yan Naung Maung Maung

8

Study on Protective Effects of Malaria Antibody among the Community in Malaria Endemic Areas……………………………………………………………………....... Moe Kyaw Myint, Khin Lin, Aung Thu, Mya Moe, Phyu Phyu Win, Zaw Lin & Kyaw Zin Thant

15

Insulin Receptor Substrate-1 Gene (G972R) Polymorphism and Metabolic Syndrome in Type-2 Diabetes Mellitus Patients……………………………………………………. Thae Nu Htwe, Myat Mon Oo, Saw Wut Hmone, Moh Moh Htun, Ohnmar Myint Thein & Myat Thandar

21

Profile of Paediatric Malignancies in Yangon Children’s Hospital: 2012-2014………… Ssu Wynn Mon, Aye Aye Khaing, Han Win, Tint Myo Hnin, Ye Myat Thu, Theingi Thwin & Kyaw Zin Thant

26

Urinary Albumin to Creatinine Ratio and the Degree of Coronary Artery Narrowing in Patients Undergoing Coronary Angiography……………………………………….... Khine Wai Mon, Saw Wut Hmone, Myint Myint Nyein & Myat Mon

32

Ki-67 Immunoexpression in Gestational Trophoblastic Diseases……………………….. Nyein Nyein Soe, Saw Wut Hmone, Myint Myint Nyein & Myat Mon

40

Molecular Detection of Human Rhinoviruses in Children with Influenza-like Illness Attending Yangon Children’s Hospital, 2016………………………………………... Htin Lin, Hlaing Myat Thu, Theingi Win Myat, Win Mar, Khaing Moe Aung, Khin Khin Oo, Khin Sandar Aye, Thida Kyaw & Ye Myint Kyaw

46

Role of Flow Cytometric Immunophenotyping in Diagnosis of Multiple Myeloma……. San San Htwe, Htun Lwin Nyein, Aye Mya Khine, Khin La Pyae Tun, Win Pa Pa Naing, Ni Ni Win, Moe Thuzar Min, Sein Win & Khin Saw Aye

52

Physical Fitness of the Elderly People from Home for the Aged (Hninzigone), Yangon………………………………………………………………………………... Khin Mi Mi Lay, Nway Htike Maw, Pyae Phyo Kyaw, Sandar Win, Khin San Lwin, Htet Htet Lwin, Yi Yi Mon, Lei Lei Win Hlaing & Theingi Thwin

58

Genotypic Analysis of Epstein-Barr Virus (EBV) in Nasopharyngeal Carcinoma Patients………………………………………………………………………………... Moh Moh Htun, Chaw Su Hlaing, Myat Mon Oo, Kay Thi Aye, Aung Zaw Latt, Soe Aung, Hlaing Myat Thu, Khin Pyone Kyi & Kyaw Zin Thant

64

Plasma Malondialdehyde Level and Vibration Perception Threshold in Non-exposed Subjects and Lead-exposed Battery Workers………………………………………… Thura Tun Oo, Zaw Linn Thein, Zarli Thant & Ohnmar

71

Are They Disclosed?: Situation of HIV Status Disclosure among Adolescents in Four Townships of Myanmar………………………………………………………………. Kyaw Min Htut, Myo Myo Mon, Lwin Lwin Ni, Aung Soe Min & Ni Ni Htay Aung

76

Short Reports: Applications of Alumina Nanoparticles from Coal Fly Ash……………………………..

Myat Myat Thaw & Khine Thin Thin Aye

83

Iodine Deficiency in Pregnant Women Living in the Coastal Area of Mon State.............. Theingi Thwin, Moh Moh Hlaing, Mya Ohnmar, Sandar Tun, Thidar Khine, Wah Wah Win, Su Su Hlaing & Hla Phyo Lin

85

i

EDITORIAL

The 46th Myanmar Health Research Congress, organized by the Ministry

of Health and Sports was successfully held in Department of Medical

Research from 8th to 12th January, 2018 with the theme, "To prevent,

Detect, Treat & Live with Cancer" in order to promote the awareness on

increasing burden of the cancer in our country. A total of 110 research

papers and 80 posters were presented by local and international

researchers. During the academic session, not only cancers, but also the

various disciplines of research areas including malaria, tuberculosis,

hepatitis, dengue, maternal and child health, snake bite, food and drug

administration, traditional medicine, reproductive health and environ-

mental health, were presented in the congress to achieve the highest

quality in health care by promoting research capacity strengthening of

the healthcare personnel. Apart from the paper and poster session,

12 symposia and 4 scientific talks on current health issues in

Myanmar were presented and discussed by the researchers to share and

exchange their knowledge, comprehensive views, opinion and valuable

experiences.

Although presentation in the Research Congress is one of the routes for

disseminations of the research findings, the researchers should be aware

of the importance of contribution in way of the academic publications in

local as well as international peer-reviewed journals. We have warmly

invited all researchers who presented their research findings in Myanmar

Health Research Congress to submit their papers to our Journal,

Myanmar Health Sciences Research Journal.

At the same time, the annual meeting of the MHSRJ editorial board

members including the participation of International Editorial Board

Members was held on January 10, 2018. In this meeting, we decided to

promote the status of the journal by linkage to PubMed in the coming

years by encouraging the researchers to submit the original as well as

ii

review articles to MHSRJ taking the advantages of open-accessed and

"free-of-charge" journal and ensuring the highly qualified peer review

process.

In this issue, we would like to highlight the important considerations on

optimizing the use of antibiotics, as the pharmacologists' perspective

with a leading article, "Evaluation of Pharmacokinetics and Pharmaco-

dynamics of Levofloxacin in Patients with Acute Exacerbation of

Chronic Obstructive Pulmonary Disease". This hospital based analytical

study indicated that the adequacy on pharmacokinetic and pharmaco-

dynamics of levofloxacin in patients with acute exacerbation of chronic

obstructive pulmonary disease was found to be low to achieve the

targeted value even in clinically cured cases. Rational use of antibiotics

with correct dose is crucial to prevent antimicrobial resistance.

Moreover, a total of 13 original research articles and 2 short reports

were published in this issue providing the relevant up-to-date research

findings on various topics such as vector control, serological aspect of

malaria, metabolic syndrome in diabetes, paediatric and adult

malignancies, cardio-vascular diseases, gestational trophoblastic

diseases, influenza-like illness, multiple myeloma, physical fitness of the

elderly people, lead poisoning and disclosure in HIV status. Moreover,

short reports on applications of alumina nanoparticles from coal fly ash

and iodine deficiency among pregnant women living in coastal region in

Myanmar, are also reported.

As every article in this issue provides considerable value in their

respective research area, we believe that knowledge of the researcher

would be improved to utilize the research findings in daily practice in

health care with innovative ideas in various ways. Moreover, we would

like to invite all readers to visit our MHSRJ web page,

“www.myanmarhsrj.com” to access the archive of previous published

articles.

Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Evaluation of Pharmacokinetics and Pharmacodynamics of Levofloxacin in Patients with Acute Exacerbation of Chronic Obstructive Pulmonary Disease

Khin Hnin Pwint1*, Mar Mar Kyi2, Khin Chit1, Yamin Ko Ko1,

Moe Moe Aye1, Phyu Phyu Aye1, Phyo Wai Aung1, Mya Mya Moe1 & Thizar Myo1

1Department of Medical Research

2University of Medicine 2 (Yangon)

Certain antimicrobial therapy should target not only clinical success but prevention of resistance in future. In modern antimicrobial therapy, in addition to a measure of the potency of the drug for the pathogen (Minimum Inhibitory Concentration-MIC), a measure of drug exposure of the individual patient (pharmacokinetic data) becomes essential component in rational dosage regime. The aim of the study was to assess thepharmaco-kinetic/pharmacodynamic (PK/PD) adequacy of levofloxacin in the treatment of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Hospital-based analytical study was done in 20 patients with AECOPD admitted to three teaching hospitals. Serial blood collection was done to determine pharmacokinetics of oral and intravenous infusion levofloxacin in these patients. Culture and sensitivity of infecting pathogens were done and PK/PD indices were calculated by integrating pharmacokinetic data(Cmax or AUC) with microbiological data (MIC). Pharmacokinetic para-meters were not significantly different between different routes indicating interchangeability of the route of administration of levofloxacin. Pathogens isolated were Klebsiella spp. (20%), Escherichia coli (10%), Haemophilus influenzae (10%), Staphylococcus aureus (5%), Streptococcus pneumoniae (5%) and Pseudomonas spp. (5%). Although the resulted PK/PD indices were found to be low to achieve the targeted PK/PD values (Cmax/MIC of ≥8-10 and AUC/MIC of ≥87) in most patients, 90% (18/20) achieved clinical cure. The results indicated that although patients got clinical cure, microbiological eradication was uncertain and risk of emergence of resistance was high. This study highlighted the need of efficacy indices (PK/PD indices) for optimizing antimicrobial therapies in various infections for prevention and reduction of antimicrobial resistance problem in Myanmar.

Key words: Levofloxacin, PK/PD, COPD patients, Myanmar

INTRODUCTION

Lower respiratory tract infections (LRTIs) namely acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and pneumonia are the leading infectious causes of death in most countries. In Myanmar, pneumonia and acute lower respiratory tract infections are listed among the leading causes of morbidity and mortality.1 Evolving drug resistance makes treating lower respiratory tract infections a major challenge.

In practice, most empirical LRTI therapy is clinically based and bacterial eradication is usually considered secondarily. Failure to kill or eradicate causative bacteria is most likely to result from sub-optimal therapy which predispose to resistance emergence. Spontaneous clinical recovery may mask differences in bacteriological effectiveness of antibiotics and allow suboptimal agents to continue to be prescribed.2, 3 Successful ____________________________________ *To whom correspondence should be addressed. Tel: +95-95251323 E-mail: khinhninpwint@gmail.com

2

antimicrobial therapy should target not only clinical success but prevention of resistance in future. In modern antimicrobial therapy, in addition to a measure of the potency of the drug for the pathogen (MIC), a measure of drug exposure of the individual patient (pharmacokinetic data) becomes essential component in rational dosage regime. Integration of MIC with pharmacokinetic (PK) parameter provides pharmacokinetic/ pharmacodynamic indices, which are valuable tools with which to predict clinical outcome, microbiologic outcomes and optimal drug dose.4, 5 Animal and clinical data indicated that the development of resistance is correlated with dosing regimens that do not achieve sufficiently high PK/PD indices.6 These indices include time above MIC (T>MIC); the ratio between peak serum concentration (Cmax) and MIC (Cmax/MIC) and relation between drug exposure i.e. area under the serum 24 hours concentration-time curve (AUC0-24) and MIC (AUC0-24/MIC).

Levofloxacin is a fluoroquinolone anti-bacterial agent characterized by a broad spectrum of antibacterial activity against aerobic microorganisms, both gram-negative and gram-positive, which may cover most of the aetiological agents frequently responsible for AECOPD. As levofloxacin has a good bioavailability, early and simple change of route from intravenous to oral treatment is possible. Moreover, because of being given without interference with other drugs, having fewer side effects and an excellent and rapid bactericidal activity, levofloxacin offers the clinician and patient an excellent therapy for AECOPD.

Studies on pharmacokinetics of quinolones like levofloxacin in correlation with MIC of commonly found pathogens (PK/PD indices) in Myanmar are urgently needed to assess whether the currently used regimens for LRTIs are really effective or not. Therefore, this study aimed to evaluate pharmacokinetic and pharmaco-dynamic indices of the standard regimen of

levofloxacin 500 mg once daily, frequently used in routine clinical practice for the treatment of patients with AECOPD. The most preferable treatments in AECOPD are those that are effective and minimize the amount of time that the patient spends in hospital. Oral or intravenous infusion levofloxacin has been prescribed frequently as either monotherapy or in combination therapy of AECOPD in Myanmar. There were some studies indicated that intra-venous therapy incurred longer hospital stay, higher cost and more discomfort to patient than oral therapy.7, 8

Therefore, the second objective was to study pharmacokinetic profiles of oral and intravenous infusion levofloxacin to expedite a sequential timely conversion from intravenous to oral therapy, or replacing intravenous with oral treatment for enabling both a cost-effective treatment of infections and an early hospital discharge.

MATERIALS AND METHODS

The study was prospective, non-blinded pharmacokinetic-pharmacodynamic study. Patients were recruited from North Okkalapa, Thingangyun and Insein general and teaching hospitals. Twenty patients with AECOPD (10 in oral group and 10 in intravenous infusion group) admitted to study hospitals participated in the study. Laboratory tests, sputum culture and sensitivity test and assay for levofloxacin were carried out at Department of Medical Research. Adult patients admitted to medical ward of study hospitals who were ≥18 years of age, diagnosed as AECOPD by physician in charge, prescribed oral or intravenous infusion levofloxacin as part of their required medical care, were eligible for inclusion in this study and agreed to sign a written informed consent. Patients receiving therapy with any drugs capable of causing pharmacocokinetic drug interactions with levofloxacin and quinolone antibacterials within two weeks were excluded.

3

Identifications of organisms and suscepti-bility testing for levofloxacin

The aetiological agents were assessed by cultures of sputum before administration of drug and, whenever isolated, in vitro susceptibility (by Modified Kirby-Bauer method) to levofloxacin were tested. The results were interpreted by comparing with standard zone size for each drug from zone size interpretation chart as resistant, intermediate or susceptible.

Pharmacokinetic study The intravenous infusion and oral pharma-cokinetic evaluations were carried out on 3rd day under steady-state conditions. From each patient, blood sample (3 ml) was taken from forearm vein through the cannula at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6 and 24 hours after dosing. Levofloxacin plasma concentrations were analyzed by means of a high per-formance liquid chromatography using the method of Wong et al.9 with some modifications. Analysis of pharmacokinetic parameters and PK/PD indices Data entry and analysis were done using Microsoft Excel 2007 and SPSS version 22. Model dependent pharmacokinetic analysis was done for each patient. PK/PD indices were also analyzed by Microsoft Excel 2007. The level of statistical significance for all statistical tests was defined as a ‘p’ value less than 0.05. Ethical consideration This study was reviewed and approved by Ethics Review Committee of University of Medicine 2 (Yangon).

RESULTS

Patient characteristics Mean age of the patients was 71.4±9.63 years and male to female ratio was 1:2.3. The average weight was 48.23±11.45 kg with the mean BMI and creatinine clearance of 0.44±4.64 kg/m2 and 7.68±21.58 ml/min, respectively.

Bacteriological identification from sputum of AECOPD patients

Only 11 out of 20 patients (55%) had a microbiologically confirmed bacterial etiology. The infection was monomicrobial in all cases and 6 different species of micro-organisms were isolated. The most common pathogen was Klebsiella spp. (n=4, 20%), followed by Escherichia coli (n=2, 10%), Haemophilus influenzae (n=2, 10%), Staphylococcus aureus (n=1, 5%), Strepto-coccus pneumonia (n=1, 5%) and Pseudo-monas spp. (n=1, 5%).

More than 80% of isolates were shown to be sensitive in vitro to levofloxacin except one Klebsiella spp. and Streptococcus pneumonia (Fig. 1). As most of the organisms isolated were gram-negative ones, azithromycin was not sensitive in most cases. Ceftriaxone was found out to be resistant to one Klebsiella isolate, Staphylococcus aureus, Streptococcus pneumoniae and Pseudo-monas isolates (resistance percent - 36.4%). Co-amoxiclav resistance was found in one Klebsiella spp., one Haemophilus influenzae isolate and Pseudomonas isolate (resistance percent - 27.3%) (Fig. 1).

Fig. 1. Sensitivity pattern of microorganisms iso-lated from AECOPD patients

The mean plasma concentration-time profiles of oral and intravenous infusion levofloxacin in AECOPD patients are compared (Table 1 & Fig. 2). Plasma levo-floxacin concentration rapidly rose within 1 hour and reached the peak at 1 hour in

A =Azithromycin C=Coamoxiclav B =Ceftriaxone D=Levofloxacin

A B C D

Per

cent

age

Sensitive Resistance

A B C D

4

both oral and intravenous infusion groups with the highest levels of 11.74±6.45 and 19.22±6.07 µg/ml, respectively. Table 1. Comparison of pharmacokinetic para-

meters of oral and intravenous infusion levofloxacin in AECOPD patients

Para- meters Unit

Oral (n=10)

IV infusion (n=10) P

value Mean SD Mean SD A µg/ml 21.80 9.29 31.70 18.02 B µg/ml 10.42 6.18 8.98 2.88 Kab h-1 2.06 1.25 2.83 1.26 0.2 t1/2ab h 0.55 0.41 0.27 0.08 0.05 Alpha h-1 0.47 0.10 0.6 0.19 0.07 Beta h-1 0.06 0.02 0.05 0.02 0.3 t1/2α h 2.04 0.72 1.62 0.79 0.2 t1/2β h 12.73 3.85 17.1 6.19 0.07 AUC µg/ml.h 212.64 93.57 265.24 103.71 0.2 CL L/h 2.79 1.2 3.58 1.36 0.2 Vd L 51.27 25.73 50.88 19.37 0.9 Cmax µg/ml 11.74 6.45 19.22 6.07 0.02* Tmax h 2.1 1.41 1.05 0.16 0.03*

A=Concentration at time “0” which was back extra- polated from distribution phase

B=Concentration at time “0” which was back extrapolated from elimination phase

Alpha=Distribution rate constant of two compart-ment model,

Beta=Elimination rate constant of two compartment model

Fig. 2. Comparison of plasma concentration-time profiles of oral and intravenous levofloxacin in AECOPD patients

Plasma levofloxacin concentration then grdually decreased within 6 hours to 24 hours after administration in oral group but in intravenous infusion group, there was a steep fall within 1 hour and then a gradual fall up to 24 hours. Though the mean plasma concentrations of levofloxacin in intravenous

infusion group of patients were higher than oral group at most time points, significant increase in concentration was seen only at 1 hour sample (peak) (Table 1 & Fig. 2).

Creatinine clearance (CLcr) of the patients ranged from 12.2 to 90.1 ml/min with the mean of 39.69±18.78 ml/min. Correlation by linear regression analysis showed positive correlation between estimated creatinine clearance (CLcr) and levofloxacin clearance (CL) of the COPD patients (r2=0.8041, p<0.05) (Fig. 3).

Fig. 3. Relationship between levofloxacin

plasma clearance (CL) and estimated creatinine clearance (CLcr) by means of

the Cockcroft and Gault formula Pharmacokinetic-pharmacodynamic target attainment in AECOPD patients

Infecting bacteria sensitive to levofloxacin were assumed to have MIC ≤2 µg/ml.10

Studies have shown that both the Cmax/MIC and AUC0-24/MIC ratios are good predictors of fluoroquinolone efficacy. All patients achieved the desired Cmax/MIC at MICs of ≤1 µg/ml. However, only 11.11% (1 out of 9) achieved desired Cmax/MIC at 2 µg/ml. All patients achieved the desired AUC0-24/ MIC of ≥50 for treatment of infections caused by gram-positive pathogens with MICs of ≤1 µg/ml. For gram-negative organisms, only 66.67% of patients achieved desired AUC0-24/MIC ratio of ≥87 at a MIC of 1 µg/ml. At MIC of 2 µg/ml, only 33.33% achieved lower threshold of targeted PK/PD index (AUC0-24/MIC ≥50), 22.22% achieved PK/PD index of 87 and no patient achieved higher PK/PD index of 125 (Table 2).

Levofloxacin clearance (ml/min/kg)

Est

imat

ed C

L cr (

ml/m

in/k

g) 2.50

2.00

1.50

1.001.500.501.500.001.500.00

1.500.501.50

1.001.50

1.50 2.00

y=1.348x-0.2724 r2=0.8041

5 10 15 20 25 300

201816141210

68

42

0

IV infusion Oral

Time (hours)

Pla

sma

conc

entra

tion

(µg/

ml)

IV infusion Oral

5

Table 2. Percentage of AECOPD patients achieving targeted PK/PD indices with administration of levofloxacin 500 mg once daily dose

PK/PD indices % of patients achieving target PK/PD indices at different MIC

0.25 µg/ml (Low)

1 µg/ml (Medium)

2 µg/ml (High)

Cmax/MIC 10 100.0 100.0 11.1

AUC0-24/MIC 50 100.0 100.0 33.3 87 100.0 66.7 22.2 125 100.0 22.2 0.0

For sensitive organism, MIC levels of 0.25, 1 and 2 µg/ml were used to evaluate achievement of target AUC0-24/MIC and Cmax/MIC indices for each patient. The targeted PK/PD indices for levofloxacin are Cmax/MIC ratios of ≥10; AUC0–24/MIC ratio of ≥87 to 125 for gram-negative bacterial infections; AUC0-24 /MIC ratio of ≥35 to 50 for gram-positive pathogens.5, 11-13

Outcome of therapy

Median length of hospital stay was 5 and 6 days for oral and IV treatment, respect-tively. In patients who responded, clinical symptoms subsided in 2.50±0.52 days. At the end of levofloxacin therapy, the overall clinical success rate was 90% since 18 out of 20 cases were cured.

DISCUSSION

In teaching hospitals where the present study was done, macrolides, second or third generation cephalosporins, co-amoxiclav and levofloxacin were found to be commonly used antibiotics either single or in combination therapy of AECOPD. In this study, sensitivity of pathogens to levofloxacin was the highest followed by co-amoxiclav and ceftriaxone (Fig. 1). The difference in sensitivity might be due to antibiotic usage pattern of the study hospitals.

Furlanut, et al.14 reported that peak serum concentration of levofloxacin after oral and intravenous infusion were 7.93±3.44 µg/ml and 10.71±3.30 µg/ml, respectively and the values were lower than those found in Myanmar AECOPD patients. AUC0-α was 74.97±29.29 µg.hr/ml and 85.60± 38.21 µg.hr/ml, respectively and these

values were also lower than the present study. Lower clearance and longer elimination half-life were found in Myanmar AECOPD patients than other studies. The difference between Myanmar patients and other studies may be due to difference in weight (48.23±11.45 kg vs. 71±15 kg) and difference in renal function (creatinine clearance of 37.68±21.58 ml/min vs. 73.13±6.0 ml/min). The results showed that Myanmar patients had a larger drug exposure to the test drug (levofloxacin).

Most of the pharmacokinetic parameters were not different statistically between different routes of administration except higher Cmax and shorter Tmax in intravenous infusion group and the results of this study agreed with the study of Furlanut, et al.14

The differences may be because of food effect in oral group as patients had to be allowed to take levofloxacin whether they were fasted or not. This study also clearly demonstrates that great inter-patient variability is present and high degree of variability was observed to depend on difference in renal function which was described by creatinine clearance. Saito, et al.15 and Kiser, et al.8 also reported that inter-individual variation in levofloxacin pharmacokinetics was largely related to estimated creatinine clearance since the drug is excreted unchanged from kidney.

The results of present study showed that duration of hospital stay was 1 day shorter in oral group than intravenous group with no difference in pharmacokinetic parameters which determined the efficacy of levoflo-xacin (AUC, Cmax) between the routes. Although intravenous levofloxacin should be considered the optimal route for initial empirical therapy in hospitalized patients with AECOPD, the oral route may represent both an effective and cost saving regimen in mild to moderate AECOPD outpatients or as a continuation therapy.

In this study, almost all patients achieved the PK/PD target for gram-positive organisms (AUC/MIC of 35-50) showing

6

that AECOPD patients in the study hospitals would be adequately treated with 500 mg of levofloxacin once daily regime. However, because of the high incidence of infections caused by gram-negative organisms in AECOPD patients, higher targeted AUC0-24/ MIC ratios (≥87) are necessary for effective empirical therapy. Higher doses of levoflo-xacin (i.e. greater than 500 mg/day) would be necessary to reliably achieve higher PK/PD indexes for treatment of infection caused by such organisms.

Use of levofloxacin as part of combination regimens would be the most appropriate clinical approach for the empirical treatment of severe systemic infections, especially gram-negative bacterial infections in this population. Some studies suggested the use of high dose, short course therapy (750 mg)16 or twice daily regime of levo-floxacin in the view of clinical and prevention of resistance.17, 18

Although some patients in this study got clinical cure despite their low PK/PD indices, further emergence of resistance in those patients was questionable. Most of the patients in this study were elderly with decreased clearance resulting in increased plasma concentration. Even if in these patients, microbiological eradication and emergence of resistance was doubtful, the treatment outcome in younger patients with severe LRTIs (e.g., community acquired pneumonia) may be questionable with 500 mg once daily regime. Conclusion

This is the first study conducted in Myanmar regarding the pharmacokinetic disposition of levofloxacin in AECOPD patients. Despite several limitations, this study nevertheless provides the pharmaco-kinetic profiles of oral and intravenous routes of levofloxacin in Myanmar people. Moreover, this study will support to increase awareness in the medical community of the importance of PK/PD indices in optimal dosage regime and prevention of antibiotic resistance.

REFERENCES

1. Health in Myanmar, Ministry of Health, 2014. 2. Mlynarczyk G, Mlynarczyk A & Jeljaszewicz J.

Epidemiological aspects of antibiotic resistance in respiratory pathogens. International Journal of Antimicrobial Agents 2001; 18: 497-502.

3. Dagan R, Klugman KP, Craig WA & Baquero F. Evidence to support the rationale that bacterial eradication in respiratory tract infection is an important aim of antimicrobial therapy. Journal of Antimicrobial Chemotherapy 2001; 47(2): 129-40.

4. Andes D. In vitro pharmacokinetic and pharma-codynamic properties of antimicrobials in the therapy of respiratory tract infections. Current Opinion in Infectious Disease 2001; 14(2): 165-72.

5. Craig W. In vitro pharmacokinetic/pharmaco-dynamic parameters: Rationale for antibacterial dosing of mice and men. Clinical Infectious

Diseases 1998; 26: 1-12. 6. Ball P, Baquero F, Cars O, File T, Garau J,

Klugman K, et al. Antibiotic therapy of community respiratory tract infections: strategies for optimal outcomes and minimized resistance emergence. Journal of Antimicrobial Chemo-therapy 2002; 49(1): 31-40.

7. Chien SC, Chow AT, Natarajan J, Williams RR, Wong FA, Rogge MC, et al. Absence of age and gender effects on the pharmacokinetics of a single 500-milligram oral dose of levofloxacin in healthy subjects. Antimicrobial Agents and Chemotherapy 1997; 41(7): 1562-5.

8. Kiser TH, Hoody DW, Obritsch MD, Wegzyn CO, Bauling PC & Fish DN. Levofloxacin pharmacokinetics and pharmacodynamics in patients with severe burn injury. Antimicrobial Agents and Chemotherapy 2006; 50(6): 1937-45.

9. Wong FA, Juzwin SJ & Flor SC. Rapid stereospecific high-performance liquid chromato-graphic determination of levofloxacin in human plasma and urine. Journal of Pharmaceutical and Biomedical Analysis 1997; 15(6): 765-71.

10. DeFife R, Scheetz MH, Feinglass JM, Postelnick MJ & Scarsi KK. Effect of differences in MIC values on clinical outcomes in patients with bloodstream infections caused by gram-negative organisms treated with levofloxacin. Anti-microbial Agents and Chemotherapy 2009; 53(3): 1074-9.

11. Cazzola M, Matera MG, Donnarumma G, Tufano MA, Sanduzzi A, Marchetti F, et al. Pharmacodynamics of levofloxacin in patients with acute exacerbation of chronic bronchitis. CHEST 2005; 128(4): 2093-8.

7

12. Drusano GL & Craig WA. Relevance of pharmacokinetics and pharmacodynamics in the selection of antibiotics for respiratory tract infections. Journal of Chemotherapy 1997; 9(3): 38-44.

13. Schentag JJ. In vitro correlation of pharma-cokinetic parameters to efficacy of antibiotics: relationships between serum concentrations, MIC values, and bacterial eradication in patients with gram-negative pneumonia. Scandinavian Journal of Infectious Diseases 1991; 74: 218-34.

14. Furlanut M, Brollo L, Lugatti E, Di Qual E, Dolcet F, Talmassons G, et al. Pharmacokinetic aspects of levofloxacin 500 mg once daily during sequential intravenous/oral therapy in patients with lower respiratory tract infections. Journal of Antimicrobial Chemotherapy 2003; 51(1): 101-6.

15. Saito A, Oguchi K & Harada Y. Pharmacokinetics of levofloxacin in patients with impaired renal

function. Chemotherapy 1992; 40(3): 188-95. 16. Dunbar LM, Wunderink RG, Habib MP, Smith

LG, Tennenberg AM, Khashab MM, et al. High- dose, short-course levofloxacin for community-acquired pneumonia: A new treatment paradig. Clinical Infectious Diseases 2003; 37(6): 752-60.

17. Holland ML, Chien SC & Corrado ML. The pharmacokinetic profile of levofloxacin following once- or twice-daily 500 mg oral administration of levofloxacin hemihydrates. 5th International Symposium on New Quinolones, Programme and Abstracts; 1994 August 25-27; Singapore.

18. Jensen KM & Paladino JA. Cost-Effectiveness of abbreviating the duration of intravenous antibacterial therapy with oral fluoroquinolones. Pharmaco Economics 1997; 11(1): 64-74.

8

Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Repellency Effect of Artemisia vulgaris Essential Oil against Aedes aegypti Mosquitoes in Laboratory Condition

Maung Maung Mya1*, Nwe Nwe Oo2, Aye Win Oo1,

Sein Thaung1 & Yan Naung Maung Maung1

1Medical Entomology Research Division Department of Medical Research

2Yangon University

The mosquito species Aedes aegypti L. is a major vector of dengue fever (DF), dengue haemorrhagic fever (DHF), Chikungunya, yellow fever and Zika fever. Dengue virus transmission (serotypes 1-4) by Aedes mosquitoes is a public health problem that principally affects tropical countries. Myanmar is one of the DF and DHF endemic areas in Southeast Asia Region. In mosquito control programs, botanical origin insecticides (pyrethrum, rotenone, sabadilla etc.) have the potential to be used as ovicidal, larvicidal and adulticidal of eggs, larvae and adult mosquitoes. Artemisia vulgaris (derived from Taunggyi Township, Shan State) dried leaves powder was subjected to hydro-distillation in a Clevenger-type distilling apparatus. The resulting oil was dried over anhydrous sodium sulphate and resulting 8 gm of essential oils were obtained from 100 gm of dried leaves powder. Mosquito repellency assay was done to test the efficacy of the essential oil against Aedes aegypti on human volun-teers. Artemisia vulgaris essential oil applied on average 342 cm2

area of

arm for 100% protection against Aedes aegypti mosquitoes landing to probe the skin was found with the dose 0.08 g/ml or 0.0002 g/cm2. Repellency activity of complete protection time of Artemisia vulgaris oil dose 0.0002 g/cm2 provided 97.62% protection for 30 minutes, 92.86% protection for 60 minutes and 85.71% protection for 120 minutes against Aedes aegypti. Artemisia vulgaris leaves essential oil did not cause dermal irritation when applied to human skin. No adverse effects on human volunteers were observed after application. Essential oil of Artemisia vulgaris leaves was found to have very effective protection from biting of Aedes aegypti.

Key words: Repellency, Dilution, Protection, Artemisia vulgaris, Essential oil

INTRODUCTION Each year, an estimated 50 to 100 million new cases of dengue occur around the world, of these, 500,000 cases correspond to dengue haemorrhagic fever with mortality of 5% (25000 cases).1, 2 The WHO initiative for vaccine Research (IVR), in collaboration with a wide range of partners, aims to facilitate the development and future introduction of safe, effective and affordable dengue vaccines.3 The mosquito species Aedes aegypti L. is a major vector of dengue fever (DF), dengue haemorrhagic fever

(DHF), Chikungunya, Yellow fever and Zika fever. Dengue virus transmission (serotypes 1-4) by Aedes mosquitoes is a public health problem that principally affects tropical countries. Dengue haemor-rhagic fever outbreaks were recognized as new diseases first in Myanmar in 1970 caused by dengue type one.4 Dengue fever and dengue haemorrhagic fever are increasingly becoming serious public health ___________________________________ *To whom correspondence should be addressed. Tel: +95-9402751359 E-mail: dr.mgmgmya@gmail.com

9

problems especially among the 5-10 and 11-15 years old age groups and now noted 15 years above, a vast majority of the cases occur in 5-8 years old age group.5, 6 Almost half of the global population lives in high-risk areas and currently more than 100 countries experience dengue fever and dengue haemorrhagic fever.7

Rapid, poorly planned urbanization in association with weak regulatory policies for discharge of solid waste has resulted in the accumulation of discarded con-tainers in most developing countries. These accumulations have favored the establish-ment and geographic spread of this mosquito. The strategies to control dengue transmission used by the public vector control programs have not been adequate in most countries. The emergence of insec-ticide resistance, the difficulty of eliminating larval population through environmental sanitation and lack of efficacy of ultra-low volume insecticide spraying to control adults are factors which have limited the effectiveness of vector control programmes.8 The indiscriminate use of synthetic insecticides is creating multifarious problems like environmental pollution, insecticide resistance, and toxic hazards to humans.

Globally, there have been conscientious efforts to overcome these problems, and great emphasis has been placed recently on environment friendly and economically viable methodologies for pest control. Phytochemicals obtained from the huge diversity of plant species are important source for safe and biodegradable chemicals, which can be screened for mosquito repellent, larvicidal and insecti-cidal activities. Large numbers of plant products have been reported to have mosquito larvicidal and repellent activity against adult mosquitoes.9 In recent years essential oils have received much attention as potent bioactive compounds against various mosquitoes species.10 Artemisia vulgaris L. is a member of the Asteraceae family. It is a tall (1-1.5 meter) aromatic,

threatened perennial herb distributed throughout the northern temperature regions of Africa, Asia, Europe, India, Myanmar and North America. In Myanmar Artemisia vulgaris L. plants are abundantly present in Chin, Kachin and Shan State. In traditional medicine, this plant is widely used for the treatment of diabetes and extract of the whole plant is used for epilepsy and in combination for psychoneurosis, depression, irritability, insomnia and anxiety states.11

It is necessary to find out more potential natural product in plants pest. Synthetic insecticides are toxic and adversely affect the environment by contaminating soil, water and air.12 Therefore, there is a need to find out the alternative ways for environ-mental safety, biodegradable, cost effective and indigenous methods to prevent mosquito bites. Recently increased interest is developing plant origin repellency effects as an alternative to chemical repellency. The study was undertaken to assess the repellent potential of essential oil of Artemisia vulgaris L. against Aedes aegypti mosquitoes.

MATERIALS AND METHODS Aedes aegypti mosquito larvae and adult Aedes mosquitoes emerged from pupae were collected from Hlinethaya Township were reared in laboratory of Medical Entomology Research Division, Department of Medical Research. Larvae were fed on DMR larva food. Adults were provided with 10% sucrose solution and 8 weeks old mouse for blood meal. Mosquitoes were held at 26±2ºC, 65-75% relative humidity with a photo period of 12-hour light and 12-hour dark. Mosquitoes were reared continuously in laboratory for repellency test. Laboratory-reared 3-5 days old mos-quitoes were used for repellency of essential oil of Artemisia vulgaris. Mosquitoes species identification

Larvae and adult mosquitoes emerged from larva survey were identified by morpho-logical methods.13

10

Collection and preparation of extraction of Artemisia vulgaris leaves essential oil Artemisia vulgaris (Myanmar name: Shanmanaing leaves) were collected from Taunggyi Township, Shan State. A total of 10 kilograms of Artemisia vulgaris leaves were cleaned and dried in shed place at room temperature for 30 days. Dried leaves were ground by grinding machine to make powder and finely ground 100 grams of dried leaves powder were mixed with 1000 ml distilled water and subjected to hydro-distillation in a Clevenger-type distilling apparatus for two hours. The resulting oil was dried over anhydrous sodium sulphate and resulting 8 gm of essential oils was obtained from 100 gm of dried leaves powder. The essential oil was stored in airtight fuscous glassware in a refrigerator at 4ºC until use. The extraction was done in Department of Botany, Yangon University. Repellent activity testing The repellent study was done according to the method of World Health Organization.14

Three to five days old blood starved 50 numbers of female Aedes aegypti mos-quitoes were kept in a steel net cage (59 x 59 x 59 cm). The volunteer had no contact with lotions, perfumes, or perfumed soaps on the day of the assay. Using a pipette, 1 ml of ethyl alcohol (95%) diluent used in the preparation of the test repellent was applied evenly to average 342 cm2

of

forearm skin between the wrist and elbow of the volunteer and allowed to dry 1 minute. Before insertion of arm into the cage containing 50 Aedes female mosquitoes, the hands were protected by plastic gloves to protect mosquitoes bite. This procedure was done according to WHO.14 The first step, ethyl alcohol applied forearm was inserted into the cage and the number of mosquitoes that landed on the skin during a 30-second period was counted. The control forearm was carefully withdrawn and this arm was then treated with 1 ml of 0.1 g/10 ml of Artemisia vulgaris essential oil solution and allowed to dry. The treated arm was placed in the cage for another 30 seconds

period and observed for mosquito landing. This procedure was repeated for each additional incremental of Artemisia vulgaris oil dose. The tests were carried out one after the other without delay and Artemisia vulgaris oil dose at each test was calculated as the sum of the doses applied to arrive at the cumulative dose for each test. Test was proceeded until the mosquito landing rate on the exposed forearm was less than 10 females in 30 seconds. Two trained technicians recorded the number of landings. At the conclusion of the dose response experiment, 1 ml of ethyl alcohol was applied on the other forearm and allowed to dry. This forearm was inserted in the cage for 30 seconds to verify that the number of landings was more than 10 per 30 seconds as was observed at the beginning of the experiment.

Estimation of complete protection time

The complete protection time of Artemisia vulgaris oil extract was determined when 99 to 100% protection dose (0.08 g/ml) was used on 342 cm2 area of forearm skin between the wrist and elbow. Four mosquito cages (size 59 x 59 x 59 cm) each containing 50 non blood fed 5 days old Aedes aegypti female mosquitoes were normally used. Two cages were used for testing two female volunteers and another two cages were used for testing two male volunteers. Same procedure was followed as above. In the first step, ethyl alcohol applied forearm was inserted into the cage and the number of mosquitoes that land on the skin during a 3-minute period was counted. The control forearm was carefully withdrawn from the cage. Then, 0.08 g of Artemisia vulgaris oil extract, prepared in 1 ml of ethyl alcohol solution was applied evenly on 380 cm2 of another forearm skin between the wrist and elbow. The treated arm was placed in and mosquito landing was observed.

After 30 minutes, the 3-minute period Artemisia vulgaris oil repellent treated arm was inserted again into the cage and exposed for 3 minutes to determine landing activity. This procedure was repeated at

11

30 minutes intervals for 180 minutes and the procedure was used consistently throughout the experiment. The mosquitoes that landed on the hand were recorded and then shaken off before imbibing any blood. Complete protection time was estimated after experi-ment. Same repellency test procedure was followed on other three volunteers. As Aedes aegypti is a day time biter, test was done between 08:00 hours and 16:00 hours. Tests were done in 15 x 10 x 10 ft room, at 25-27ºC and relative humidity of 60-80%.

Data analysis

Data entry and processing was made using Microsoft Excel software. The average repellency data were subjected to calcula-ting percentage protection of mosquito bite. P=1-(T/C)=(C-T)/C

Protection (P) was expressed as a proportion of the number of mosquitoes landing on treated arm, (T) in relation to the number of landings on the control arm, (C) of the same individual. C was the average landing on two untreated arms.

RESULTS

Table 1. Experiment of successive Artemisia vulgaris oil doses applied to arrive at a cumulative dose for Aedes aegypti

A B C D E Left arm control average area 342 cm2

1 ml ethyl

alcohol 0 gm 18±1.83 Average

control (Left+Right)

17.5 (C) Left arm dose 1 0.01 0.01 4±0.82 77.14 Left arm dose 2 0.01 0.02 3±0.82 82.86 Left arm dose 3 0.02 0.04 2±0.82 88.57 Left arm dose 4 0.04 0.08 0 100 Right arm control

1 ml ethyl alcohol

only

0 17±1.41

A=Application sequence, 4 replicates B=Repellent solution concentration to be applied in

1 ml (g/ml), frequently added C=Cumulative amount of repellent (g/342 cm2 area)

(Total amount) D=Average mosquito landing to probe the skin E=% protection [P=1-(T/C)=(C-T)/C] Successive cumulative dose of Artemisia vulgaris essential oil applied on 342 cm2

area of arm for 100% protection of Aedes

aegypti mosquito landing to probe the skin was found to be dose of 0.08 g/ml or 0.0002 g/cm2 as shown in Table 1. Table 2. Repellency test of Artemisia vulgaris

oil 0.08 g/ml on 4 volunteers’ arms in laboratory

Replicates (Repellent

applied areas)

Control*

Duration of repellency test (Artemisia vulgaris oil 0.08 gm

in 1 ml of ethylalcohol) & No. of mosquito bite

A B C D E F Male 1 (362 cm2)

16 1 2 3 5 7 9

Male 2 (359 cm)

17 0 1 2 2 4 7

Female 1 (325 cm2)

22 1 2 2 3 6 7

Female 2 (321 cm2)

29 0 1 2 2 4 7

Total bite (Average 342 cm2)

84 ±5.94

2 ± 0.58

6 ±0.58

10 ±0.5

12 ±1.14

19 ±1.5

30 ±1

A=30 minutes (No. of mosquito bite) B=60 minutes (No. of mosquito bite) C=90 minutes (No. of mosquito bite) D=120 minutes (No. of mosquito bite) E=150 minutes (No. of mosquito bite) F=180 minutes (No. of mosquito bite) *=3 minutes (No. of mosquito bite) Table 3. Estimation of complete protection time

of Artemisia vulgaris essential oil repellent against Aedes aegypti

Dose 0.0002 g/cm2 A B C D E F G

Control* 84 - - - - - - Percentage** 0 2

(97.62) 6

(92.86)10

(88.10) 12

(85.71)19

(17.38)30

(64.29)A=0 minute (No. of mosquito bite) B=30 minutes (No. of mosquito bite) C=60 minutes (No. of mosquito bite) D=90 minutes (No. of mosquito bite) E=120 minutes (No. of mosquito bite) F=150 minutes (No. of mosquito bite) G=180 minutes (No. of mosquito bite) *=Total number of bite (4 replicates) **=Percent protection of mosquito bites (4 replicates) Repellency activity of complete protection time of Artemisia vulgaris oil dose 0.0002 gm/cm2 provided 85.71% protection for 120 minutes against Aedes aegypti and over 90% pro-tection was found for 60 minutes, i.e. 97.62% protection for 30 minutes and 92.86% protection for 60 minutes (Table 2 & 3). The essential oil of Artemisia vulgaris leaves was found to have very effective protection from biting of Ae. aegypti mosquitoes.

12

DISCUSSION

Insecticide residues in the environment as a result of chemical insecticide usage have turned the researcher’s attention towards natural products. In the past years, the plant kingdoms synthesize a variety of secondary metabolite which plays a vital role in defense of plants against insects/ mos-quitoes. Mosquito repellents may be one of the most effective tools for protecting human from vector-borne diseases and nuisance caused by mosquitoes. Natural products are safe for humans when compared to that of synthetic compounds. Other researchers revealed that the screening of locally available medicinal plants for mosquito control would generate local employment, reduce dependence on expensive imported products and stimulate local efforts to enhance public health.15 Different parts of the plants contain a complex of chemicals with unique biological activity16, 17 which is thought to be due to toxins and secondary metabolites which act as mosquitocidal agent.18 Smoke is still, the most widely used common methods of repelling biting insects that is used throughout the world.19 Most households in the developing world rely on personal protection measures of limited effectiveness, such as burning mosquito coils or leaves. The bundles of dried Artemisia vulgaris L. were burned to repel biting insects since they contain insect repellents that can be released by combustion. The larvicidal, pupicidal, adulticidal, and repellent activities of Artemisia nilagirica against the mosquitoes, Anopheles stephensi and Aedesa egypti, were suggested that the leaf extract was used as a potent larvicidal agent.20 Artemisia extracts contain secondary metabolites mainly monoterpenoids such as vulgarole, spathulenol, vulgarin, triterpenoide: α-amyrin, α-amryin acetate and fernenol.21

The insecticidal properties of Artemisia vulgaris and other plants of the genus Artemisia have been attributed to the

presence of these secondary metabolites. The compounds isolated from Artemisia vulgaris were mainly monoterpenoids such as linalool, camphor, isoborneol, borneol, terpinen-4-ol, isobornyl, nonanone-3, (α+β)-thujone, bornyl acetate, β-pinene, myrcene, α-terpinene, limonene, and cineote. These compounds were reported as effective repellent compounds against Aedes aegypti.22

Repellency activity of complete protection time of Artemisia vulgaris oil possessed significant repellent activity against Aedes aegypti. The dose 0.0002 g/cm2 provided 85.71% protection for 120 minutes against Aedes aegypti and over 90% protection was found for 60 minutes i.e. 92.86% protection for 30 minutes and 97.62% protection for 60 minutes, respectively. Although it exerted an effective biting protection time against Aedes aegypti, it is lower than the protection time of currently used synthetic compounds such as DEET, A13-37220, A35765 and CIC-4.23, 24

These chemical compounds provide better and longer protection against many biting insects (ED50 and ED95 level=0.37-25.37 µg/cm2, 3-8 hours). Repellent protection time in laboratory may change depending on the biological characteristics of the mosquito test population. Different in species and body size, sugar water availability, adult density in test cages, and mosquito age can affect test results.25-27

Mixture of 5% vanillin and essential oil of Curcuma longa found effective repellent activity for 8 hours against Aedes aegypti, Anopheles dirus and Culex quinque-fasciatus.28 Other researchers also observed that the repellent activity of methanol extract of Ferronia elephantum leaves against Aedes aegypti activity at 1.0 and 2.5 mg/cm2 concentrations gave 100% pro-tection up to 2.14±0.16 hours and 4.00± 0.2.4 hours, respectively and the total per-centage protection was 45.8% at 1 mg/cm2 and 59.0% at 2.5 mg/cm2 concentrations for 10 hours.29 These results revealed that essential oil of Artemisia vulgaris plant

13

extracts was very effective to prevent Aedes aegypti mosquitoes bite i.e. 85.71% protect-ion for 120 minutes and over 90% protection for 60 minutes in day time.

Conclusion From these results, the study concluded that the essential oil of Artemisia vulgaris leaves exhibits repellent activities against dengue vector mosquitoes. More studies are needed to elucidate the ovicidal activity against a wide range of mosquito species. The active compound responsible for repellent activity should be identified, which could be used to control different mosquito species in the future. These results could encourage the search for new active natural compounds offering an alternative to synthetic repellents and insecticides from other medicinal plants.

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a global health problem. Emerging Infectious Diseases 1998; 4(3): 442-450.

2. Gubler DJ. The changing epidemiology of yellow fever and dengue, 1900 to 2003: Full circle? Comparative Immunology, Microbiology & Infectious Diseases 2004; 27(5): 319-330.

3. World Health Organization. Dengue Vaccine Research. WHO Report: Global strategy for dengue research [Internet]. 2016 Available from: http:// www.who.in.research.development.

4. Ohn Khin. Epidemiological situation of Dengue Haemorrhagic Fever in Rangoon, Burma. Dengue News WHO SEARO and Western Pacific Region, 1985.

5. Prasittsuk C, Andjaparidze AG & Kumar V. Current status of Dengue/ Dengue Haemorrrhagic fever in WHO Southeast Asia Region. Dengue Bulletin 1998; 22: 1-10.

6. Hlaing Myat Thu. Virology report. In: Annual Report of Department of Medical Research (Lower Myanmar), 2009.

7. Guha-Sapir D & Schimmer B. Dengue fever: New paradigms for a changing epidemiology of yellow fever. Emerging Themes in Epidemiology 2005; 2: 1.

8. Gubler DJ & Clack GG. Dengue/Dengue Haemorragic Fever. The emergence of a global health problem. Emergence of Infectious Diseases 1995; 1: 55-57.

9. Maung Maung Mya, Sein Min, May Aye Than, Sein Thaung, Chit Thet Nwe & Zar Zar Aung Larvicidal effects of Garlic bulb (Allium

sativum) extract on larvae of Aedes aegypti and Culex quinquefasciatus under laboratory condition. Myanmar Health Sciences Research Journal 2014; 26(3): 189-194.

10. Tripathi AK, Upadhay S, Bhuiyan M & Bhattacharya PR. A review on prospects of essential oils as biopesticide in insect-pest management. Journal of Pharmacognosy and Phytother 2009; 1(5): 52-63.

11. Lewis WH & Elvin MPF. Medical Botany: Plants Affecting Human Health, 2nd ed., John Wiley and Sons Inc. New Jersey 2003; 345.

12. Kalu IG, Ofoegbu U, Eroegbusi J, Nwachukwu & Ibeh B. Larvicidal activities of ethanol extract of Allium sativum (garlic bulb) against the filarial vector, Culex quinquefasciatus. Journal of Medicinal Plants Research 2010; 4(6): 496-498.

13. Rampa R & Prachong P. Illustrated keys to the medically important mosquitos of Thailand, Southeast Asian Journal of Tropical Medicine and Public Health 1994; 25(1): 1-66.

14. World Health Organization. Guidelines for efficacy testing of mosquito repellents for human skins. Institutional Repository for Information Sharing, WHO Geneva, 2009.

15. Bowers WS, Sener B, Evans PH, Bingol F & Erdogan I. Activity of Turlish medicinal plants against mosquitoes Aedes aegypti and Anopheles gambiae. Insect Science and Application 1995; 16(3/4): 339-342.

16. Govindarajan M, Jebanesan A, Pushpanathan T & Samidurai K. Studies on effect of Acalyoha indica L. (Euphorbiaceae) leaf extract on the malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Parasitology Research 2008a; 103(3): 691-695.

17. Govindarajan M, Jebanesan A & Pushpanathan T. Larvicidal and ovicidal activity of Cassia fistula Linn. leaf extract against filarial and malarial vector mosquitoes. Parasitology Research 2008b; 102(2): 289-292.

18. Niraimathi S, Balaji N, Venkataramanan N & Govindarajan M. Larvicidal activity of alkaloid from Sida acuta against Anopheles subpictus and Culex tritaenrhynchus. International Journal of Current Research 2010; 11:34-38.

19. Vernede R, Ven Meer Mm & Alpers MP. Smoke as a form of personal protection against mosquitoes: A field study in Papua New Guinea. South East Asian Journal of Tropical Medicine & Public Health 1994; 25(4):771-775.

20. Panneerselvam C, Murugan K, Kovendan K & Kumar PM. Mosquito larvicidal, pupicidal, adulticidal, and repellent activity of Artemisia nilagirica (Family: Compositae) against Anopheles stephensi and Aedes aegypti. Parasitology Research August 2012; DOI 10.1007/s00436-012-3073-9.

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21. Glasby JS. Dictionary of Plants Containing

Secondary Metabolites. Taylor and Francis e-Library, 1644, 2005.

22. Hwang YS, Wu KH, Kumamoto J, Axelrod H & Mulla MS. Isolation and identification of mosquitoes repellents in Artemisia vulgaris. Journal of Chemical Ecology 1985; 11(9): 1297-1306.

23. Schreek CE & Mc Govern TP. Repellent tests in the field and laboratory against wild populations of Monsonis titllans (Diptera: Culicidae). Journal of Medical Entomology 1985: 22: 658-662.

24. Coleman RE, Robert LL, Robert LW, Glass JA, Seeley DC, Laughinghouse A, et al. Laboratory evaluation of repellents against four Anopheline mosquitoes (Diptera: Culicidae and two phebotomine sand flies (Diptera: Psychodidae). Journal of Medical Entomology 1993; 30: 499-502.

25. Gouck HK & Smith CN. The effect of age and

time of day on the avidity of Aedes aegypti. Fla. Entomology 1962; 45: 93-94.

26. Khan AA, Maibach HI & Skidmore DL. Insect Repellent: Effect of mosquito and repellent related factors on protection time. Journal of Economic Entomology 1975; 68: 43-45.

27. Xue RD, Barnard DR & Schreck CE. Influence of body size and age of Aedes albopictus on human host attack rates and the repellency of Deet. Journal of Mosquito Control Association 1995; 11: 50-53.

28. Tawatsin A. Wratten SD, Scott RR, Thavara U & Techadamrongsin Y. Repellency of volatile oils from plants against three mosquito vectors. Journal of Vector Ecology 2001; 26: 76-82.

29. Venketachalam MR & Jebanesan A. Repellent activity of Ferronia elephantum Corr. (Rutaccae) leaf extract against Aedes aegypti. Bioresource Technology 2001; 76(3): 287-288.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Study on Protective Effects of Malaria Antibody among the Community in Malaria Endemic Areas

Moe Kyaw Myint1*, Khin Lin1, Aung Thu1, Mya Moe1,

Phyu Phyu Win1, Zaw Lin2 & Kyaw Zin Thant3

1Department of Medical Research (Pyin Oo Lwin Branch)

2Department of Public Health 3Department of Medical Research

Malaria antibodies have been associated with transmission intensity and antibody responses are not much varied in seasonal condition. Antibody assessment is probable to provide a useful epidemiology tool. This study aimed to detect prevalence of antibody in different risk areas in different seasons. Community-based, cross-sectional descriptive study was conducted at high and moderate risk areas during rainy and dry seasons in 2012-2013. Rapid diagnostic testing was used for examination of antigens and antibodies. Microscopic examinations were done. Among total 414 participants, malaria antigens and antibodies (P. falciparum/ P. vivax) were detected in 17.9% (74) and 19.1% (79) of participants, respectively. Participants with older age (35±12.7 years) had more prevalence of antibody than younger ones (31.4±14.4 years). Mean difference was 3.6 years (p=0.040). Antibody prevalence was higher in participants of high risk areas (20.4%) than those of moderate risk areas (17.6%). Variation of antibody prevalence between rainy and dry season was less than that of antigen prevalence. In high risk areas, antibody was 25.5% in rainy season and 10.4% in dry season. Antigen prevalence had much variation with 32.9% in rainy season compared to 5.2% in dry season in high risk area. However, in moderate risk areas, antibody prevalence was 17.7% in rainy season and it remained with 16.7% in dry season. The antigen prevalence was 13.3% in rainy season and it was (0%) not found in dry season in moderate risk areas. Therefore, less seasonal variation of antibody prevalence between rainy and dry season was observed in moderate risk areas. The study concluded that protective effects of malaria antibody were observed in older age and associated with transmission intensity. Therefore, antibody assessment can probably provide useful epidemiology tool as it has less seasonal variation.

Key words: Malaria antibody and antigen, Seasonal variation, Endemic area

INTRODUCTION

Malaria is one of the most severe public health problems worldwide. It is not only a leading cause of death but also a disease in many developing countries, where young children and pregnant women are mostly affected. According to the World Health Organization’s World Malaria Report (2009) and the Global Malaria Action Plan; 3.3 billion people (half the world’s population) live in areas at risk of malaria transmission in 109 countries worldwide,

35 countries (30 in sub-Saharan Africa and 5 in Asia) account for 98% of global malaria deaths. In 2008, there were estimated 190- 311 million clinical malaria episodes, and 708,000-1,003,000 deaths. Malaria is the 5th cause of death from infectious diseases worldwide (after respiratory infections, HIV/AIDS, diarrheal diseases, and tuber-culosis) in low-income countries.1 ___________________________________ *To whom correspondence should be addressed. Tel: +95-9402751359 E-mail: dr.myintmoekyaw@gmail.com

16

In Myanmar, according to Myanmar Health Statistics (2010), malaria morbidities were 10.75, 10.00 and 11.28 (per 1,000 popula-tions) in 2008, 2009 and 2010, respectively. Mortalities were 1.84, 1.64 and 1.33 (per 100,000 populations) in 2008, 2009 and 2010, respectively. In 2010, percentage of populations living under malaria high risk, moderate risk, low risk and no risk areas were 22%, 25%, 16% and 37%, respectively.2 Malaria infection caused by five species of Plasmodium (P. falciparum, P. vivax, P. ovale, P. malariae & P. knowlesi), produces malaria antibodies in one or two weeks after initial infection.3 These anti-bodies persist for three to six months after parasite clearance and remain months or years in semi-immune persons in endemic areas where re-infection is frequent. However, in a naive patient, antibody levels fall rapidly.4

Antibody prevalence was different in areas with stable and unstable malaria. In stable malaria area, antibody was more commonly found than unstable malaria area.5 Accordingly, malaria epidemics could be expected to occur in areas with unstable malaria due to lack of antibody protection in those areas. This antibody protection might also be varied with epidemiological charac-teristics of the community people such as age, occupation, past history of malaria, migration, high risk group, bed net utilization, past history of treatment, geography of residing areas and existing activities of preventive and control measures in the community. Therefore, by using antibody surveys, antibody prevalence resulting different antibody protection may be analyzed in various epidemiologic conditions of the communities.

Malaria antibody responses are long lasting in human blood and those are not much varied in seasonal conditions. Therefore, cumulative responses can be detected in the malaria areas. In addition, antibody detection may facilitate to get accurate findings for malaria situation of the area

more easily than antigen detection and microscopic examination for malaria parasites as these examinations cannot detect the infection for long duration, in low parasite density and in areas with low transmission. Moreover, microscopic examination results rarely reveal valid results and which may not reflect situation of malaria in the area. Thus, antibody detection may reveal not only endemicity situation of the area but also reveal the antibody protection among the community.6

Malaria antibody assessment by ELISA is probable to provide a useful epidemiology tool which can detect malaria endemicity in transmission areas.7, 8 Malaria antibody to circumsporozoite antigen has been asso-ciated with malaria transmission intensity9 and also related with repeated exposure to infection in studies done at Brazil and Siri Lanka.10, 11 General objective of the study was to detect the protective effects of malaria antibody among the community in malaria endemic areas in selected townships of Mandalay region. This study detected the levels of antibodies acquired against Plasmodium falciparum (P.f) and Plasmodium vivax (P.v) in residents living in selected townships of Mandalay Region. The study provided the serological data showing anti-body protection in different epidemiological information. In addition, malaria situations of different areas were explained by anti-body survey. Thus, this valuable information may be useful to apply in the stratification of malaria endemicity of the areas for pre-vention and control activities.

MATERIALS AND METHODS

A community-based, cross-sectional descrip-tive study was conducted during wet season, June to November in 2012 and dry season, February to March in 2013 at two villages (Chaung Gyi and Milann) of Thabeikkyin Township and two villages (Gaelong and Banpon) of Pyin Oo Lwin Township in Mandalay Region. In those villages, Milann, and Banpon were high risk transmission

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areas (Stratum 1a; according to stratification done by VBDC) because those are moun-tainous and located in the forest. These areas also have migrant population in development project sites which are gold mine and dam construction.

The other two villages were clarified as moderate risk transmission areas (Stratum 1b) because those are 1-2 miles away from the edge of forest and able to access to nearest health center within 1-3 hours by means of travel available.

Sample populations A total of 414 participants were recruited in the study. Among these, 188 were from the villages of moderate risk and the others 226 were enrolled from the villages of high transmission areas.

Data collection and data management Pre-tested pro-forma was used to collect the data concerning epidemiological charac-teristics of the residents in study townships. Total 30 microliters of blood samples were collected to do (a) microscopic examinations for malaria parasites (b) rapid diagnostic tests for malaria antigens (c) rapid diagnostic tests for malaria anti-body. Binocular microscope was used for microscopic examinations of malaria parasites in field surveys.

Parasite density was calculated as multiplication of parasite count by 8000 per number of WBC counted. Rapid diagnostic test (RDT) kits (SD Standard Diagnostics, INC) were used for examination of antigens and antibodies of P.f and P.v. Malaria Antigen P.f/P.v Test is one step, rapid, qualitative and differential test for the detection of HRP-II (Histidine-rich protein II) specific to P.f and pLDH (Plasmodium lactate dehydrogenase) specific to P.v. Diagnosis of antibody is the immuno-chromatographic (rapid) test for the detection of all isotypes of antibodies (IgG, IgM, IgA) specific to P.f/ P.v antigen, Merozoite Surface Protein (MSP). Sensiti-vity (95% Confidence Interval (CI)) of the RDT for malaria antigen P.f and P.v are

99.7% (98.5-100%) and 95.5% (90-98.1%). Specificity (95% CI) for both antigen is 99.5% (97.2-99.9%). Sensitivity of the RDT for malaria antibody P.f and P.v are 87% and 86%. Specificity of both antibodies is 99.5%. The data relating epidemiological characteristics and serological results were collected with proforma during surveys. The recorded data were compiled, coded, entered into SPSS 20.0 software and analyzed.

Ethical consideration Ethical approval was obtained from Department of Medical Research (Pyin Oo Lwin Branch) Ethics Committee to collect the blood samples and epidemiological statements for interview questionnaires from the participants.

RESULTS

A total of 414 participants were included in the study. Among them, 307(74.2%) were studied in rainy season and 107(25.8%) in dry season. Males were 279(67.4%) and females were 135(32.6%). Mean age (in year) of participants was 32.1±14.1. The youngest was 2 and eldest was 75 years. Age grouping were made as: young for below 10 years, teen age for 10 to 19, working group for 20 to 49 and old age for 50 and above. According to age group, 29(7%), 40(9.7%), 304(73.4%) and 41(9.9%) were distributed in below 10, 10-19, 20-49 and 50 and above years of age group, respectively.

Forty percent (40.1%) of them were farmers. The occupants such as fishermen, wood cutters, gold mine workers, laborers and charcoal makers were observed in 13.3%, 12.6%, 9.7%, 6.5% and 6.3% of the participants, respectively. More than half of respondents i.e. 232(56%) were not bed net users absolutely. Nearly half of the res-pondents, 188(45.4%) were from moderate risk areas and 226(54.6%) participants were from high risk areas. Malaria antigen for either P.f or P.v was found in 74(17.9%) and antibodies were observed in 79(19.1%) of total participants.

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Table 1. Antibody and antigen prevalence by malaria risk area (n=414)

Ab(+)=Antibody positive, Ag(+)=Antigen positive, Ab+Ag=Both antibody and antigen positive

Antibody prevalence among age group in high risk areas showed as 13.3%, 13%, 12.1% and 8.7% in below 10, 10-19, 20-49 and 50 and above years of age group, respectively. In moderate risk areas, antibody positive were found in 0.0%, 5.9%, 18.7% and 22.1% in below 10, 10-19, 20-49 and 50 and above years of age group, respectively (Table 1). Participants with older age (35±12.7 years) had more prevalence of antibody than younger aged participants (31.4±14.4 years). Mean difference was 3.6 years (p=0.040). Among total 414 participants, by RDT testing, antibody positive for P.f, P.v and mixed cases were found in 51(12.3%), 19(4.6%) and 9(2.2%) participants, respec-tively. Antigen positive for P.f, P.v and mixed were found in 38(9.1%), 31(7.5%) and 5(1.2%) participants, respectively. Those are shown in Table 2 and Table 3. On microscopic examination, all antigen positive cases i.e. 74(17.9%) were micro-scopically detected with parasitaemia. On the other hand, all antigen negative cases were not detected with parasitaemia on microscopic examination in this study.

Antibody prevalence by endemic areas and by seasons

In high endemic area, 27 had antibody positive and 19 had mixed with both antibody and antigen among 226 tested population. In moderate endemic area, 31 had antibody and 2 had mixed with antigen and antibody. Therefore, antibody prevalence was higher in high risk areas with 20.4% (27+19) of 226 tested partici-pants residing in those areas and it was

Table 2. Antibody and antigen prevalence by

season in high risk area (n=226) Rainy(n=149) Dry(n=77)

AB(+) (%) Ag(+) (%) AB(+) (%) Ag(+) (%) P.f P.v Mix P.f P.v Mix P.f P.v Mix P.f P.v Mix 20

(13.4) 14 (9.4)

4 (2.7)

23 (15.4)

21 (14.1)

5 (3.4)

5 (6.5)

3 (3.9)

0 (0)

2 (2.6)

2 (2.6)

0 (0)

Ab(+)=Antibody positive, Ag(+)=Antigen positive, P.f=P. falciparum, P.v=P. vivax Table 3. Antibody and antigen prevalence by

season in moderate risk area (n=188) Rainy(n=158) Dry(n=30)

AB(+) (%) Ag(+) (%) AB(+) (%) Ag(+) (%) P.f P.v Mix P.f P.v Mix P.f P.v Mix P.f P.v Mix 22

(13.9) 1

(0.6) 5

(3.2) 13

(8.2) 8

(5.1) 0 (0)

4 (13.3)

1 (3.3)

0 (0)

0 (0)

0 (0)

0 (0)

Ab(+)=Antibody positive, Ag(+)=Antigen positive, P.f=P. falciparum, P.v=P. vivax

lower in moderate risk areas with 17.6% (31+2) of 188 tested participants residing in respective areas. Similarly, antigen pre-valence was higher in high risk areas with 23.5% (53 of 226 tested populations) and it was lower in moderate area with 11.2% (21 of 188 tested populations).

Antibody prevalence was 25.5% (38 of 149 tested participants) in rainy season and it was only 10.4% (8 of 77 tested participants) in dry season in high risk area. However, in moderate risk areas, antibody prevalence was 17.7% (28 of 158 tested participants) in rainy season and it remained with 16.7% (5 of 30 tested participants) in dry season. There was large difference of antigen prevalence between rainy and dry season in both areas. The antigen prevalence was 32.9% (49 of 149 tested populations) in rainy season and it was only 5.2% (4 of 77 tested populations) in dry season of high risk area. Similarly in moderate risk area,

High risk area (n=226) Moderate risk area (n=188) Total (%) Ab(+) (%) Ag(+) (%) Ab+Ag (%) Tested Ab (+) (%) Ag (+) (%) Ab+Ag (%) Tested

Age (Year)

<10 2(13.3) 2(13.3) 1(6.7) 15 0(0) 1(7.1) 0(0) 14 29(7) 10-19 3(13) 6(26.1) 0(0) 23 1(5.9) 1(5.9) 0(0) 17 40(9.7) 20-49 20(12.1) 23(13.9) 16(9.7) 165 26(18.7) 15(10.8) 1(0.7) 139 304(73.4) 50 + 2(8.7) 3(13) 2(8.7) 23 4(22.1) 2(11.1) 1(5.6) 18 41(9.9)

Sex Male 17(11.3) 20(13.3) 15(10) 150 19(14.7) 15(11.6) 2(1.6) 129 279(67.4) Female 10(13.2) 14(18.4) 4(5.3) 76 12(20.3) 4(6.8) 0(0) 59 135(32.6)

Season Rainy 19(12.8) 30(20.1) 19(12.8) 149 26(16.5) 19(12) 2(1.3) 158 307(74.2) Dry 8(10.4) 4(5.2) 0(0) 77 5(16.7) 0(0) 0(0) 30 107(25.8)

Total (%) 27(11.9) 34(15) 19(8.4) 226 31(16.5) 19(16.1) 2(1.7) 188 414

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the antigen prevalence was 13.3% (21 of 158 tested population) in rainy season and it was (0%) not found in dry season. According to prevalence of antibody and antigen for P.f and P.v by seasonal distribution in both moderate and high risk areas, more percentage of antibody and antigen were found in rainy season than dry season. However, antibody prevalence variation between rainy and dry season was less than antigen prevalence variation between rainy and dry season. Those are described in Table 2 and Table 3.

DISCUSSION

Studies on malaria antibody showing in seasonal and endemic area difference are very few in Myanmar. This study provides prevalence of malaria antibody not only in dry and wet season but also in different endemic areas. Seroprevalence of malaria antibody among females was lower with 6.2% (14/226) in high risk regions. It was higher among males with 14.2% (32/226) in high risk areas. However, it was lower in males in dry season with 2.1% than females with 9%. Antibody prevalence was higher among older aged participants (35±12.7 years) than younger aged participants (31.4±14.4 years). Mean difference was 3.6 years (p=0.040). Study done in Gambia revealed that sero-prevalence was increased with age and lower in males 17.5% than females 21.3%.12

In this study, malaria antibody assessment showed difference in antibody prevalence between high and moderate risk areas. Antibody prevalence was higher in high risk areas (20.3%) than moderate risk areas (17.6%). Antibody prevalence was also associated with antigen positive rate. The antigen was higher in high risk areas with comprising 23.5% (53 of 226 tested populations) than moderate risk areas which comprised 11.2% (21 of 188 tested populations). Therefore, this study revealed that malaria antibody assessment could estimate malaria endemicity in malarious

areas. A biochemical study done in Tanintharyi Region reported that malaria antibody assessment was a useful tool for rapid detection of malaria transmission intensity. It revealed that malarial antibodies reflect cumulative exposure and is less influenced by seasonality due to the longer duration of specific antibody responses. In the study, malaria antibody was found in 121(27.07%), 32(5.77%) and 23(5.08%) for Plasmodium falciparum and in 62(13.87%), 20(3.60%) and 21(4.63%) for P. vivax malaria in high, moderate and low transmi-ssion microstratified areas, respectively. Malaria antigen for P. falciparum was discovered in 39 (11.47%), 4 (0.72%) and 1(0.23%) and for P. vivax 11(3.24%), 1(0.18%) and 4(0.93%) in high, moderate and low transmission areas, respectively.13 This study also revealed different antibody prevalence between rainy and dry season in both areas. According to prevalence of antibody and antigen for P.f and P.v by seasonal distribution in both moderate and high risk areas, more percentage of antibody and antigen were found in rainy season than dry season. However, antibody prevalence variation between rainy and dry season was less than that of antigen. Antibody level remains in sera for longer period after parasite clear. Therefore, antibody level showed less seasonal variation. A study done in Mandalay also revealed as malaria antibody responses are long lasting in human blood and those are not much varied in seasonal conditions. Therefore, cumula-tive responses can be detected in the malaria areas.6 Conclusion According to the results of this study, it was concluded that malaria antibody was more prevalent in elder age group than younger age group. Antibody was higher in high risk areas than moderate risk areas. Another point to be considered was that malaria antibody prevalence in studied area had less seasonal variation. Antibody prevalence variation between rainy and dry season was less than that of antigen.

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Therefore, it was also concluded that transmission endemicity could be estimated by antibody assessment in the malaria endemic areas.

ACKNOWLEDGEMENT

The authors would like to express the great thanks to official concerns of WHO Country Office for giving support to conduct this study. Our thanks also go to community people for their participation in this study. We also thank to Research Assistants of Parasitology Research Division, Department of Medical Research (Pyin Oo Lwin Branch) for their kind hard-working.

REFERENCES

1. World Malaria Report 2009 and the Global Malaria Action Plan. Geneva, World Health Organization, 2009.

2. Myanmar Health Statistics, 2010. Ministry of Health, Myanmar, 2010.

3. Garraud O, Mahanty S & Parraut R. Malaria-specific antibody subclasses in immune individual: A key source of information for vaccine design. Trends in Immunology 2003; 24(1): 30-35.

4. Seed CR, Kitchen A & Davis TME. The current status and potential role of laboratory testing to prevent transfusion-transmitted malaria. Trans-fusion Medicine Reviews 2005; 19(3): 229-40.

5. Saw Lwin, et al. Unstable malaria (epidemic prone) vs. Stable malaria. Paper presented in National Seminar on Malaria; 2011 Dec 26, Nay Pyi Taw, Myanmar.

6. Khin Lin, Moe Kyaw Myint & Kyaw Zin Thant. Prevalence of malaria antibodies in the rural areas of Mandalay Region. Programme and Abstracts of the Myanmar Health Research

Congress; 2012 Jan 9-13; Yangon, Myanmar. p. 3-4.

7. Esposito F, Lombardi S, Modiano D, et al. Prevalence and levels of antibodies to the circumsporozoite protein of Plasmodium falciparum in an endemic area and their rela-tionship to resistance against malaria infection, Transactions of the Royal Society of Tropical Medicine and Hygiene 1988; 82(6): 827-32.

8. Ramasamy R, Nagendran K & Ramasamy MS. Antibodies to epitopes on merozoite and sporozoite surface antigens as serologic markers of malaria transmission: Studies at a site in the dry zone of Sri Lanka. American Journal of Tropical Medicine and Hygiene 1994; 50(5): 537-47.

9. Druilhe P, Pradier O & Marc JP. Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection. Infection and Immunity 1986; 53(2): 393-97.

10. Carvalho ME De, Ferreira MU, De Souza MR, et al. Malaria seroepidemiology: Comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates, Memorias do Instituto Oswaldo Cruz 1992; 87(2): 205-208.

11. Mendis C, Del Giudice G, Gamage-Mendis AC, et al. Anti-circumsporozoite protein antibodies measure age related exposure to malaria in Kataragama, Sri Lanka. Parasite Immunology 1992; 14(1): 75-86.

12. Oduro AR, Conway DJ, Schellenberg D, et al. Seroepidemiological and parasitological evalua-tion of the heterogeneity of malaria infection in the Gambia. Malaria Journal 2012; 12: 222.

13. Khin Myo Aye, Myat Phone Kyaw, Thaung Hlaing, et al. Malaria Antibody: Is it an alternative tool for estimation of local malaria transmission in malaria micro-stratified areas? Myanmar Medical Journal 2012; 55(1): 19-26.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Insulin Receptor Substrate-1 Gene (G972R) Polymorphism and Metabolic Syndrome in Type-2 Diabetes Mellitus Patients

Thae Nu Htwe1*

, Myat Mon Oo2, Saw Wut Hmone3,

Moh Moh Htun2,Ohnmar Myint Thein1

& Myat Thandar4

1Physiology Department, University of Medicine 1 (Yangon)

2Department of Medical Research 3University of Medicine 1 (Yangon)

4University of Nursing (Yangon)

Metabolic syndrome (MS) is one of the fastest growing health problems worldwide. It is major risk factor for both diabetes and cardiovascular disease. Many candidate gene studies have identified the linkage between MS and a number of genes. The insulin receptor substrate-1 (IRS-1) gene especially at codon 972 (G972R) is also a candidate for insulin resistance in type 2 diabetes and MS. The aim of the present study was to determine the insulin receptor substrate-1 gene (G972R) polymorphism and MS in type 2 diabetes mellitus (T2DM) patients. The genomic DNA of the 100 subjects from Diabetes Out-patients Department of YGH was amplified by polymerase chain reaction (PCR) and digested by restriction fragment length polymorphism (RFLP) with BstN1 used for codon 972. Metabolic syndrome was defined as in IDF (International Diabetes Federation) criteria. Metabolic syndrome was found in 84% (16/19) of carrier patients and 82% (67/81) of non-carrier patients. It was not significantly associated with presence or absence of IRS-1 (G972R) mutation. There was a significant difference (p=0.005) in proportion of MS between IRS-1 (G972R) non-carriers with family history of diabetes mellitus and those without. It, therefore, seems reasonable to argue that family history of T2DM does not play an important role in the development of MS in the IRS-1 (G972R) non-carrier.

Key words: Insulin receptor substrate-1, Metabolic syndrome

INTRODUCTION

Metabolic syndrome (MS) is a very common, multi-component condition charac- terized by insulin resistance, dyslipidaemia, abdominal obesity and hypertension, that is associated with an increased risk of T2DM, cardiovascular disease and artherosclerosis.1

Insulin resistance is present in the majority of people with MS. Thus, metabolic syndrome is strongly associated with risk for incident diabetes, likely because some of its components are more strongly associated with diabetes risk.2 Hong Kong has one of the highest prevalence of diabetes (8.6%) and various components of MS

(11.6-23.6%) in Asia.3 Metabolic syndrome was detected in 39.6% of patients with acute ischaemic stroke and 50% of patients with acute myocardial infarct in Myanmar subjects.4, 5 The etiology is complex, determined by the interplay of both genetic and environmental factors.6 Candidate gene studies have identified linkage between MS and a number of genes such as PPAR gamma, adiponectin, and beta-adrenergic receptors.7 Moreover, De la Cruz-Mosso, et al.8 suggested that the -844 G/A PAI-1 polymorphism was related with the risk of _________________________________________________*To whom correspondence should be addressed. Tel: +95-95044707 E-mail: thaenushinthant@gmail.com

22

developing metabolic syndrome, obesity and atherogenic dyslipidemia, and the HindIII C/G PAI-1 polymorphism was associated with the increase of total cholesterol levels in Mexican children. In addition to these genes, in a previous insulin receptor substrate-1 (IRS-1) proteins have been identified and IRS-1 functions as one of the key downstream signaling molecules pathway.9, 10

The binding of insulin to its receptor triggers a complex signaling cascade of protein phosphorylation and dephospho- rylation culminating in the metabolic and mitogenic effects of insulin. Elucidation of insulin signaling pathway has been central to understanding the mechanisms underlying insulin resistance in diabetes. The insulin receptor is a tetrameric protein composed of two α- and two β-subunits. The β-subunit cytosolic domain possesses tyrosine kinase activity. Insulin binding to the α-subunit extracellular domain activates the β-subunit tyrosine kinase, resulting in both autophosphorylation of the receptor and phosphorylation of downstream signal transduction elements. The autophospho- rylation of the insulin receptor tyrosine residues starts up a protein phosphorylation cascade. First out are a set of proteins known as insulin-receptor substrates (IRS-1-4). These are coupled to several additional protein kinase signal systems: Pathways signaling through PI3-kinase and phosphati-dylinositol (3, 4, 5) P3 (PI-3 kinase and protein kinase B/Akt), Mitogen-activated protein kinases (MAPKinases).11 Thus, genetic changes in IRS-1 may potentially contribute toward the development of insulin resistance, the most common of these being a glycine to arginine change at codon 972 (G972R).9

And also, the G972R polymorphism of IRS-1 gene was associated with insulin resistance, salt sensitivity and non-dipper hypertension.12 However, not many studies exist concerned with relationships between IRS-1 gene polymorphism and MS. Therefore, the aims of the study were to

determine IRS-1 gene (G972R) polymer-phism and proportion of MS in T2DM patients and also examine the relationships between gene polymorphism and MS.

MATERIALS AND METHODS

After getting informed consent, history taking and physical examinations including anthropometric measurement were done and 5 ml of blood samples were collected from diabetic patients attending Diabetes Out-patients Department of YGH. Fasting blood sugar, lipid profile, polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) for IRS-1 gene were measured at Pathology Research Division, Department of Medical Research. Data were analyzed by SPSS (version 16.0) statistical software. Metabolic syndrome was defined as in IDF (International Diabetes Federation) criteria. The disease association with proportions of sample variables was tested by ῾Chi’ square test with 95% confidence interval. Ethical consideration

The study including proforma and written informed consent form was submitted to the Ethical Review Committee, UM (I), Yangon to obtain ethical approval study.

RESULTS

Among 100 T2DM patients, 81 patients were of homozygous (G/G) genotype, 18 patients were of heterozygous (G/A) and only one patient of homozygous (A/A) genotype. The allele frequencies of ῾G’ was 90% and that of “A” was 10% (Fig. 1).

Fig. 1. Homozygous A/A genotype of codon 972 IRS-1 gene at lane 5

108bp

51bp

108 bp

51 bp

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Table 1. Distribution of metabolic syndrome in the IRS-1 (G972R) carrier and non- carrier groups

Metabolic syndrome

Carrier (n=19) (%)

Non-carrier (n=81) (%)

Remark

Presence Absence

16(84) 3(16)

67(82) 14(17) NS

NS=not significant, P value=0.876 Metabolic syndrome distribution in the IRS-1 (G972R) carrier and non-carrier groups

Metabolic syndrome was found in 84% (16/19) of carrier patients, 82% (67/81) of non-carrier patients. Metabolic syndrome was not significantly associated with presence or absence of IRS-1 (G972R) mutation (Table 1). Table 2. Metabolic syndrome between IRS-1

(G972R) non-carriers with and without family history of diabetes mellitus (DM)

Metabolic syndrome

IRS-1(G972R) non-carrier

With family history of DM

Without family history of DM

Total Remark

Presence 15 52 67 S Absence 4 10 14

19 62 81 S=Significant, P value=0.005 Metabolic syndrome between IRS-1 (G972R) non-carrier with and without family history of diabetes mellitus

There was significant difference (p=0.005) in proportion of MS between IRS-1 (G972R) non-carriers with family history of diabetes mellitus and those without (Table 2).

DISCUSSION

Prevalence of IRS-1 (G972R) polymorphism

The prevalence of G972R polymorphism in the present study was much higher than other studies in Asia region. The BMI of the present study was 28.35±4.36 which was much higher than 24±3.0 (Japan),13 25.69±5.27 (Northern Indian)14 and 25.1±4.3 (South Indian).15 But the prevalence of G972R does not seem to be related to

BMI since the prevalence was quite high 15.8% in the Orkunoglu, et al.16 study in which BMI is 22.14±3.98. Polymorphism plus other environmental and metabolic risk factors could increase one’s chance of developing T2DM. Metabolic syndrome distribution in the IRS-1 (G972R) carrier and non-carrier groups The prevalence of MS seems to vary among different study populations based on the presence of risk factors including BMI, life styles, ethnicity, race, age and sex. It was 83% in the present study, 85% in Scott, et al. study,17 59.5% in Ranjith, et al.,18 54.2% in Rojas, et al.,19 64.6% in Chung-Hua.20 The prevalence of MS was varied when the criteria used for the diagnosis of MS were different. Using the clinical definitions, namely the original NCEP-ATP III, the prevalence of MS in the Philippines in 2003 was 11.9%. It became 18.6% when the modified AHA/NHLBI criteria were used.21 Similarly, the prevalence of MS as defined by the NCEP ATP III criteria was 60.4% whereas it was close to it, but not exactly the same, 59.5% in young Indian patients when the IDF criteria were used.18 In the present study, MS was not signi-ficantly associated with IRS-1 (G972R) polymorphism (84% in carrier vs. 82% in non-carrier). Such absence of association between MS and insulin resistance was reported also by Ranjith, et al.18

But Sarac, et al. study showed there was statistically significant association of IRS-1 and IRS-2 polymorphisms with MS.22 One reason for absence of association between MS and polymorphism is the present study included 100 overweight and obese subjects with T2DM and Sarac, et al.22 study had 100 MS subjects and 30 normal control subjects. In addition, the present study determine the relationship between MS and G972R carrier and non-carrier group and that of Sarac, et al.22 study showed relationship between gene and 100 MS and control groups.

24

Metabolic syndrome between IRS-1 (G972R) non-carrier with and without family history of diabetes mellitus In the present study, the proportion of IRS-1 (G972R) non-carrier without family history of diabetes mellitus was higher than that with family history of diabetes mellitus in the MS patients, so there was a significant association between MS and without family history of diabetes mellitus in IRS-1 (G972R) non-carrier. In a nationally representative sample of United State adults without diabetes, family history of diabetes shows a significant, independent association with metabolic syndrome.23

Although family history of diabetes associated with MS,24 the prevalence of chronic diabetic complications and metabolic syndrome is not associated with maternal type 2 diabetes in Scheffel, et al. study.25 It, therefore, seems reasonable to argue that family history of T2DM does not play an important role in the development of MS in the IRS-1 (G972R) non-carrier.

REFERENCES

1. Mooler DE & Kaufman KD. Metabolic syndrome: A clinical and metabolic perspective. Annual Review of Medicine 2004; 56: 45-62.

2. Ford ES, Li C & Satter N. Metabolic syndrome and incident diabetes. Diabetes Care 2008; 31(9): 1898-1904.

3. Chan JCN, Cheung SL & Jih IY. The metabolic syndrome in Hong Kong Chinese: The inter-relationships among its components analyzed by structural equation modeling. Diabetes Care 1996; 19: 953-959.

4. Yin Nwe Tun. Clinical profile of metabolic syndrome in patients with acute ischaemic stroke. [MMedSc thesis]. University of Medicine 1: Yangon; 2007.

5. Cho Mar Lwin. The clinical profile of metabolic syndrome in patients with acute myocardial infarction. [MMedSc thesis]. University of Medicine 1: Yangon; 2005.

6. Goodyear LJ, Giorgino F, Sherman LA, Carey J, Smith RJ & Dohn GL. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. Journal of Clinical Investigation 1995; 95: 2195-2204.

7. Qing S, Shaoshan SW & Zafari AM. Genetics of the metabolic syndrome. Hospital Physician 2006; 51-56.

8. De la Cruz-Mosso U, Munoz-valle JF, Salgado-Goytia L, Garcia-Carreon A, Illades-Aguiar B, Castaneda-Sauccedo E & Parra-Rojas I. Relationship of metabolic syndrome and its components with-844 G/A and HindIII C/G PAI-I gene polymorphism in Mexican children. BMC Pediatrics 2012; 29: 41.

9. Almind K, Bjorbaek C, Vestergaard H, Hansen T, Eshwald SM & Perterson O. Amino acid polymorphism in insulin receptor substrate-1 in non-insulin dependent diabetes mellitus. Lancet 1993; 342: 828-832.

10. Celi SF, Negri C, Tanner K, Raben N, Pablo FD, Rovira A, et al. Molecular scanning for mutations in insulin receptor substrate-1 (IRS-1) gene in Maxican Americans with type 2 diabetes mellitus. Diabetes Metabolism Research Reviews 2000; 16: 370-377.

11. Pirola L, Johnston AM, & Obberghen EV. Modification of insulin action. Diabetologica 2004; 47: 170-184.

12. Dziwura J, Binczak-Kuleta A, Miazgowski T, Ziemak J & Widecka. The association between G972R polymorphism of the IRS-1 gene, insulin resistance, salt sensitivity and non-dipper hypertension. Hypertension Research 2011; 34: 1082-1086.

13. Yamada K, Yuan X & Ishiyama S, et al. Codon 972 polymorphism of the insulin receptor substrate-1 gene in impaired glucose tolerance and late-onset NIDDM. Diabetes Care 1998; 21(5): 753-756.

14. Neena S, Achyut P, Jai P, Agarwal CG, Pant DC & Balraj M. Association of β2-adrenergic receptor and insulin receptor substrate-1 polymorphism with obesity in a Northern Indian population. Indian Journal of Human Genetics 2008; 14(2): 48-54.

15. Bodhini D, Venkatesan R & Viswanathan M. Association study of IRS-1 gene polymorphisms with type-2 diabetes in South Indians. Diabetes Technology and Therapeutics 2011; 13(7): 767-772.

16. Orkunoglusuer FE, Mergen H, Bolu E & Ozata M. Molecular scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in Turkish with type-2 diabetes mellitus. Endocrine Journal 2005; 52: 593-598.

17. Scott R, Donoghoe M, Watts GF, Obrien R, Pardy C, Taskinen M, et al. Impact of metabolic syndrome and its components on cardiovascular disease event rates in 4900 patients with type 2 diabetes assigned to placebo in the field

25

randomized trial. Cardiovascular Diabetology 2011; 10: 102- 109.

18. Ranjith N, Pegoraro RJ, Naidoo DP, Shanmugam R & Rom L. Genetic variants associated with insulin resistance and metabolic syndrome in young Asia Indians with myocardial infarction. Metabolic Syndrome and Related Disorder 2008; 6: 209-214.

19. Rojas R, Aguilar-Salinas CA, Jimenez-Corona A, Shamah-Levy T, Rauda J, Avila-Burgos L, et al. Metabolic syndrome in Mexican adults. Results from the national health and nutrition survey 2006. Salud Publica de Mexico 2010; 52. [Internet]. 2010. [updated 2010 May 2; 2013]. Available from: htpp:// www. dx. doi. org/ 10.1590/ S0036-363420100 00700004.

20. Chung-Hua H. Different impacts of metabolic syndrome components on insulin resistance in type 2 diabetes. International Journal of Endocrinology 2013. [Internet]. [Cited 2 May 2013] Available from: http:// dx.doi.org/ 10.1155/ 2013/740419.

21. Morales DD, Punzalan FE, Paz-pacheco E, Rody GS & Duante CA. Metabolic syndrome in the Philippine general population: Prevalence and

risk for atherosclerotic cardiovascular disease and diabetes mellitus. Diabetes and Vascular Disease Research 2008; 5: 36-43.

22. Sarac F, Berdeli A, Sarac S, Savas S, Atan M & Akcicek F. Insulin receptor substrate gene polymorphism are associated with metabolic syndrome but not with its components. Journal of Diabetes Mellitus 2013; 3(4): 214-220.

23. Ghosh A, Liu T, Khoury MJ & Valdez R. Family history of diabetes and prevalence of the metabolic syndrome in U.S. adults without diabetes: 6-year results from the National Health and Nutrition Examination Survey (1999-2004). Public Health Genomics 2010; 13(6): 353-359.

24. Mithun D, Pal S & Ghosh A. Family history of type-2 diabetes and prevalence of metabolic syndrome in adult Indians. Journal of Cardiovascular Disease Research 2012; 3(2): 104-108.

25. Scheffel RS, Kramer CK, Rdos DV, Pinto LC, Crispim D, Gross JL , et al. The prevalence of chronic diabetic complications and metabolic syndrome is not associated with maternal type 2 diabetes. Brazillan Journal of Medical and Biological Research 2008; 41(12): 1123-1128.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Profile of Paediatric Malignancies in Yangon Children’s Hospital: 2012-2014

Ssu Wynn Mon1*, Aye Aye Khaing2, Han Win1, Tint Myo Hnin2, Ye Myat Thu2,Theingi Thwin1 & Kyaw Zin Thant1

1Department of Medical Research

2Haematology-Oncology Unit Yangon Children’s Hospital

Although the incidence of paediatric malignancy is on rise, little is known about the epidemiology of pediatric cancer especially in low and middle income countries. In Myanmar, reports on the pattern and incidence of childhood cancer are very few. Reliable paediatric cancer data are essential for assessing the magnitude of this problem and for paediatric cancer care. Therefore, this study was aimed to describe the relative frequencies of various paediatric malignancies in Yangon Children’s Hospital (YCH) and their distributions according to age and sex. This hospital-based retrospective study covered all children aged 0 to 14 years with confirmed malignancies. We reviewed hospital records of Haematology-Oncology Unit, YCH from January 2012 to December 2014. A total of 609 patients were diagnosed as cancer during study period. Among them, 379(62.2%) were haematological malignancies and 230(37.8%) were solid tumours. Acute lymphoblastic leukemia (27.5%), acute myeloblastic leukemia (14.3%), Non-Hodgkin’s lymphoma (14%) and retinoblastoma (9.7%) were four most common childhood malignancies. Wilm’s tumour (6.8%), neuroblastoma (6.7%), germ cell tumour (4.1%) and rhabdomyosarcoma (3.6%) were also common. Less common were brain tumours (2.1%), Hodgkin’s lymphoma (1.5%) and Burkitt’s lymphoma (1.0%). Prevalence was higher in boys (58.6%) than girls (41.4%) with male to female ratio 1.5:1. About half (50.9%) of the patients were younger than 5 years and prevalence was less in more than 10 years age group (15.7%). As data were collected from hospital, not from population, findings from this study might not be the representation of our national statistics. But as Haematology-Oncology Unit in YCH is one of the two paediatric oncology centers in Myanmar and patients from most regions of the country attend this hospital for specialist care, these findings might reflect the burden of paediatric malignancy in Myanmar.

Key words: Paediatric malignancies, Yangon Children’s Hospital

INTRODUCTION

The incidence of childhood tumour and type vary greatly throughout the world.1 Although it is relatively rare, comprising less than 1% of cases of malignant disease,2 its incidence is on rise.3 Surveillance, Epidemiology, and End Results (SEER) reports from the National Cancer Institute (NCI) indicated that approximately 164 cases of cancer occur per 1 million populations less than 20 years of age.4 Paediatric malignancies come next to accident as the leading cause of

death among children in the developed world, remaining one of the major causes of death in children between the ages of 0 to 14 years.1 In developing countries like India childhood mortality is still due to malnu-trition and infections, but paediatric tumors are also rising in number.5

With the major advances in the diagnosis, multi-modality therapy, development of ____________________________________ *To whom correspondence should be addressed. Tel: +95-9260985020 E-mail: ssuwinmon@gmail.com

27

rational use of combined chemotherapy and improved supported care, the cure rate in childhood cancer has increased tremendously and over 60% of all childhood cancers are now curable.6 In developed countries, childhood cancer cure rate increased up to 80%7 and five-year survival for certain cancers for example, Hodgkin disease and retinoblastoma, was 95% in 2007.3 However, over 80% of the world’s children with cancer live in less privileged countries,7 where the cure rate is much lower. Moreover, very little is known about the epidemiology of pediatric cancer in low- and middle-income countries (LMICs). Cancer registries are almost nonexistent, and underdiagnosis and underregistration are additional barriers; the development of childhood cancer registries in LMICs should be prioritized.8 There is a lack of nationwide population based cancer registries on the incidence and patterns of childhood cancer in developing countries9 and this also limits our knowledge of the epidemiology of pediatric cancer. Additionally, good quality-population level statistics on the occurrence of cancer at young age have been more difficult to obtain than in adult.10 Serious under reporting, even in western countries, has been documents.11, 12

Due to these difficulties, costs, and uncer-tainties associated with accurate population based cancer registries, hospital registries are the only available source of information for assessing the disease pattern in community, also serving as a practical first step toward a possible population-based assessment of cancer incidence rate.13, 14 As reduction of child mortality is one of the Millennium Development Goals; initiatives aimed at reducing the burden of non-communicable diseases, including child-hood cancer, are needed to be developed.8

Myanmar is the second largest country in Southeast Asia with an estimated total population in 2010 of 59.13 million of which 18.84 million were children <15- years- old.15 There are two pediatric oncology and hematology centers in

Myanmar located at Yangon and Mandalay Children's Hospitals serving the southern and northern regions of the country, respectively but there is no national population based pediatric cancer registry.16 Reliable paediatric cancer data is essential for assessing the magnitude of this problem and for qualified paediatric cancer care.8 Although many papers have been published on this in some developing countries,10 reports on the pattern and incidence of childhood cancer in Myanmar are very few. The aim of this study was to describe the relative frequencies of various paediatric malignancies and their distribution according to age and sex. It can help assessing the burden of paediatric malignancies in Myanmar and the information collected has the potential to assist scientific analysis, planning and research.

MATERIALS AND METHODS

In this retrospective study, 3-year hospital records of Haematology-Oncology Unit, Yangon Children's Hospital, from January 2012 to December 2014 were used. All children with cancer, aged 0 to 14 years diagnosed by means of histological or cytological examination during that period were included in the study. Tumour types were classified according to SEER (Surveillance, Epidemiology and End Results) modification of ICCC (International Classification of Childhood Cancer) by National Cancer Institute (NCI).17 Data were extracted from the hospital records using the predesigned proforma. The profile of childhood cancer was studied focusing on the prevalence of tumors according to age, sex and type of tumors.

RESULTS

From January 2012 to December 2014, a total of 609 children were admitted to Haematology-Oncology Unit, Yangon Children's Hospital, with confirmed malig-nancies, of which 379 cases (62.2%) were haematological malignancies and 230 cases (37.8%) were solid tumours.

28

Leukemia constituted 43.5% (265 cases) of all childhood malignancies; among them Acute lymphoblastic leukaemia was the most frequent leukemia, accounted for 63% (167 cases) of all leukemia cases. The next most common leukemia was acute myeloid leukemia accounted for 32.8% (87 cases) of total leukaemia patients. Among lym-phomas, Non-Hodgkin lymphomas were more prevalent (85%, 85 cases) than Hodgkin’s lymphoma (9 cases, 9%) and Burkitt’s lymphoma (6 cases, 6%) (Fig.1). Retinoblastoma was the most frequently found solid tumour (59 cases, 9.7%) of

Fig.1. Common haematological malignancies in YCH during the study

A = Others G = Pancreaticoblastoma B = Ovarian cancer H = Hepatoblastoma C = Wilm’s tumour I = Germ cell tumour D = Colon cancer J = Neuroblastoma E = Osteosarcoma K = Brain tumour F = Rhabdomyosarcoma L = Retinoblastoma

Fig. 2. Common solid tumours in YCH during the study

all cancer patients admitted to Yangon Children’s Hospital. Second common solid tumours were Neuroblastoma (41 cases, 6.7%) and Wilm’s tumour (41 cases, 6.7%) followed by germ cell tumour (25 cases, 4.1%), rhabdomyosarcoma (22 cases, 3.6%) and brain tumour which accounted for only 2.1% of all childhood malignancies, 13 cases (Fig. 2). The prevalence of paediatric malig-nancies in Yangon Children’s Hospital became higher in 2013 than 2012 but it was slightly lower down in 2014 (Table 1). Table 1. Number of childhood cancer cases in

YGH (2012-2014) Year Male Female Total 2012 103 78 181 2013 138 88 226 2014 115 87 202

Table 2. Gender distribution of paediatric cancer

patients admitted to Yangon Children’s Hospital during the study period

Tumour types F % M % Total % Acute lymphoblastic

leukemia 68 11.2 99 16.3 167 27.5

Acute myeloblastic leukemia

40 6.6 47 7.7 87 14.3

Chronic myeloid leukemia 4 0.6 5 0.8 9 1.5 Juvanile myelomonocytic

leukemia 1 0.2 1 0.2 2 0.3

Non-Hodgkin's lymphoma 23 3.8 62 10.2 85 14.0 Hodgkin's lymphoma 0 0 9 1.5 9 1.5 Burkitt's lymphoma 0 0 6 1.0 6 1.0 Langhan cell histiocytosis 3 0.5 6 1.0 9 1.5 Haemophagocystic

lymphohisteocytosis 4 0.6 1 0.2 5 0.8

Brain tumour 8 1.3 5 0.8 13 2.1 Primitive neuroecto-

dermal tumour 0 0 2 0.3 2 0.3

Neuroblastoma 17 2.8 24 4.0 41 6.7 Malignant nerve sheath

tumour 0 0 1 0.2 1 0.2

Retinoblastoma 31 5.1 28 4.6 59 9.7 Wilm's tumour 16 2.6 25 4.1 41 6.7 Heptoblastoma 5 0.8 7 1.2 12 2.0 Osteosarcoma 3 0.5 1 0.2 4 0.7 Rhabdomyosarcoma 11 1.8 11 1.8 22 3.6 Liposarcoma 0 0 1 0.2 1 0.2 Germ cell tumour 13 2.1 12 2.0 25 4.1 Others malignant epithelial neoplasms Adrenal carcinoma 1 0.2 0 0 1 0.2 Paranasal sinus tumour 1 0.2 0 0 1 0.2 Squamous cell carcinoma 0 0 1 0.2 1 0.2 Colon cancer 1 0.2 1 0.2 2 0.33 Pancreatoblastoma 0 0 2 0.3 2 0.33 Ovarian cancer 2 0.3 0 0 2 0.33 Total 252 41.4 357 58.6 609 100

M=Male, F=Female, %=Percentage

A = Juvanile myelomonocytic leukemia B = Bukitt’s lymphoma C = Langerhan cell histiocytosis D = Haemophagocytic lymphohistiocytosis E = Chronic myeloid leukemia F = Hodgkin’s lymphoma G = Non-Hodgkin’s lymphoma H = Acute myeloblastic lymphoma I = Acute lymphoblastic

18016014012010080604020

0A B C D E F G H I

A B C D E F G H I J K

Type

s of

pae

diat

ric c

ance

r

Total number of cases

Type

s of

tum

or

Total numbers of case

A B C D E F G H I J K L Total numbers of case

29

Paediatric malignancies were affected more in boys (58.6%) than girls (41.4%) with the ratio of 1.5:1 (Table 2). Table 3. Age distributions of paediatric cancer types among 609 patients in YGH

Tumour types Age (Years) Total % <5 5 to 10 >10 Acute lymphoblastic leukemia

77 57 33 167 27.4

Acute myeloblastic leukemia

29 40 18 87 14.3

Chronic myeloid leukemia 1 4 4 9 1.5 Juvanile myelomonocytic

leukemia 1 0 1 2 0.3

Non-Hodgkin's lymphoma 26 41 18 85 14 Hodgkin's lymphoma 2 5 2 9 1.5 Burkitt's lymphoma 6 0 0 6 1 Langhan cell histiocytosis 8 1 0 9 1.5 Haemophagocystic

lymphohisteocytosis 4 1 0 5 0.8

Brain tumour 1 11 1 13 2.1 Primitive neuroectodermal

tumour 0 2 0 2 0.3

Neuroblastoma 34 5 2 41 6.7 Malignant nerve sheath

tumour 0 1 0 1 0.2

Retinoblastoma 51 7 1 59 9.7 Wilm’s tumour 34 7 0 41 6.7 Heptoblastoma 9 0 3 12 2 Osteosarcoma 0 1 3 4 0.6 Rhabdomyosarcoma 12 9 1 22 3.6 Liposarcoma 0 0 1 1 0.2 Germ cell tumour 14 6 5 25 4.1 Adrenal carcinoma 1 0 0 1 0.2 Paranasal sinus tumour 0 0 1 1 0.2 Squamous cell carcinoma 0 1 0 1 0.2 Colon cancer 0 0 2 2 0.3 Pancreatoblastoma 0 2 0 2 0.3 Ovarian cancer 0 2 0 2 0.3 Total (%) 310

(50.9) 203

(33.3) 96

(15.8) 609 100

About 51% (309/607) of all patients belonged to below 5 years age group, one third (34%, 203/607) were aged 5-10 years and 15% (95/607) were more than 10 years. Acute lymphoid leukaemia (ALL) was peaked (25%, 77/309) before the age of 5 years, followed by retinoblastoma (16.5%, 51/309) and neuroblastoma (11.0%, 34/309). From 5 to 10 years, ALL (28%, 57/203), NHL (20.2%, 41/203) and AML (14.2%, 29/203) were three most prevalent malignancies. After the age of 10 years, 133 the most frequent neoplasm remained ALL (34.7%, 33/95) but among solid tumours, epithelial neoplasm like ovarian cancer, bone tumour and colon cancer became more frequent (Table 3).

DISCUSSION

The three most common malignancies in this study were leukemia (43.5%), lymphoma (16.5%) and retinoblastoma (9.7%). Jabeen, et al. also revealed in their hospital based retrospective study that lymphoma (24.2%), retinoblastoma (17.4%) and leukaemia (14.3%) were commonest childhood cancer in their centre.18 It was observed that these hospital-based data differed from population-based data as Cancer Facts & Figures 2014 by American Cancer Society described that cancers most common in children ages 0-14 are acute lymphocytic leukemia (26%), brain and CNS (21%), neuroblastoma (7%), and non-Hodgkin lymphoma (6%).1

Brain tumour is the third most common childhood malignancy and commonest malignancy among solid tumours in USA, UK and also in some centers from India.19 But, it stood as sixth commonest tumour in the centre (13 cases, 2.1%). This might be related to the referral pattern. In Myanmar, children with brain tumour are usually referred to Neurosurgical Department and Radiotherapy Department of Yangon General Hospital. After surgery and/or radiotherapy most of the patients failed to seek further treatment with chemotherapy. Similar finding was seen in a Turkish study done in 2013 by Kutluk, et al.7where they described that some of the children with CNS tumour who required chemotherapy were not referred to Paediatric Oncology Clinic.

Hodgkin’s lymphoma was commonest in children between age 5 to 10 year and most of the adolescent cases were expected to be taking treatment at adult oncology centers. Burkitt’s lymphoma is the least common lymphoma, constituted only 3% of total lymphoma cases and 1% of all malignant cases as our center is not in endemic area. Although osteosarcoma was top eight paediatric malignancy, encompassing 2% of the total cases, other bone tumours like chondrosarcoma, Ewing’s sarcoma were not seen in this study.

30

Retinoblastoma occurs in children under 5 years (86.4%) and this proportion seems to be high. This could also be associated with the pattern of referral as there is a good relationship between paediatric oncologists and paediatric eye surgeons from Yangon Eye Hospital. This result is almost similar to paediatric malignancies profile study carried out by Jabeen, et al. where they found that 17.4% of all solid tumours were retino-blastoma which stood the first among all solid tumours.18

Hepatoblastoma is listed as a rare disease by the Office of Rare Diseases (ORD) of the National Institutes of Health (NIH). The incidence of hepatocellular carcinoma in the United States is 0.8 in children between the ages of 0 and 14 years.20 But, it was not an uncommon cancer in Yangon Children’s Hospital and 2% (12 cases) were reported as hepatoblastoma during study period. No hepatocellular carcinoma case was seen during this study. Due to incomplete data record, information regarding the stage, family history and referred regions could not be described in this study.

Conclusion

As data were collected data from hospital, not from population, findings from this study may not be the representation of the national statistics. However, as Haematology-Oncology Unit in Yangon Children’s Hospital is one of the two major academic paediatric oncology centers in Myanmar and patients from most regions of the country attend this government hospital for specialist care and cure, these findings could reflect the burden of paediatric malignancy in Myanmar. These basic data could be a support for health planning, resource allocation and further paediatric oncology researches.

It also highlighted the importance of careful documentation and completeness of data in hospital based cancer registry and moreover, it can serve as a potential platform for a nationwide population-based cancer registry.

ACKNOWLEDGEMENT

The authors would like to thank all the staff and patients of Haematology-Oncology Unit, Yangon Children’s Hospital. The study was supported by Department of Medical Research.

REFERENCES

1. American Cancer Society. Cancer Facts and Figures, 2014 [Internet]. Available from: https://www.cancer.org/content/dam/cancer-org/ research/ cancer- facts- and- statistics/ annual- cancer-facts-and-figures/2014/cancer-facts-and-figures-2014.pdf

2. Steliarova-Foucher E, Stiller C, Kaatsch P, et al. Geographical patterns and time trends of cancer incidence and survival among children and adolescents in Europe since the 1970s (the ACCIS project): An epidemiological study. Lancet 2004; 364(9451): 2097-2105.

3. Aurora RS, Eden T & Kapoor G. Epidemiology of childhood cancer in India. Indian Journal of Cancer 2009; 46: 264-273.

4. Casciato DA. Cancer in Chilhood. In: Manual of Clinical Oncology, Theodore B. Moore & Carole GH Hurvitz. 7th ed, 2012; 397-407.

5. Rathi AK, Kumar S, Ashu A, Singh K & Bahadur AK. Epidemiology of paediatric tumours at a tertiary care centre. Indian Journal of Medical and Paediatric Oncology 2007; 28(2): 33-35.

6. Ayra LS. Childhood cancer challenges and opportunities. Indian Journal of Paediatric 2003; 70: 159-162.

7. Kutluk MT, Yesilipek A, et al. Turky. Turkish National Paediatric Cancer Registry 2002-2008. Poster presentation at 2013 ASCO Annual Meeting; May 31-June 4 2013; Chicago, Illinois, USA, 8.

8. Carlos Rodriguez-Galindoa, Paola Friedricha, Lisa Morrisseya, & Lindsay Fraziera. Global challenges in pediatric oncology. Current Opinion in pediatrics 2013; 25(1): 3-15.

9. Akhtar SS, Abu Bakr MA, Dawi SA & Huq IU. Cancer in Libya- a retrospective study (1981-1985). African Journal of Medicine and Medical Science 1993; 22(1): 17-24.

10. Parkin DM, Kramarova E, Draper GJ, et al. International Incidence of Childhood Cancer. Vol II. International Agency for Research on Cancer, WHO 1998; 1-391.

11. Draper GJ, Bower BD, Darby SC & Doll R. Completeness of registration of childhood

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leukaemia near nuclear installations and elsewhere in the Oxford region. British Medical Journal 1993; 299(6705): 592.

12. Cook-Mozaffari PJ, Vincent T, Forman D, Ashwood FL & Alderson M. Cancer incidence and mortality in the vicinity of nuclear Installations, England and Wales 1959-1980. Study on Medical and Population Subjects No. 51, London HMSO 1987; 4(1): 959-980.

13. Valsecchi MG, Steliarova-Foucher E. Cancer registration in developing countries: Luxury or necessity? The Lancet Oncology 2008; 9(2): 159-167.

14. Howard SC, Metzger ML, Wilimas JA, Quintana Y, Pui CH, Robison LL & Ribeeiro RC. Childhood cancer epidemiology in low income countries. Cancer 2008; 112: 461-472.

15. Ministry of Health. Health in Myanmar, 2011.

16. Jay Halbert & Aye Aye Khaing. Overview of pediatric oncology and hematology in Myanmar. South Asian Journal of Cancer 2014; 3(1): 78-82.

17. International Classification of Childhood Cancer (ICCC). [Internet]. 2008 [updated 2008]. Available from: https://seer.cancer.gov/iccc/

18. Jabeen S, Hanque M, Islam M & Talukder MH. Profile of paediatric malignancies: A five-year study. Journal of Dhaka Medical College 2010; 19(1): 33-38.

19. Joshi V & Kumar A. Paediatric Haemato-oncology in India. Epidemiologic differences Agrwal MB. Haematology Today 2004.

20. Howlader N, Noone AM, Krapcho M, et al. Childhood cancer by the ICCC. In: SEER Cancer Statistics Review, 1975-2009 (Vintage 2009 Populations). National Cancer Institute, 2012, Section 29.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Urinary Albumin to Creatinine Ratio and the Degree of Coronary Artery Narrowing in Patients Undergoing Coronary Angiography

Khine Wai Mon1*, Saw Wut Hmone, Myint Myint Nyein & Myat Mon

1Department of Pathology

University of Medicine 1 (Yangon)

Coronary artery disease (CAD) is one of the major causes of death and disability in both developed and developing countries. Microalbuminuria is regarded not only an indicator of global endothelial dysfunction but also an antecedent to atherosclerotic coronary artery disease. A cross-sectional study was done on 57 patients of both sexes undergoing coronary angiography at Cardiac Medical Unit, Yangon General Hospital in order to associate urinary albumin to creatinine ratio (UACR) and the degree of coronary artery narrowing. The mean age was 57.9±9.8 years with M:F ratio of 1.7:1. UACR was measured by immunoturbidimetry method using UACR 30mg/g as cut- off value for having microalbuminuria. The severity of coronary artery narrowing was recorded as normal, one vessel narrowing, two vessels narrowing and three vessels narrowing. Microalbuminuria was present in 31.6% of total cases. There were 21.1% CAD negative (normal) patients and 78.9% CAD positive (stenosis ≥50%) patients. Microalbuminuria was detected in 16.7% of patients with one vessel narrowing, 22.2% with two vessels narrowing and 61.1% with three vessels narrowing. None of the patient with normal angiogram result had microalbuminuria. There was a significant association between UACR and degree of coronary artery narrowing (p=<0.001). This showed that patients with microalbuminuria (raised UACR) have greater atherosclerotic burden and more severe coronary artery disease than those without microalbuminuria.

Key words: Urinary albumin to creatinine ratio, Coronary artery disease, Coronary angiography

INTRODUCTION

Coronary artery disease (CAD) is a well-known cause of death and disability worldwide. Atherosclerosis is responsible for almost all cases of CAD. It is a multifactorial disorder with several different risk factors. Advancing age, male sex, hypertension, diabetes mellitus, cigarette smoking and dyslipidemia are the major and independent well-known risk factors for CAD.1 There are also many new biomarkers relating cardiovascular risk, including C-reactive protein (CRP) levels,2 B-type natriuretic peptide,3 fibrinogen,4 D-dimer5 and homocystine.6

The high volume of fluid filtered across the glomerular endothelium (140-180 L/day)

markedly amplifies the functional con-sequence (increase albumin filtration) of early endothelial injury in the glomerulus in contrast to the systemic endothelial bed in which early atherosclerotic injury is un-detectable. The emergence of micro-albuminuria unmasks systemic endothelial injury likely occurring simultaneously in other vascular beds, progressively from silent to overt disease later years.7 It is thought to be an indicator of glomerular endothelial dysfunction and of macro-molecular hyperpermeability.8 Increasing prevalence of microalbuminuria in various clinical conditions, such as _____________________________________________________ *To whom correspondence should be addressed. Tel: +95-95107279 E-mail: khaiwaimon@gmail.com

33

diabetes mellitus, acute ischemic heart disease, acute stroke9, arterial hypertension10 and carotid atherosclerosis,11 strengthens the link between microalbuminuria and vascular diseases. Although a 24-hour urine collection is the gold standard for the detection of microalbuminuria, several studies have found that a urinary albumin to creatinine ratio (UACR) is equally sensitive, specific, and can be easily utilized on a daily basis.12

In this study, urinary albumin to creatinine ratio was done in patients undergoing coronary angiography aiming to detect the association of microalbuminuria in terms of UACR and the degree of narrowing of coronary artery.

MATERIALS AND METHODS The study was a cross-sectional, hospital- and laboratory based analytical study. Fifty- seven patients admitted to Cardiac Medical Unit (CMU) of Yangon General Hospital (YGH) for coronary angiography were included.

The patients with overt proteinuria, hematuria, urinary tract infection detected by routine urinalysis and renal insufficiency with abnormal urea and creatinine result were excluded. Patient with blood pressure above 140/90 mmol/l or patient taking anti-hypertensive drug was recorded as having hypertension.13 Patient with fasting blood sugar ≥126 mg/dl (7.0 mmol/l) or 2HPP (two-hour post-prandial glucose) ≥200 mg/dl (11.1 mmol/l) or random blood sugar ≥200 mg/dl (11.1 mmol/l) in patient with symptoms of hyperglycaemia or HbA1C ≥6.5%14 or patient taking anti-diabetic drugs was recorded as having diabetes mellitus. Patient with fasting lipid profile showing one of the following values: Total cholesterol ≥240 mg/dl, Triglyceride ≥200 mg/dl, LDL cholesterol ≥160 mg/dl, HDL cholesterol <60 mg/dl was recorded as having dyslipidemia. Patient who had history of smoking; either current smoker or ex-smoker was recorded as smoker.

Five milliliters of random urine sample were taken for determination of microalbuminuria in terms of urinary albumin to creatinine ratio. It was measured by DCA 2000+ Microalbumin/Creatinine assay with: albumin reagent: 3.3-10% purified poly-clonal goat anti-human albumin antiserum in 50 mM TRIS; 8.6% w/v non-reactive ingredients (15 ul dried in each cartridge), creatinine alkaline reagent: 28% potassium hydroxide, 5% non-reactive ingredients (30 ul dried in each cartridge) and buffer solution: 0.22% w/v 3, 5-dinitro-benzoic acid, 4% polyethylene glycol in 25 mM HEPPS buffer, with 2% non-reactive ingredients (0.57 ml in each cartridge).

The ratio of urinary albumin to creatinine ratio (UACR) was used to define micro-albuminuria. The patients with urinary albumin to creatinine ratio of 30-300 mg/g were defined as having microalbuminuria. In this study, the stenosis ≥50% in a main coronary artery or in one of the other branches was recorded as having significant coronary disease.15 The <50% stenosis was recorded as non-significant or normal.

Statistical analysis was performed using Statistical Package for Social Science (SPSS) 16.0. The data were described in terms of mean and standard deviation, while categorical variables were expressed as numbers and percentages when appropriate. Associations between two categorical variables were tested using Pearson Chi-Square, as appropriate. A ‘p’ value of ≤0.05 was considered statistically significant.

RESULTS Demographic characteristics Within the study group, the youngest patient was found to be 30 years and the oldest age was 77 years. The mean age was 57.91±9.86 years. The majority of the patients (52%) were in 56-70 years age group. There were 36 men (63.2%) and 21 women (36.8%). Male to female ratio was found to be 1.7:1.

34

Urinary albumin to creatinine ratio (UACR) among patients undergoing coronary angiography In this study, UACR was studied with cut- off value at 30-300 mg/g as raised UACR and 18(31.6%) out of 57 patients had raised UACR. Degree of coronary artery narrowing in patients undergoing coronary angiography The results of coronary angiography showed that there were 12(21.1%) CAD negative (normal) patients and 45(78.9%) CAD positive (stenosis ≥50%) patients. Among the CAD positive patients, 21(46.7%) had one vessel involvement, 9(20.0%) had two vessels and 15(33.3%) had three vessels involvement. Association between UACR and degree of coronary artery narrowing in patients undergoing coronary angiography

In the patients with normal UACR, 12(30.8%) patients had normal angiogram result, 18(46.2%) had one vessel narrowing, 5(12.8%) had two vessels narrowing and 4(10.2%) had three vessels narrowing. On the other hand, in patients having raised UACR, there was no normal angiogram result and 11(61.1%) patients had three vessels narrowing while 3(16.7%) and 4(22.2%) had one vessel and two vessels narrowing, respectively.

Association between CAD risk factors and UACR in patients undergoing coronary angiography

In the study population, the mean age in the group of patients with normal UACR was 55.69±9.91 years ranged from 30-77 years, and in those with raised UACR was 62.72±8.07 years ranged from 46-72 years. In normal UACR group, there were 25 males (64.1%) and 14 females (35.9%). In raised UACR group, there were 11 males (61.1%) and 7 females (38.9%). The association between smoking, DM, hyper-tension, dyslipidemia and UACR in studied population were not found to be significant as shown in Table 1.

Table 1. Association between CAD risk factors and UACR in patients undergoing coronary angiography

Number of patients p

value Normal UACR Raised UACR n=39(68.4%) n=18(31.6%)

Age Range Mean SD

30-77 55.69 9.91

46-75 62.72 8.07

0.101

Sex Male Female

25(64.1%) 14(35.9%)

11(61.1%) 7(38.9%)

0.828

Family history of CAD Yes No

18(46.2%) 21(53.8%)

3(16.7%)

15(83.3%)

0.032

Smoking Yes No

24(61.5%) 15(38.5%)

8(44.4%)

10(55.6%)

0.227

Diabetes mellitus Yes No

12(30.8%) 27(69.2%)

7(38.9%)

11(61.1%)

0.546

Hypertension Yes No

27(69.2%) 12(30.8%)

14(77.8%) 4(22.2%)

0.504

Dyslipidemia Yes No

14(35.9%) 25(64.1%)

10(55.6%) 8(44.4%)

0.162

Fig 1. Association between UACR and degree

of coronary artery narrowing in patients undergoing coronary angiography

Association between traditional CAD risk factors and degree of coronary artery narrowing in patients undergoing coronary angiography The mean age of patients with normal or <50% narrowing was 54.67±12.34 years and in those with ≥50% narrowing in one

A= Normal B= >50% narrowing in one vessel C= >50% narrowing in two vessels D= >50% narrowing in three vessel

A B C D Degree of coronary artery narrowing

20

18

16

14

12

10

8

6

4

2

0

Num

ber o

f pat

ient

s 12

18

5 4

3 4

11

0

Normal UACR Raised UACR

35

Table 2. Association between traditional CAD risk factors and degree of coronary artery narrowing in patients undergoing coronary angiography

vessel was 55.71±8.42 years, ≥50% narrowing in two vessels was 58.89±11.25 years and ≥50% narrowing in three vessels was 63±7.19 years. In each group the per-centage of male was apparently higher than female (Fig. 1). The association between family history of CAD, smoking, DM, hypertension, dyslipidemia and the degree of coronary artery narrowing were not found to be significant as shown in Table 2. Association between UACR and degree of coronary artery narrowing in patients undergoing coronary angiography

Table 3. Association between UACR and degree of coronary artery narrowing in patients undergoing coronary angiography

A= Normal or <50% narrowing B=≥50% narrowing in one vessel C=≥50% narrowing in two vessels D=≥50% narrowing in three vessel

UACR was raised in none of the patient with normal coronary artery. Only 3(14.3%) of patients with one vessel disease had raised UACR while UACR was raised in 4(44.4%) of patients with double vessel disease and 11(73.3%) of patients with triple vessel disease. There was a significant association between UACR and the degree of coronary artery involvement. The higher the vessel involvement, the more is the chance of having raised UACR (Table 3).

DISCUSSION

The traditional risk factors of coronary artery disease do not entirely explain the variation of CAD incidence and mortality in individual and populations. This fact has led to studies on nontraditional cardiovascular risk factors and microalbuminuria appears as one of these factors. In this study, 31.6% of patients had raised UACR (microalbuminuria). This finding was more or less comparable to the studies done by Sadaka, et al.,16 Rein, et al.,17 Deveci, et al.18 and Hashim, et al.,21 in which the prevalence were 34%,16 24%,17 28%18 and 37%,21 respectively.

Number of patients p

value Normal or <50%

narrowing ≥50% narrowing in

one vessel ≥50% narrowing in

two vessels ≥50% narrowing in

three vessels n=12(21.1%) n=21(36.8%) n=9(15.8%) n=15(26.3%)

Age Range Mean SD

30-77 54.67 12.34

41-75 55.71 8.42

41-71 58.89 11.25

46-72

63 7.19

0.115

Sex Male Female

7(58.3%) 5(41.7%)

14(66.7%) 7(33.3%)

6(66.7%) 3(33.3%)

9(60%) 6(40%)

0.952

Family h/o CAD Yes No

5(41.7%) 7(58.3%)

8(38.1%) 13(61.9%)

2(22.2%) 7(77.8%)

6(40%) 9(60%)

0.795

Smoking Yes No

7(58.3%) 5(41.7%)

14(66.7%) 7(33.3%)

3(33.3%) 6(66.7%)

8(53.3%) 7(46.7%)

0.404

DM Yes No

4(33.3%) 8(66.7%)

6(28.6%) 15(71.4%)

3(33.3%) 6(66.7%)

6(40%) 9(60%)

0.916

Hypertension Yes No

7(58.3%) 5(41.7%)

16(76.2%) 5(23.8%)

7(77.8%) 2(22.2%)

11(73.3%) 4(26.7%)

0.693

Dyslipidemia Yes No

4(33.3%) 8(66.7%)

6(40.0%) 9(60.0%)

24(42.1%) 33(57.9%)

6(40%) 9(60%)

0.876

Total A B C D P

value n=12 (21.1)

n=21 (36.8)

n=9 (15.8)

n=15 (26.3)

UACR Normal Raised

12(100)

0(0)

18(85.7) 3(14.3)

5(55.6) 4(44.4)

4(26.7) 11(73.3)

<0.001

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In the study population, the coronary angiographies were done in cases with post-acute myocardial infarction (57.9%), post-NSTEMI (10.5%), unstable angina (14.1%) and positive exercise tolerance test (17.54%). The results of coronary angiography showed that 21.1% of patients had normal coronary artery while 36.8% had one vessel narrowing, 15.8% had two vessels narrowing and the rest (26.3%) had three vessels narrowing.

Association between UACR and degree of coronary artery narrowing

In the patients having normal UACR, 12(30.8%) had normal angiogram result, 18(46.2%) had one vessel narrowing, 5(12.8%) had two vessels narrowing and 4(10.2%) had three vessels narrowing. On the other hand, in patients having raised UACR, there was no normal angiogram result and most 11(61.1%) patients had three vessels narrowing while 3(16.7%) and 4(22.2%) had one vessel and two vessels narrowing, respectively. This finding was statistically significant with the p value of <0.001. According to the study, the patients with microalbuminuria had a greater athero-sclerotic burden and a more severe coronary artery disease in the forms of total number of vessels affected than those without microalbuminuria.

These results supported many studies. In the study of Sadaka, et al., microalbuminuria was observed in 34% of patients and these patients were found to have more severe angiographic CAD compared to those without microalbuminuria. That study also concluded that microalbuminuria could be utilized as an independent risk factor for CAD. Similar results were presented by Hoseini and Rasouli who performed a study consisting of 153 non diabetic patients who underwent coronary angiography.19 In the study of Sukhija, et al., the patients with microalbuminuria have more severe angiographic CAD showing microalbu-minuria is independent of other risk factors and is particularly evident in patients with DM.22

The study performed by Parvizi, et al., showed that the urinary albumin/creatinine ratio of the patients with confirmed coronary atherosclerotic lesion was higher than that of the control (p=0.00). It indicates the existence of a significant correlation between the extension of atherosclerotic lesions and UACR.23

Hashim, et al., found that the frequency of microalbuminuria was elevated in the study population (37%) which is significantly higher as compared to the general population which ranges from 2.2% to 10.2% in various studies. That study also highlights that MA is more frequent in non-diabetic patients with CAD than the general population and thus may be an important emerging risk marker for CAD.21 According to the study, although statistically not significant, microalbuminuria (Raised UACR) was seemed to be more common in older age group (mean age 62.72±8.06 years vs. 55.69±9.91 years) (p=0.101). This finding was in accordance with the findings of Rein, et al.,19 Ei Ei Shwe24 and Sandar Soe.25 Among 18 patients with raised UACR, 11(61.1%) patients were men and 7(38.9%) women. Therefore, microalbuminuria was found to be common in male although statistically not significant (p=0.828). It was found to be similar to the result of Sadaka, et al., in which raised UACR was accounted for 73.5% men and 26.5% women.16 Yin Mon Thant26 stated that the occurrence of microalbuminuria between men and women had ratio of 1.77:1 when studied in Myanmar patients with AMI.26 In the study of Parsa, et al., among 16 patients with microalbuminuria, there were 12 men (35.3%) and 4 women (9.3%) and there was a significant difference between the genders regarding the prevalence of micro-albuminuria (OR of male/ female=5.3, 95% CI: 1.53-18.5, p<0.005).20 Patients with diabetes mellitus are at high risk of suffering renal damage. Early detection of diabetic nephropathy is one of

37

the important steps in the management of diabetic patients. Microalbuminuria is common in diabetic patients than those without diabetes. In HOPE study, 32.6% of participants with DM and 14.8% of participants without DM were detected to have microalbuminuria at baseline.27 In the study of Ei Ei Shwe, 43% of patients with DM had microalbuminuria.24

In the current study, 19 patients (33.3% of study population) had diabetes and 7 diabetic patients (12.9% of the study population and 36.8% of diabetic patients) were found to have microalbuminuria. This might be due to the small study population and the selected diabetic patients under-going coronary angiography had good glycemic control and renoprotective drugs like ACEI and ARB were already given to the patients. The patients with advanced nephropathy and macroalbuminuria were also excluded from the study by exclusion criteria. Out of 57 patients, 21 patients had family history of CAD. Microalbuminuria was present in 3 patients among those having family history. There were 32 patients who had smoking history, in which 8 patients had microalbuminuria. Hypertension was observed in 41 patients out of 57 and 14 hypertensive patients had microalbuminuria. Dyslipidemia was present in 24 patients and 10 of those had microalbuminuria. There was no obvious significant association between the CAD risk factors and UACR in the study population except with family history of CAD. In the study of Sadaka, et al., there was no significant difference in the prevalence of hypertension and hypercholesterolemia between the patients with or wi thout microalbuminuria.16 Sukhija, et al., studied the relationship of microalbuminuria and coronary artery disease in patients with or without DM and stated that there were no significant differences in the prevalence of hypertension, hypercholesterolemia and current smoking across the 4 groups of patients (DM+ MA+, DM+ MA-, DM-

MA+, DM- MA-).22 The patients were categorized according to the degree of coronary artery narrowing as following: patients with normal coronary artery (normal or <50% narrowing), those with ≥50% narrowing in one vessel, two vessels and three vessels.

The mean age of study population according to status of coronary artery narrowing were 54.67±12.3 years in patients with normal artery while 55.71±8.4 years, 58.89±11.2 years and 63±7.1 years in patients with one vessel, two vessels and three vessels narrowing, respectively. It could be concluded that the older the age, the more is the possibility of having multiple vessels involvement although not significant statistically (p=0.115). In the study of Bildirici, et al., as well, those with CAD were older than those without CAD (59±11years vs. 55±9 years).28

In each category, the number of male exceeded that of female (p=0.952). This observed finding strengthened the fact that male sex is the risk factor for CAD although not significant statistically. This finding was consistent with the findings of Sadaka, et al.,16 Hoseini, et al.,19 Bildirici, et al.,28 and Rein, et al.17

Family history of CAD was present in 41.7% of normal patients, 38.1% of patients with one vessel disease, 22.2% of patients with double vessel disease and 40% of patients with triple vessel disease. There was no statistically significant association between the present or absent of family history of CAD and degree of coronary artery narrowing in this study (p=0.795).

Smokers were found to have more risk of having CAD than non-smokers although statistically not significant (p=0.404). This study revealed 66.7% of one vessel disease patients, 33.3% of double vessel disease patients and 53.3% of triple vessel disease patients were smokers. Similar finding were observed in the studies of Hoseini, et al.,16 Bildirici, et al.,28 and Rein, et al.,17 Diabetes was present in 28.6% patients with one

38

vessel disease, 33.3% of double vessel disease and 40% of triple vessel disease patients (p=0.916).

In this study, 76.2% of patients with one vessel disease, 77.8% of those with double vessel disease and 73.3% of triple vessel disease patients had hypertension. Although hypertensive patients are majority of the CAD patients, hypertension was not found to have statistical association with degree of coronary artery narrowing (p=0.693).

Dyslipidemia was observed in 40% of one vessel disease patients, 42.1% of double vessel disease and 40% of triple vessel disease patients (p=0.876). In the study of Sadaka, et al., there were no significant differences in the prevalence of hyper-tension and hypercholesterolemia between the patients with or without CAD.16 Furthermore, Bildirici, et al., also found out that baseline clinical and laboratory charac-teristic of patients with or without CAD, including the statuses of hypertension, DM, smoking, dyslipidemia and family history, were not different in his study.28

There are several limitations to the present study. The most important limitations are small sample size and short duration of study period. This study solely relied on only one sample of UACR and a consecutive assay could have provided additional information. Another limitation is that as most of the patients were already optimized for the confounding factors before angiography, like blood pressure optimization, sugar control and reno-protective drugs like ACEI and ARB were already given, these could have some effect on the patients’ UACR status. Since the study was conducted on a limited number of patients, there is a need for further investigations on larger group of patients in order to support these results.

ACKNOWLEDGEMENT

We would like to thank Professor Zaw Wai Soe, Rector, University of Medicine 1, (Yangon) for allowing to conduct this study;

Professor Daw Nwe Nwe, Professor and Head of Department of Cardiology, Yangon General Hospital for allowing to study the patients at Cardiac Medical Unit; Associate Professor Dr. Su Htar Lwin, Department of Preventive and Social Medicine, University of Medicine 1 (Yangon) for the kind help regarding statistical work; all staff of Department of Chemical Pathology, Yangon General Hospital for their help to get reliable results in time and all patients and their families for their willingness to help the study.

REFERENCES

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Fortmann S, Sans S, Tolonen H, et al. Estimation of contribution of changes in classic risk factor to trends in coronary event rates across the WHO MONICA project population. Lancet 2000; 355(9205): 675-87.

2. Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, et al. C-reactive protein and other circulating markers of inflam-mation in the prediction of coronary heart disease. New England Journal of Medicine 2004; 350: 1387-1397.

3. Wang TJ, Larson MG, Levy D, Benjamin EJ, Leip EP, Omland T, et al. Plasma natriuretic peptide levels and the risk of cardiovascular events and death. England Journal of Medicine 2004; 350: 655-663.

4. Danesh J, Lewington S & Thompson SG. Fibrinogen Studies Collaboration. Plasma fibri-nogen level and the risk of major cardio-vascular diseases and nonvascular mortality: An indi-vidual participant meta-analysis. Journal of the American Medical Association 2005; 294: 1799-1809.

5. Cushman M, Lemaitre RN, Kuller LH, Psaty BM, Macy EM, Sharrett AR, et al. Fibrinolytic activation markers predict myocardial infarction in the elderly: The cardiovascular health study. Arteriosclerosis, Thrombosis and Vascular Biology 1999; 19(3): 493-498.

6. Mangoni AA & Jackson SH. Homocysteine and cardiovascular disease: Current evidence and future prospects. American Journal of Medicine 2002; 112(7): 556-565.

7. Badr KF & Brenner BM. Vascular injury to the kidney. In: Harrison’s Principles of Internal Medicine. 17th ed. Fauci AS, Kasper DL, Longo DL, Braunwald E, Hauser SL, Jameson JL, et al. eds. (The McGraw-Hill Companies, Inc): 2008; 1811-1812.

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8. Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, Jensen T & Kofoed-Enevoldsen A. Albumin-uria reflects widespread vascular damage. The Steno hypothesis. Diabetologia 1989; 32: 219-226.

9. Yuyun MF, Khaw KT, Luben R, Welch A, Bingham S, Day NE, et al. Microalbuminuria and stroke in a British population: The European Prospective Investigation into cancer in Norfolk (EPICNorfolk) population study. Journal of Internal Medicine 2004; 255(2): 247-256.

10. Mimran A, Ribstein J & Du Cailar G. Microalbuminuria in essential hypertension. Current Opinion in Nephrology and Hyper-tension 1999; 8(3): 359-363.

11. Lakka TA, Salonen R, Kaplan GA & Salonen JT. Blood pressure and the progression of carotid atherosclerosis in middle-aged men. Hyper-tension 1999; 34(1): 51-56.

12. Eknoyan G, Hostetter T, Bakris GL, Hebert L, Levey AS, Parving HH, et al. Proteinuria and other markers of chronic kidney disease: A position statement of the national kidney foundation (NKF) and the national institute of diabetes and digestive and kidney diseases (NIDDK). American Journal of Kidney Diseases 2003; 42(4): 617-622.

13. JNC 8. Evidence-Based Guideline for the Management of High Blood Pressure in Adults Guidelines on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risks in adults. Journal of the American Medical Association 2014; 311(5): 507-520.

14. American Diabetes Association (ADA). Standards of medical care in diabetes. Diabetes Care 2014; 38(suppl 1): S1-S93

15. Uren NG, Melin JA, De Bruyne B, Wijns W, Baudhuin T & Camici PG. Relation between myocardial blood flow and the severity of coronary-artery stenosis. New England Journal of Medicine 1994; 330: 1782-1788.

16. Sadaka M, Elhadedy A, Abdelhalim S & Elashmawy H. Albumin to creatinine ratio as a predictor to the severity of coronary artery disease. Alexandria Journal of Medicine 2013; 49: 323-328.

17. Rein P, Vonbank A, Saely CH, Beer S, Jankovic V, Boehnel C, et al. Relation of albuminuria to angiographically determined coronary arterial narrowing in patients with and without type 2 diabetes mellitus and stable or

suspected coronary artery disease. American Journal of Cardiology 2011; 107: 1144-1148.

18. Deveci OS, Kabakci G, Tulumen E, Okutucu S, Aksoy H, Kaya EB, et al. The relationship between microalbuminuria and the presence and extent of coronary atherosclerosis. Angiology 2010; 61(2):184-191.

19. Hoseini VN & Rasouli M. Microalbuminuria correlates with the prevalence and severity of coronary artery disease in non-diabetic patients. Cardiology Journal 2009; 16(2): 142-145.

20. Parsa AFZ, Ghadirian L, Kanafi SR & Iranica 2013; 51(4): 231-235.

21. Hashim R, Nisar S, Khalil ur Rehman & Naqi N. Microalbuminuria: Association with ischemic heart disease in non-diabetics. Journal of Ayub Medical College 2006; 18(1): 40-3.

22. Sukhija R, Aronow WS, Kakar P, Garza L, Sachdeva R, Sinha A, et al. Relation of micro-albuminuria and coronary artery disease in patients with or without diabetes mellitus. American Journal of Cardiology 2006; 98(3): 279-81.

23. Parvizi R, Rahbani M, Salmasi SH & Safavi M. Relationship between microalbuminuria and extent of coronary atherosclerotic lesions. Iranian Heart Journal 2005; 6(1, 2): 20-25.

24. Ei Ei Shwe. Random urinary albumin to creatinine ratio in detection of microalbuminuria in diabetes mellitus at Yangon General Hospital. [MMedSc thesis]. University of Medicine 1: Yangon; 2010.

25. Sanda Soe. Urine albumin to creatinine ratio in patients with acute myocardial infarction. [MMedSc thesis]. University of Medicine 1: Yangon; 2011.

26. Yin Mon Thant. A study of microalbuminuria in non-diabetic ischaemic stroke. [MMedSc thesis]. University of Medicine 1: Yangon; 2005

27. Hope study investigators [The Heart Outcomes Prevention Evaluation Study]. Effects of an angiotensin converting enzyme inhibitor, ramipril, on cardiovascular events in high risk patients. New England Journal of Medicine 2002; 342(3):145-153.

28. Bildirici U, Ural E, Kilic T, Aygun F, Acar E, Cekmen M, et al. Association between documented coronary artery disease and urinary albumin, albumin to creatinine ratio. Medical Science Monitor 2010; 16(11): 545-548.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Ki-67 Immunoexpression in Gestational Trophoblastic Diseases

Nyein Nyein Soe*, Saw Wut Hmone, Myint Myint Nyein & Myat Mon

Department of Pathology University of Medicine 1 (Yangon)

Gestational trophoblastic diseases comprise of hydatidiform mole, invasive mole, choriocarcinoma and placental site trophoblastic tumor having different biological behavior. Ki-67 is a proliferative marker used to evaluate the biological behavior of different tumors. The study was aimed to determine the Ki-67 immunoexpression in gestational trophoblastic diseases and its association in different types of gestational trophoblastic diseases. A cross-sectional, descriptive study was done on 50 cases of gestational trophoblastic diseases, histologically identified as 10 cases of partial hydatidiform mole, 23 cases of complete hydatidiform mole, 9 cases of invasive mole and 8 cases of choriocarcinoma. Immunoexpression of Ki-67 was determined by PAP immunohistochemistry. All cases showed Ki-67 immunopositivity. High Ki-67 labeling index (≥50% of Ki-67 positivity) was found in 10% of partial hydatidiform mole, 73.9% of complete hydatidiform mole, 33.3% of invasive mole and 100% of choriocarcinoma. Low Ki-67 labeling index (<50% of Ki-67 positivity) was found in 90% of partial hydatidiform mole, 26.1% of complete hydatidiform mole, 66.7% of invasive mole (p=0.000). These findings indicated that high Ki-67 labeling index was observed in gestational trophoblastic diseases having worse biological behavior such as complete hydatidiform mole and choriocarcinoma. So this study supports that Ki-67 immunoexpression might predict the biological behavior and prognosis of gestational trophoblastic diseases.

Key words: Gestational trophoblastic diseases, Histological types, Ki-67 immunoexpression, Ki-67 labeling index

INTRODUCTION

Gestational trophoblastic diseases consist of a group of neoplastic disorders arising from placental trophoblastic tissue after normal or abnormal fertilization.1 They include hydatidiform mole (complete and partial), invasive mole, and frankly malignant choriocarcinoma and placental site trophoblastic tumor.2 The incidence of gestational trophoblastic disease in the Asian population was 1.95 times higher than in the non-Asian population.3 The incidence of hydatidiform mole in the UK is one in 714 pregnancies.4 Recent results indicate that the previously documented higher rates in the Far East have fallen towards the stable levels found in Europe and North America, possibly because of dietary changes.4

Hydatidiform mole is associated with increased risk of persistent trophoblastic disease or choriocarcinoma.2 That is rapidly invasive and metastasizes widely, but once identified responds well to chemotherapy.2

The risk of choriocacinoma in complete hydatidiform mole is 10%-30% and in partial hydatidiform mole is 0.5%-5%.5 Early diagnosis and biological behaviours of gestational trophoblastic diseases are important for follow up and prognosis of the patients. Ki-67 is a proliferative marker and high Ki-67 immunoexpression is also a marker of poor prognosis.6 Ki-67 has been established as a valuable reflection of the ____________________________________ *To whom correspondence should be addressed. Tel: +95-95033466 E-mail: nyeinnyeinsoe81@gmail.com

41

tissue proliferative compartment and thus could be of value in studying the biologic behavior of the gestational trophoblastic diseases.7 Ki-67 immunoexpression shows variable intensities in different subgroups of hydatidiform mole by determining the labeling index (number of positive nuclei/ total number of nuclei) in villous, cytotro-phoblasts, syncytiotrophoblasts and stroma cells. Ki-67 labeling index of villous cells, especially cytotrophoblasts, is valuable in diagnosis and differentiation between different subgroups of molar pregnancy, being the highest in complete mole (more than 50%) followed by partial mole (more than 20%).8 In this study, the immunoreactivity of Ki-67 was assessed in gestational trophoblastic diseases and its association with different types of gestational trophoblastic diseases was studied. By determining the association of the Ki-67 immunoexpression and the gestational trophoblastic diseases, it was hoped that useful marker will be found for prediction of gestational trophoblastic diseases.

MATERIALS AND METHODS A cross-sectional, descriptive study was done on 50 cases of gestational trophoblastic diseases attending Central Women’s Hospital (CWH), Yangon. The biopsied samples sent to the histological section of Pathology Department, CWH were fixed with 10% buffered para-formaldehyde. After finishing the adequate fixation, tissue processing and proper paraffin wax embedding, all the tissue sections were stained with haematoxylin and eosin. Histological grading and types of gestational trophoblastic diseases were described by using WHO classification.9 Immunohistochemical staining method The paraffin blocks were further processed for IHC staining with Ki-67 monoclonal antibody (Mouse Anti-human Ki-67 antigen, Clone: Ki88 Code: MU370-UC BioGenex, Emergo Europe) by using the Peroxidase-

antiperoxidase method. Sections 4 µm thick-ness were placed on silanised slides and then dried at 37oC overnight and incubated at 60oC for half an hour. These slides were deparaffinized through xylene-ethanol series and washed with PBS (phosphate buffered saline) for 5 minutes, 3 times, followed by washing with distilled water for 5 minutes. Antigen retrieval with 10 mM citrate buffer (pH 6.0) was done by using heat method in microwave oven. Slides were immersed with 0.3% hydrogen peroxide in absolute methanol for 15 minutes to block endogenous peroxidase activity. Then, the slides were washed with PBS for 5 minutes, 3 times and incubated in a moist chamber overnight at 4°C with the primary antibodies: Ki-67 (dilution 1:100).

After incubation is completed, these slides were washed individually with PBS (pH 7.6) for 3 times (each time for 5 minutes and incubated with secondary antibody (Horse Radish peroxidase labeled Goat Anti-mouse IgG) for an hour at room temperature. Then these slides were washed with PBS for 5 minutes, three times. The diaminoben-zidine substrate solution was freshly prepared and covered on the sections and was left at room temperature for 15-20 minutes. Then, the slides were washed with distilled water for 5 minutes, twice. Slides were counterstained with haematoxylin for 5-10 seconds and rinsed in water and proceeded in ethanol 70%, 80%, 90%, 100%, xylene III, II, I, 5 minutes each for dehydration and mounted with distene plasticizer xylene (DPX). Ki-67 immuno-positivity was observed as brown nuclear staining. In this study, Ki-67 immuno-expression was determined by semi-quantitative scoring method and Ki-67 labeling index.8

Semi-quantitative scoring method was determined by the proportion of positive nuclear staining cells over total numbers of cytotrophoblasts and syncytiotrophoblasts. The distribution of Ki-67 immunopositivity in trophoblastic cells were assessed as negative (equal or less than 10% are

42

positive cells) and positive (more than 10% are positive cells). Positive immuno-reactivity was graded as; weakly positive (1+) - (10-20%), moderately positive (2+) - (21-50%) and strongly positive (3+) - (more than 50%).8 Two senior pathologists performed to count the immunopositivity of trophoblastic cells and immunohisto-chemical staining reaction of these cells by using specific grading.

Ki-67 labeling index was determined by counting positive brown nuclear staining cells among 1000 cells of cytotrophoblasts and syncytiotrophoblasts. The labeling index was expressed as percent positive.

Ki-67 labeling index and the immuno-reactivity results were recorded and interpreted. Data analysis

Statistical analysis of data was done by using the Statistical Package for Social Science Study (SPSS), version 20. The association between Ki-67 immuno-expression and different types of gestational trophoblastic diseases was tested using Chi-square test.

RESULTS

Age distribution of gestational trophoblastic diseases

Among 50 cases of gestational trophoblastic diseases, the youngest age of patient was 16 years and the oldest age was 52 years. The highest percentage belongs to the age group between 20-29 years, i.e. 19 cases (38%) of study population followed by over 40 years, 16 cases (32%).

Among 10 cases of partial mole, the youngest age of patient was 17 years and the oldest age was 44 years. The mean age of partial mole was 26 years. In complete hydatidiform mole, the youngest age was 16 years and the oldest was 52 years. The mean age was 34.08 years. Among

9 cases of invasive mole, the youngest age was 20 years and the oldest age was 47 years. The mean age was 35 years. In choriocarcinoma, the youngest age was 29 years and the oldest age was 50 years. The mean age was 38.5 years.

Distribution of gestational trophoblastic diseases by histological types

Among 50 cases of gestational trophoblastic diseases, 23 cases (46%) were complete hydatidiform mole, 10 cases (20%) were partial hydatidiform mole, 9 cases (18%) were invasive mole and 8 cases (16%) were choriocarcinoma.

Ki-67 immunoreactivity in gestational tro-phoblastic diseases Ki-67 immunoreactivity was found in all 50 cases of gestational trophoblastic samples. The highest Ki-67 immunoreac-tivity was found in choriocarcinoma cases (77.50%) and the lowest Ki-67 immuno-reactivity was seen in partial mole (31.25%) in this study (Table 1). Table 1. Ki-67 Immunoreactivity in gestational

trophoblastic diseases

Histological types

No. of cases

Mean (%)

Standard deviation Min Max

Partial mole 10 31.25 10.09 20 50 Complete mole 23 50.78 12.01 25 80 Invasive mole 9 48.33 16.91 30 70 Choriocarcinoma 8 77.50 10.00 55 90 Total 50 50.71 18.44 20 90

Max=Maximum, Min=Minimum

The mean immunoreactivity of 50 cases of gestational trophoblastic diseases was 50.710±18.438 (%). The median value was 50.00. The minimum expression of Ki-67 immunoreactivity was 20 (%). The maxi-mum immunoreactivity was 90 (%) (Table1).

According to the mean and median value of Ki-67 immunoexpression in gestational trophoblastic diseases in this study, the best cut-off point for gestational trophoblastic diseases in labeling index score was 50 (%). The score less than 50 (%) was considered as low and more than or equal to 50 (%) as high labeling index score.

Ki-67 labeling Index= Total no. of positive

Cells x100 =LI% 1000 cells

43

Distribution of Ki-67 labeling index in gestational trophoblastic diseases

Out of 50 cases of gestational trophoblastic diseases, 29 cases (58%) showed presence of high Ki-67 index scores and 21 cases (42%) showed low index scores. Among complete hydatidiform mole, 17 cases (73.9%) showed high Ki-67 index scores and 6 cases (26.1%) showed low index scores. Out of 10 cases of partial hydatidi-form mole, only 1 case (10%) showed high Ki-67 index scores and 9 cases (90%) showed low index scores. Among invasive mole, 3 cases (33.3%) showed high Ki-67 index scores and 6 cases (66.7%) showed low index scores. All 8 cases (100%) of choriocarcinoma showed high Ki-67 index scores (Fig. 1).

Fig. 1. Distribution of Ki-67 labeling index in different types of gestational trophoblastic diseases

A=Partial H mole (H&E x 400) B=Complete H mole (H&E x 400) C=Invasive mole (H&E x 100) D=Choriocarcinoma (H&E x 100)

Fig. 2. H and E staining of different types of GTD

A=Partial H mole (Ki-67 LI=37.5%) B=Complete H mole (Ki-67 LI=80%) C=Invasive mole (Ki-67 LI=70%) D=Choriocarcinoma (Ki-67 LI=90%)

Fig. 3. IHC staining of Ki-67 (Ki-67 labeling index) in different histological types of GTD

Association of Ki-67 immunoexpression with different histological types of gesta-tional trophoblastic diseases

Among 10 case of partial hydatidiform mole, 2 cases (20%) showed the intensity of Ki-67 immunoexpression 1+ and 8 cases (80%) showed 2+ intensity. Among complete hydatidiform mole, the 2+ intensity of Ki-67 immunoexpression was found to be (52.2%) of cases and the remaining (47.8%) showed 3+ intensity. Among 9 cases of invasive mole, the intensity 2+ was seen in (66.7%) of cases and 3+ in (33.3%). All 8 cases of choriocarcinoma (100%) showed 3+ intensity of Ki-67 immunoexpression (Fig. 2 & 3).

DISCUSSION

Maternal age has an influence on the incidence of gestational trophoblastic disease. There was an excess of molar pregnancies in the extremes of reproductive age.3 In this study, the age distribution of H mole was 16-52 years. Most of the patients were found to be 20-29 years age group (38%) and the second commonest age group was 40 years and above (32%). Lurain found that advanced or very young maternal age had consistently correlation with higher rates of complete hydatidiform mole.9 There was a small difference of age incidence in this study compared with the previous studies.

A=Complete H mole B=Partial H mole

C=Invasive H mole D=Chorio CA

A B

C D

A B

C D

Types of GTD

Dis

tribu

tion

of K

i-67

(%) 20

15

10

5

0A B C D

17(73.9)

6(26.1)

1(10)

9(90)

3(33.3)

6(66.7)8(100)

>=50<50

0

44

In comparison with Ki-67 immuno-expression of partial hydatidiform mole, all cases of complete hydatidiform mole showed a higher Ki-67 intensity with mean Ki-67 immunoreactivity 50.783±12.014 (%). The study done by Ali, et al.8 showed Ki-67 immunoexpression of all villous components especially of cytotrophoblasts; being the highest in complete hydatidiform mole (68.542±11.275) followed by partial hydatidiform mole (22.5±16.611).

In this study, all 9 cases of invasive mole showed positive Ki-67 immunoexpression with the mean immunoreactivity 48.33 ±16.91 (%). In the study carried out by Makovitzky, et al.10 the invasive mole labeled strongly positive for Ki-67 antigen. All cases of chorioarcinoma showed strongly positive Ki-67 immunoexpression with the mean Ki-67 immunoreactivity 77.5±10.0 (%). The study done by Erfanian, et al.7 showed strongly positive Ki-67 immunoexpression in cytotrophoblasts 80.41±9.40 (%) and in syncytiotrophoblasts 58.75± 18.10 (%). In the current study, highest proliferative activity of Ki-67 (≥50% of Ki-67 labeling index) was found in partial mole (10%), complete hydatidiform mole (73.9%), invasive mole (33.3%) and all cases of choriocarcinoma. Therefore, it might be evident that complete hydatidiform mole was more increasing risk of chorio-carcinoma than partial hydatidiform mole.

On the other hand, among the histological subtypes of trophoblastic diseases, partial hydatidiform mole showed the lowest Ki-67 labeling index and choriocarcinoma showed the highest. In comparison with choriocarcinoma, invasive mole showed low Ki-67 labeling index score. There was significantly associated between Ki-67 labeling index and histological subtypes of gestational trophoblastic diseases (p=0.000). In the present study, Ki-67 immunoexpression was detected in all cases of partial hydatidiform mole (10 cases), complete hydatidiform mole (23 cases),

invasive mole (9 cases) and chorio-carcinoma (8 cases). According to the findings, it showed that the higher grade of histological subtypes of gestational trophoblastic diseases, the stronger the intensity of Ki-67 immunoexpression. In complete hydatidiform mole and invasive mole, there were no cases of weakly positive (1+) Ki-67 immunoexpression.

In comparison of partial hydatidiform mole with complete hydatidiform mole, there were no cases of partial hydatidiform mole showing strongly positive (3+) intensity of Ki-67 immunoexpression, whereas 47.8% of complete hydatidiform moles showed strongly positive (3+) intensity. All cases of choriocarcinoma showed strongly positive (3+) Ki-67 immunoexpression. There was a significantly association between Ki-67 immunoexpression and different histological types of gestational trophoblastic diseases (p=0.000).

Conclusion

This study was determined Ki-67 immuno-expression and Ki-67 labeling index in different types of gestational trophoblastic cases from Central Women’s Hospital, Yangon. Ki-67 is a proliferative marker and high Ki-67 immunoexpression showed a marker of bad biological behavior and poor prognosis. Early diagnosis and accurate biological behavior is important for gestational trophoblastic diseases because early effective treatment can prevent the progression of the diseases. This is the preliminary study done on association of Ki-67 immunoexpression in different histological types of gestational tropho-blastic diseases in Myanmar.

The findings of this study showed that the higher grade of histological types of the gestational trophoblastic diseases, the stronger the intensity of Ki-67 immuno-expression. The findings support that Ki-67 proliferative marker might be useful in determining the prognosis of gestational trophoblastic diseases and early treatment.

45

ACKNOWLEDGEMENT

We would like to express our sincere gratitude to Professor Dr. Zaw Wai Soe, Rector, University of Medicine 1(Yangon) for allowing us to perform this research. We want to express our thanks to Professor Dr. San San Myint, Professor and Head, Obstetrics and Gynaecology Department, University of Medicine 1 (Yangon) for allowing us to collect clinical data of the cases in Central Women’s Hospital, Yangon. We would like to thank Associate Professor Dr. Khin Shwe Mar, Department of Pathology, Central Women’s Hospital, Yangon for kind permission for specimen collection and suggestions. We would also like to thank laboratory staff of Department of Pathology, Central Women’s Hospital and Yangon General Hospital and laboratory staff of University of Medicine 1 (Yangon) for their support.

REFERENCES

1. Altieri A, Franceschi S, Ferlay J, Smith J & Vecchia CL. Epidemiology and aetiology of gestational trophoblastic diseases. The Lancet Oncology 2003; 4: 670-678.

2. Ellenson LH & Pirog EC. The Female Genital Tract. In: Robbin and Cotran Pathologic Basis of Disease, 8th ed. Elsevier Saunders, Philadelphia, 2010; 1057-1061.

3. Tham BWL, Everard JE, Tidy JA, Drew D & Hancock BW. Gestational trophoblastic disease in the Asian population of Northern England and North Wales. International Journal of Obstetrics and Gynaecology 2003; 110: 555-559.

4. Newsom-Davis T & Seckl MJ. Gestational trophoblastic tumours. In: Shaw Gynaecology, 4th ed. Elsevier, Edinburgh, 2016; 650-664.

5. Merchant SH, Amin MB, Viswanatha DS, Malhotra RK, Moehlenkamp C & Joste NE. P57kip2 immunohistochemistry in early molar pregnancies: Emphasis on its complementary role in the differential diagnosis of hydropic abortuses. Human Pathology 2005; 36: 180-186.

6. Urruticoechea A, Smith IE & Dowsett M. Proliferation marker Ki-67 in early breast cancer. Journal of Clinical Oncology 2005; 23(28): 7212-7220.

7. Erfanian M, Sharifi N & Omidi AA. P63 and Ki-67 expression in trophoblastic disease and spontaneous abortion. Journal of Research in Medical Sciences 2009; 14(6): 375-384.

8. Ali SM, Ahmed NY, Abdul Al-Hameed TT & Saeed NF. The role of Ki-67 immunoexpression in diagnosis of molar pregnancy and differen-tiating its subtypes. American Journal of Research Communication 2014; 2(4): 64-73.

9. Lurain JR. In vitro gestational trophoblastic disease 1: Epidemiology, pathology, clinical presentation and diagnosis of gestational trophoblastic disease and management of hydatidiform mole. American Journal of Obstetric and Gynaecology 2010; 531-539.

10. Makovitzky J, Radtke A, Shabani N, Friese K, Gerber B & Mylonas L. Invasive hydatidiform mole: Immunohistochemical labeling of inhibin/ activin subunits, Ki-67, p53 and glycodelin A in a rare case. Acta Histochemica 2009; 111: 360-365.

46

Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Molecular Detection of Human Rhinoviruses in Children with Influenza-like Illness Attending Yangon Children’s Hospital, 2016

Htin Lin1*, Hlaing Myat Thu1, Theingi Win Myat1, Win Mar1,

Khaing Moe Aung1, Khin Khin Oo1, Khin Sandar Aye1, Thida Kyaw1 & Ye Myint Kyaw2

1Department of Medical Research 2Yangon Children’s Hospital

Influenza-like illness (ILI) is caused not only by influenza virus but other viruses including human rhinovirus (HRV). HRV usually causes common cold and exaggerates asthmatic attack and otitis media in children. The aim of this study was to determine the prevalence and clinical severity of HRV among children with influenza-like illness. It was a cross-sectional study conducted at Out-Patient and Emergency Department of Yangon Children’s Hospital (YCH). Nasopharyngeal swab samples were obtained from a total of 153 children with ILI from January to December, 2016. Viral RNA was extracted by QIAamp® RNA Mini kits. Non-coding region of HRV gene was detected by conventional RT-PCR using Qiagen One Step RT-PCR kit. Of 153 cases, HRV was detected in 42 cases (27.5%). Males were slightly more affected than females with the ratio of male to female, 1.2:1. The maximum number of HRV cases was found in the children aged less than 5 years that accounted for 71.4%. During the study period, HRV positive cases were detected in rainy season and winter season peaking in June, November and December. Fever, cough and rhinorrhoea were observed as the main symptoms of HRV infections that were responsible for 100%, 100% and 81% of HRV cases, respectively. Gastrointestinal symptoms such as diarrhoea and vomiting were observed in 7.1% and 2.4%, respectively, of HRV-positive cases and fast breathing was observed in 2 HRV cases (4.8%). There was no HRV-positive case that presented with tightness of chest. Most of the HRV-affected children presented with low grade fever (mean=100.7ºC, SD±0.85). Clinical diagnosis of HRV cases included acute viral infection (AVI), acute respiratory infection (ARI), pneumonia and dengue haemorrhagic fever grade 1 (DHF I) accounting for 83.3%, 7.1%, 4.8% and 4.8%, respectively. This study provided baseline information about ILI cases due to human rhinovirus that would be useful for the assessment of HRV outbreak and management of children with influenza-like illness.

Key words: Human rhinovirus, Influenza-like illness, Children, Outbreak

INTRODUCTION

Influenza-like illness (ILI) is the definition used for the surveillance of influenza worldwide.1 The viral aetiology of ILI includes not only the influenza viruses but other respiratory viruses such as human rhinoviruses (HRV), adenoviruses, res- piratory syncytial virus (RSV), human enterovirus, human metapneumovirus, para-influenza viruses and human coronavirus

229E or OC43.2, 3 The frequency distri-bution of such causal viruses differs geographically. In Brazil, influenza and HRV were found to be most prevalent among ILI 39 cases. A study in Gabon stated that adenoviruses were found to be predominant among children with ILI.4 During 2010-2013, influenza virus and ___________________________________ *To whom correspondence should be addressed. Tel: +95-95004520 E-mail: drhtinlin@gmail.com

47

RSV Leyte Island of the Philippines.5 In Myanmar, 22% of ILI cases were known be caused by influenza viruses.6 According to the studies conducted at Yangon Children’s Hospital from 2013 to 2015, 6-9% of children attending YCH were due to influenza viruses. However, the aetiology of the remaining large proportion of ILI cases has not been studied.7-9 Human rhinoviruses (HRVs) are the common viruses causing ILI and respon-sible for 25% ILI cases.10 They usually cause common cold in children and adults and trigger the asthma attacks.11

Since common cold and influenza have some similar clinical features, there may be many cases of HRV in ILI cases with no laboratory-confirmed influenza virus.12

Besides, HRVs can also be regarded as the predominant viruses among children with otitis media (OM). Among the children with asymptomatic OM with effusion, HRVs were found up to 40% of the cases.13

Besides, children with HRV infection cause more severe clinical course than other respiratory viruses and cause significant co-morbidities.14 These findings highlight the important role of HRVs in respiratory diseases. In Myanmar, there were a few studies on HRVs among children with acute respiratory infection (ARI). A study conducted at Yangon Children’s Hospital in 2014-2015 found HRVs as the predominant virus in ARI cases. They accounted for 18% of all ARI cases and 71% of the ARI cases with multiple viral infections.15 However, there are very limited local data of ILI cases due to HRV in Myanmar. Therefore, the HRV should be studied among ILI cases to generate the baseline data for the assessment of the disease outbreak and management of ILI cases in clinical settings.

MATERIALS AND METHODS

Study design

It was a cross-sectional study conducted at Out-Patient and Emergency Department of Yangon Children’s Hospital (YCH).

Nasopharyngeal swab samples were obtained from a total of 153 children who attended YCH with ILI from January to December, 2016.

Inclusion criteria Children of any age, of any sex, attending Yangon Children’s Hospital with influenza like-illness were included in the study.

Working definition of influenza-like illness Influenza-like illness was defined as fever more than or equal to 38ºC with cough or sore throat or both.16

Exclusion criteria Children with cleft palate or oral thrush were excluded from this study. Specimen collection and transport

After getting the informed consent, the medical history of child was taken from the parent or guardian and medical record. Nasopharyngeal swab specimen was taken from child according to the guidelines laid down by CDC.17 The specimen was transported with Viral Transport Media (VTM) to the laboratory of Virology Research Division in cold condition. The VTM was prepared according to the guidelines laid down by WHO.18 The VTM fluid was stored at -70ºC before sample processing was done. Detection of human rhinovirus

Viral RNA was extracted from nasopharyngeal swab specimens by using RNA extraction kit (QIAmp® Viral RNA Mini Assay) according to the manu-facturer’s instructions. Non-coding region of human rhinovirus RNA was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using specific primers. The reaction mixture contained 7.5 µl of distill H2O, 5 µl of 5X buffer, 5 µl of Q solution, 1 µl of dNTP, 0.25 µl of sense primer (CCA ACA GTA GAC CTG GCA GATG), 0.25 µl of anti-sense primer (ACG GAC ACC CAA AGT AGT TGGT), 1 µl of enzyme mixture and 5 µl of RNA template. HRV positive and negative RNA templates

48

were used for quality control.19 Reverse transcription temperature was 50ºC for 45 minutes and initial denaturing temper-ature was 95ºC for 10 minutes. Cycling temperature was 94ºC for 30 seconds, 58ºC for 30 seconds and 72ºC for 1 minute for a total of 40 cycles. Amplicons were mixed with loading dye and were subjected to 2% agarose gel electrophoresis at 100 volts for 45 minutes. Reaction bands were visualized by molecular imager (Gel DocTM XR+, Bio-Rad).

Data entry and analysis

Data entry and analysis was done by using Statistical Package for Social Science (SPSS) version 15.0. Fisher’s Exact Test was applied to determine the significance of association of data. Ethical consideration

Ethical approval to conduct the study was obtained from Ethics Review Committee of Department of Medical Research.

RESULTS

When 153 ILI cases were tested with RT-PCR, 42 cases (27.5%) revealed non-coding region of HRV gene. Therefore, HRV was found to be responsible for 27.5% of children with ILI.

Among 42 HRV-positive cases, 23 cases (54.8%) were males and 19 cases (45.2%) were females. Ratio of male to female was 1.2:1. However, sex preponderance was not observed among them because males were also more affected in HRV negative cases. Regarding age group, majority of HRV-positive cases were under 5 years of age that accounted for 71.4%. There were 11 HRV-positive cases (26.2%) in the age group 5 to 9 years and one HRV-positive case in age group 10 to 12 years that included only a few ILI cases (Table 1). During the study period from January to December 2016, HRV positive cases were mainly detected in the rainy season from June to September. The maximum number

of positive cases was found in June that accounted for 46.1% (Fig. 1).

Table1. Demographic characteristics of influenza-like illness (ILI) cases

HRV cases (%) Positive Negative

Sex Male Female

23(54.8) 19(45.2)

63(56.8) 48(43.2)

Age (years) 0-4 5-9 10-12

30(71.4) 11(26.2) 1(2.4)

66(59.5) 39(35.1)

6(5.4)

Fig. 1. Monthly distribution of influenza-like

illness (ILI ) cases

Fig. 2. Gel image showing PCR bands of HRV gene

HRV-positive cases mainly presented with fever, cough and rhinorrhoea that accounted for 100%, 100% and 81%, respectively. Gastrointestinal symptoms such as diarrhoea and vomiting were observed in 7.1% and 2.4% of HRV-positive cases and fast

A=January E=May I=September B=February F=June J=October C=March G=July K=November D=April H=August L=December

6 3 0

HRV-positive case (n=42)

HRV-negative case (n=111)

10 8

12

38

9

0 0 01 0 1

18

4 3

9 7 5

A B C D E F G H I J K L Months (2016)

40

35

30

25

20

15

10

5

0

Num

ber o

f cas

es

3

5

6 4

49

breathing was observed in 2 HRV cases (4.8%). There was no HRV-positive case that presented with tightness of chest (Table 2). Table 2. Clinical presentations of influenza-

like illness (ILI) cases HRV cases (%)

Positive Negative Fever 42(100) 111(100) Cough 42(100) 111(100) Rhinorrhoea 34( 81) 101(91) Diarrhoea 3(7.1) 5(4.5) Vomiting 1(2.4) 16(14.4) Tightness of chest 0(0) 0(0) Fast breathing 2(4.8) 0(0)

Fig. 3. Clinical diagnosis of influenza-like illness (ILI) cases

Most of the HRV-positive cases had clinical diagnosis of acute viral infection (AVI) accounting for 83.3%. A few cases had been diagnosed as acute respiratory infection (ARI), pneumonia and dengue haemorrhagic fever grade 1 (DHF I) accounting for 83.3%, 7.1%, 4.8% and 4.8%, respectively (Fig. 3).

DISCUSSION

Acute respiratory infection (ARI) including ILI is a common cause of frequent visit to Out-Patient Setting of Yangon Children’s Hospital.20A total of 153 children who attended the hospital due to ILI were recruited in the study. When their NP swab

specimens were tested for HRV gene, 26 cases (27.5%) showed HRV positive. In other studies, HRV was found to be responsible for 16% of ILI cases aged up to 25 years and 26% of ILI cases aged less than 5 years.20, 21 This study included the children population aged less than 13 years and prevalence of HRV was found to be 27.5%. It was observed that the prevalence of HRV depends on the age range of ILI cases. According to the previous studies conducted at YCH, influenza virus was detected in 6-9% of children with ILI attending the hospital.7-9

Therefore, the proportion of HRV cases was found to be larger than that of influenza cases among ILI cases attending YCH. The remaining 72.5% ILI cases may be due to other respiratory viruses such as influenza virus, adenoviruses, respiratory syncytial virus, human enterovirus, human meta-pneumo virus, parainfluenza viruses and human corona virus 229E or OC43 that were not tested in this study.2, 3

Among HRV-positive children, 23 children (54.8%) were males and 19 children (45.2%) were females. The ratio of male to female was 1.2:1. In agreement with this study, Miller and scientists have also found that males were slightly more affected by HRV than the females.21 In this study, a large number of HRV cases were detected in children under 5 years of age and the detection rate was found to decrease in the older age groups. A study in New York also stated that HRV detection rate increased with the decreasing age of children.22

In a study in Latin America, overall HRV positivity rate was greater in the children under 5 years of age than children above 5 years which is in agreement with this study.21

Seasonal pattern of HRV varies geo-graphically. Peak of HRV infection is seen in early fall and spring in template regions. In tropical regions, HRV showed distinct seasonality in rainy season.23 However, HRV infection cases were detected through-out the year in Trinidad which is a country

AVI=Acute viral infection ARI=Acute respiratory infection Pneu=Pneumonia DHFI=Dengue haemorrhagic fever infection

Num

ber o

f cas

es

7.1

20.4

4.8

4.8

100 90

80

70

60

50

40

30

20

10

0 AVI ARI Pneu DHFI Clinical diagnosis

83.3

98.9

HRV-negative case (n=111)

HRV-positive case (n=42)

50

with tropical weather.24 This study in Myanmar demonstrated that HRV cases were detected in both rainy season and winter season especially in June, November and December highlighting the timing for preventive measures of ILI cases due to HRV infection. Regarding the clinical presentations of HRV, fever, cough and rhinorrhoea were found as the main presenting features of children with HRV. In previous studies conducted in YCH, laboratory-confirmed influenza cases were also found to present with these symptoms as their main symptoms.7-9

Therefore, these findings highlight the importance of laboratory confirmation for definite diagnosis finding of viral respiratory infection. However, fever presented by HRV cases is usually lower than influenza cases.25 Similar finding was also observed by this study in which most HRV-positive children (172) presented with low grade fever (mean=100.7°C, SD ±0.85). A few HRV cases (4.8%) in this study were clinically diagnosed as dengue infection. This may be due to the constitutional symptoms such as headache, myalgia and vomiting that can be found in both HRV infection and dengue infection.26, 27 Apart from such cases, HRV cases were mostly diagnosed in the hospital setting as acute viral infection. So, HRV should also be considered in the management of AVI cases especially presenting with ILI. Conclusion This study provided baseline information about ILI cases due to human rhinovirus that would be useful for the assessment of HRV outbreak and management of children with influenza-like illness.

REFERENCES

1. WHO. WHO surveillance case definitions for

ILI and SARI [Internet]. 2016 [updated 2016 Jan; cited 2016 Jan] Available from: http://www. who.int/ influenza surveillance-case-definition/en

2. Kelly H & Birch C. The causes and diagnosis of influenza-like illness. Australian Family Physician 2004; 33(5): 305-309.

3. Lekana Douki SE, Nkoghe D, Drosten C, Ngoungou EB, Drexler JF & Leroy EM. Viral etiology and seasonality of influenza-like illness in Gabon, March 2010 to June 2011. BMC Infectious Diseases 2014; 14: 373. doi:10.1186/1471-2334-14-373.

4. Bellei N, Carraro E, Perosa A, Watanabe A, Arruda E & Granato C. Acute respiratory infection and influenza-like illness viral etiologies in Brazilian adults. Journal of Medical Virology 2008; 80(10): 1824-1827. doi: 10.1002/jmv.21295.

5. Otomaru H, Kamigaki T, Tamaki R, Opinion J, Santo A, Daya E, et al. Influenza and other respiratory viruses detected by influenza-like illness surveillance in Leyte Island, the Philippine, 2010-2013. PLoS ONE 10(4): e0123755. doi: 10.1371/ journal. pone.0123755.

6. Hasegawa G, Yadana Kyaw, Hla Myat Nwe, Danjuan L, Saito R, Suzuki H, et al. Epidemiological study on influenza virus infections in Yangon, Myanmar. Tropical Medicine and Health 2006; 34(1): 3-6.

7. Htin Lin, Hlaing Myat Thu, Khin Thet Wai, Khin Mar Aye, Mo Mo Win, Kay Thi Aye, et al. Influenza viruses in children attending Yangon Children’s Hospital, Myanmar during influenza season in 2013. Outbreak, Surveillance and Investigation Reports 2014; 7(2): 6-10.

8. Htin Lin, Hlaing Myat Thu, Mo Mo Win, Khin Mar Aye, Khin Thet Wai, Win Mar, et al. Determination of predominant subtype of influenza virus among children attending Yangon Children’s Hospital, 2014. Myanmar Health Sciences Research Journal 2015; 27(2): 111-117.

9. Htin Lin, Hlaing Myat Thu, Mo Mo Win, Khin Mar Aye, Win Mar, Hla Myo Thu, et al. Molecular detection of influenza viruses circulating among children attending Yangon Children’s Hospital, 2015. Myanmar Health Sciences Research Journal December 2016; 28(3): 109-114.

10. Department of Medical Research. Annual Report 2015; 146-147.

11. Galindo-Fraga A, Ortiz-Hernandez AA, Ramírez-Venegas A, Vázquez RV, Moreno-Espinosa S & Llamosas-Gallardo B. Clinical characteristics and outcomes of influenza and other influenza-like illnesses in Mexico City. International Journal of Infectious Diseases 2013; 17(7): e510-e517.

12. CDC. Common Colds: Protect yourself and others [Internet]. 2016 [updated 2016 Feb 8; cited 2016 Feb 23]. Available from: http://www. cdc.gov/ features/ rhinoviruses

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13. CDC. Cold Versus Flu [Internet]. 2016 [cited 2016 Feb 12] Available from: http://<www. cdc.gov/ flu/about/coldflu

14. Asner SA, Petrich A & Smieja M. Clinical severity of rhinovirus/ enterovirus compared to other respiratory viruses in children. Influenza and other Respiratory Viruses 2014; 8(4): 436-442.

15. Chantzi FM, Papadopoulos NG, Bairamis T, Tsiakou M, Bournousouzis N, Constantopoulos AG, et al. Human rhinoviruses in otitis media with effusion. Paediatric Allergy Immunology 2006; 17(7): 514-518.

16. CDC. Influenza-like illness case definition [Internet]. 2016 [updated 2016 Feb 8] Available from: http://www.acha.org/ ILI_ Project/ ILI_ case_definition _CDC.pdf

17. CDC. Interim guidelines for specimen collection, processing and transfer for patients with suspected infection of novel influenza A virus associated severe diseases in human [Internet]. 2016 [cited 2016 Jan 15] Available from: http://www.cdc.gov/ flu/ a vianflu/ h7n9/specimen_collection.html

18. WHO.Viral Transport Medium (VTM) [Internet]. 2015 [cited 2015 April 9] Available from: http://www.who.int/influenza/human_avian_interface/virology_laboratories_and_vaccine/guidelines_collection_humans/en

19. Yoshida LM. Personal communication. November 5, 2015.

20. Yangon Children’s Hospital. Basic Fact, 2015.

21. Garcia J, Espejo V, Nelson M, Sovero M, Villaran MV, Gomez J, et al. Human rhino-viruses and enteroviruses in influenza-like illness in Latin America. Virology Journal 2013; 10: 305. doi: 10.1186/1743-422X-10-305.

22. Miller EK, Lu X, Erdman DD, Poehling KA, Zhu Y, Griffin MR, et al. Rhinovirus-associated hospitalizations in young children. Journal of Infectious Diseases 2007; 195: 773-81.

23. Hay CM. The Respiratory Viruses: Influenza, RSV, and Rhinoviruses [Internet]. 2016 [cited 2016 Nov] Available from: http://www. columbia.edu/itc/hs/medical/pathophys/id/2008/respiratoryvirusesNotes.pdf

24. Matthew J, Pereira LMP, Pappas TE, Swenson CA, Grindle KA, Roberg KA, et al. Distri-bution and seasonality of rhinovirus and other respiratory viruses in a cross section of asthmatic children in Trinidad, West Indies. Italian Journal Pediatrics 2009; 35: 16.

25. Allen PJ. Home care Fact Sheet: Influenza [Internet]. 2016 [cited 2016 Nov] Available from: http://www.emedicine. medscape. com

26. Camargo CN, Carraro E, Granato CF & Bellei N. Human rhinovirus infections in sym-ptommatic and asymptomatic subjects. Brazilian Journal of Microbiology 2012; 43(4). [Internet]. 2016 [cited 2016 Nov] Available from: http:// dx. doi.org/ 10.1590/ S1517-83822 0120 004 000049)

27. WHO. Clinical Course of Dengue [Internet]. 2016 [cited 2016 Jan] Available from: http://www.wpro.who.int>module4-ro

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Role of Flow Cytometric Immunophenotyping in Diagnosis of Multiple Myeloma

San San Htwe1*, Htun Lwin Nyein2, Aye Mya Khine1, Khin La Pyae Tun1, Win Pa Pa Naing1, Ni Ni Win1, Moe Thuzar Min1, Sein Win2 & Khin Saw Aye1

1Department of Medical Research

2Department of Clinical Hematology, Yangon General Hospital

Immunophenotyping has become an invaluable tool in the management of hematological malignancies and is increasingly finding a role in the diagnosis and monitoring of plasma cell disorders. The aim of the study was to provide an accurate diagnosis of multiple myeloma by application of flow cytometry immunophenotyping. A cross-sectional descriptive study was conducted at Department of Clinical Hematology, Yangon General Hospital from September 2015 to August 2016. Multiparametric flow cytometry immunophenotyping was performed using monoclonal antibodies against CD56, CD19, CD138, CD38 and CD45. A total of 40 clinically suspected cases of multiple myeloma were included for the study. Among the 40 cases, 25 cases (62.5%) were diagnosed as multiple myeloma by the positive expression of CD138 or CD38, negative CD19 expression, weak or negative CD45 expression and positive or negative CD56 expression. Age of the patients ranged from 49 years to 89 years. The male to female ratio was 1.1:1. Serum protein electrophoresis and densitometer reading were performed in all cases for detection of monoclonal band and monoclonal protein concentration. Monoclonal band was detected visually and estimation of monoclonal protein was done by densitometer. Among 40 cases, 25 (62.5%) had monoclonal gammopathy. Among these cases, 20 (80%) had monoclonal band in the gamma () region and 5 (20%) had in the beta () globulin region. The mean concentration of monoclonal protein was 4.50 g/dl, with a range of 2.28 to 7.95 g/dl. The remaining 15 (37.5%) were not detected monoclonal band. In conclusion, flow cytometry immuno-phenotyping is useful tool for the diagnosis of multiple myeloma and it should be included as a routine assay in monoclonal gammopathy patients.

Key words: Multiple myeloma, Flow cytometry, Immunophenotyping, Diagnosis

INTRODUCTION

Multiple myeloma is a clonal B-cell disorder in which malignant plasma cells accumulate in the bone marrow, producing lytic lesions, excessive amounts of monoclonal protein in the serum or urine, and evidence of end-organ damage (hyper-calcemia, renal insufficiency, anemia, or bone lesions).1 It accounts for 1% of all malignancies and 10% of hematological malignancies.2, 3 Evaluation of multiple myeloma disease is based on a variety of laboratory techniques, including bone marrow morphology and immunophenotyping, analysis of serum and

urine M- component and free light chains, hematological and biochemical parameters, cytogenetics, DNA ploidy, and measure-ment of plasma cell proliferative activity. These investigations are important to support the diagnosis of multiple myeloma, to guide the therapy, to provide prognostic information, and to monitor treatment efficacy.4 Immunophenotyping has become an invaluable tool in the management of hematological malignancies and is increasingly finding a role in the diagnosis and monitoring of plasma cell disorders.5 _______________________________________________________________________

*To whom correspondence should be addressed. Tel: +95-95506954 E-mail: sshtwe01@gmail.com

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Immunophenotyping by flow cytometry is a sensitive method that is used for the diagnosis and clinical monitoring of the disease. Flow cytometry in multiple myeloma is beneficial in detecting malignant plasma cells and prognostic markers and monitoring the development and differentiation of myeloma cells.6 Flow cytometry has many advantages: distinguishing among normal, reactive,

and malignant plasma cells;7-11 evaluating the risk of progression from

monoclonal gammopathy of unknown significance (MGUS) to multiple myeloma;12-14

detecting prognostic markers;10, 15-17 evaluating minimal residual disease

(MRD);7, 18, 19 identifying new targets for myeloma

therapy.10, 17, 20

Multiple myeloma is not uncommon globally as well as in Myanmar. Being limited availability of laboratory diagnostic facility, there had been no studies using flow cytometry immunophenotyping for diagnosis of multiple myeloma in Myanmar. The study was aimed to provide an accurate diagnosis of multiple myeloma by application of flow cytometry immuno-phenotyping.

MATERIALS AND METHODS

Study population A cross-sectional, descriptive study was conducted at Department of Clinical Haematology, Yangon General Hospital from September 2015 to August 2016. A total of 40 clinically suspected cases of multiple myeloma were included in the study. After getting informed consent, relevant clinical history, physical exami-nation and laboratory results were recorded in case report form. Then, 2 ml of bone marrow aspirate with EDTA tube and 2 ml of venous blood with plain tube were collected and sent to Blood Research Division, Department of Medical Research within 2 hours.

Flow cytometry immunophenotyping Plot configuration and optimization including isotype control, fluorescent (colour) control, calibration beads and compensation were firstly performed. After getting the standard optimized setting of machine, the patient’s samples were analyzed throughout the process. Accurate and con-sistent test results were checked and standardized over time regardless of variables.

Immunophenotypic evaluation was per-formed using multicolour flow cytometry CyFlow Cube 8 (Sysmex Partec). Multi-parametric flow cytometry immunopheno-typing was performed using monoclonal antibodies against CD56, CD19, CD138, CD38 and CD45 conjugated with phyco-erythrin (PE), allophycocyanin (APC), fluorescein isothiocyanate (FITC), and phycoerythrin (PE-Dy).

Data were analyzed by CyView™ software. The plasma cells were initially gated using CD138 and side scatter, following which, CD138+ gated cells were analyzed for CD56, CD19 and CD45. In cases of CD138-specimens, the bone marrow aspirate was restrained with a CD56/CD19/CD38/CD45 panel. In this case, plasma cells were gated using CD38 and side scatter and CD38+ cells were analyzed for CD56, CD19 and CD45.

Serum protein electrophoresis Visual detection of a monoclonal band following serum protein electrophoresis was used to confirm the presence of monoclonal protein. The concentration of monoclonal protein was quantified by using a densitometer. Statistical analysis

Data entry was performed and checked for double entry, incorrectness and incompleteness to validate the data. Data were analyzed by using SPSS 16.0. Simple descriptive analysis for each variable was done.

Ethical consideration This study was approved by Ethics Review Committee, Department of Medical Research.

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RESULTS

A total of 40 clinically suspected cases of multiple myeloma were included for the study. Among the 40 cases, 25 cases (62.5%) were diagnosed as multiple

myeloma by the positive expression of CD138 or CD38, negative CD19 expression, weak or negative CD45 expression and positive or negative CD56 expression (Fig. 1-3). Fifteen cases (37.5%) had no evidence of multiple myeloma by

Fig.1. Phenotype of neoplastic plasma cells showing expression of CD56+ CD19- CD138+ CD45+

Fig. 2. Phenotype of neoplastic plasma cells showing expression of CD56- CD19- CD138+ CD45-

Fig. 3. Phenotype of neoplastic plasma cells showing expression of CD56+ CD19- CD38+ CD45+

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flow cytometry. Age of the patients ranged from 49 years to 89 years. The male to female ratio was 1.1:1. In 23 out of 25 cases (92%), plasma cells could be sufficiently identified through initial CD138 gating. The positive expression rates of CD56, CD19, CD138 and CD45 in neoplastic plasma cells were 84% (21/25), 0% (0/25), 92% (23/25) and 32% (8/25), respectively (Table 1). Two of 25 cases (8%) were negative for CD138 expression following initial CD138 gating and were subsequently restained and gated using CD 38. Table 1. Antigenic expression rates in multiple

myeloma cases

Antigen Expression Multiple myeloma patients (%) CD138 Positive

Negative 23(92) 2(8)

CD56 Positive Negative

21(84) 4(16)

CD19 Positive Negative

0(0) 25(100)

CD45 Positive Negative

8(32) 17(68)

Serum protein electrophoresis and densitometer reading were performed in all cases for detection of monoclonal band and monoclonal protein concentration. Monoclonal band was detected visually and estimation of monoclonal protein was done by densitometer. Among 40 cases, 25 cases (62.5%) had monoclonal gammopathy. Among them, 20 cases (80%) had monoclonal band in the gamma () region and 5 cases (20%) had in the beta () globulin region. The mean concentration of monoclonal protein was 4.50 g/dl, with a range of 2.28 to 7.95 g/dl. The remaining 15 cases (37.5%) were not detected monoclonal band.

DISCUSSION

Immunophenotyping has become an invaluable tool in the management of hematological malignancies and is increasingly finding a role in the diagnosis and monitoring of plasma cell disorders. The major advantage of flow cytometry when compared to other methods is the

possibility to discriminate between normal polyclonal and abnormal clonal plasma cells. In addition to establishing the diagnosis of plasma cell disorders, several studies have reported an association between the phenotype of neoplastic plasma cells and prognosis.

This study demonstrated the four-color flow cytometry using monoclonal antibodies against CD56, CD19, CD138 (CD38) and CD45 to detect the neoplastic plasma cells in patients with multiple myeloma.

The European Myeloma Network has recommended using CD38, CD138 and CD45 together with CD19 and CD56 to identify multiple myeloma cells.7 Minimum of 4 markers is recommended for basic plasma cell analysis so that expression of CD56, CD19, CD138 (CD38) and CD45 should be analyzed in every monoclonal gammopathy case to identify CD138+ CD38+ plasma cells and to discriminate normal or reactive plasma cells (CD19+ CD56 - CD45+) and abnormal plasma cells (CD19 - CD56 + or - CD45 - or +).7, 21

In all multiple myeloma cases, a single antigen cannot be used to distinguish neoplastic plasma cells. Although CD138 is expressed at high levels on plasma cells, 22 this marker cannot be used to discriminate neoplastic myeloma cells from reactive plasma cells. In our study, CD138 was used for the initial identification of plasma cells because plasma cells are the only cells in the bone marrow that express CD138. Subsequent selection using CD38 was analyzed only for samples with dim to negative CD138 expression. In this study, 23 of 25 cases (92%) showed CD138+ neoplastic plasma cells, with only 2 cases (2/25,8%) with CD138- neoplastic plasma cells. However, this later group showed CD38+ neoplastic plasma cells and therefore, in these cases, CD38 was used for selection. No difference was noted in the immunophenotypic profile between CD38+ cells and cells gated using the CD138 marker.

56

CD56 is expressed mainly on neoplastic plasma cells although not in all cases. CD56 expression was reported in 70 to 80% of multiple myeloma patients. 23 This study showed that CD56 expression was 84%. Lack of CD56 expression in myeloma has been associated with a worse prognosis.24

Similarly, both CD45- and CD45+ neoplastic plasma cell populations have been described. Because of the varied expression patterns of CD45 on neoplastic plasma cells, there was some limitation in identifying them using CD45 alone, unlike the case with CD56 and CD19. However, the presence of CD45- neoplastic plasma cells has been associated with poor clinical outcome.25 In this study, CD45 expression was found in 32% of multiple myeloma patients. Reactive plasma cells express CD19; however, neoplastic plasma cells show no or only a dim CD19 expression.12, 26 CD19 represents the most valuable antigen to identify neoplastic plasma cells in patients with multiple myeloma. These findings suggest that CD19 expression was negative in all patients.

Conclusion

This study revealed the diagnostic role of flow cytometry immunophenotyping in patients with multiple myeloma. As an adjunct to morphologic evaluation of marrow aspirate smear, histology, immuno-histochemistry of marrow biopsy and protein electrophoretic analyses, flow cytometry immunophenotyping is useful tool for the diagnosis of multiple myeloma and it should be included as a routine assay in monoclonal gammopathy patients.

ACKNOWLEDGEMENT

This project was supported by World Health Organization. We would like to express our deepest gratitude to Sysmex Partec Company for technical assistance and reagents support to develop flow cytometry

laboratory in Department of Medical Research. We wish to express our sincere gratitude and thanks to all the patients for their kind participation in this study.

REFERENCES

1. Munker R, Hiller E, Glass J & Paquette R. Modern Hematology: Biology and Clinical Management. 2nd ed. Totowa; NJ: Humana Press; 2007; 250.

2. Kyle RA & Rajkumar SV. Multiple myeloma. The New England Journal of Medicine 2004; 351: 1860-1873.

3. Rajkumar SV & Kyle RA. Multiple myeloma: Diagnosis and treatment. Mayo Clinic Proceedings 2005; 80: 1371-1382.

4. Cannizzo E, Bellio E, Sohani AR, Hasserjian RP, Ferry JA, Dorn ME, et al. Multiparameter immunophenotyping by flow cytometry in multiple myeloma: The diagnostic utility of defining ranges of normal antigenic expression in comparison to histology. Cytometry B Clinical Cytometry 2010; 78(4): 231-8.

5. Kumar S, Kimlinger T & Morice W. Immunophenotyping in multiple myeloma and related plasma cell disorders. Best Practice Research Clinical Haematology 2010; 23(3): 433-451.

6. Albayrak M, Balcik OS, Dagdas S, Yilmaz M, Ceran F, Yokus O, et al. Role of flow cytometry in multiple myeloma and the prognostic significance of CD87 (uPAR) expression. Turkish Journal of Hematology 2010; 27: 182-9.

7. Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008; 93: 431-438.

8. Bataille R, Robillard N, Avet-Loiseau H, Harousseau J-L & Moreau P. CD221 (IGF-IR) is aberrantly expressed in multiple myeloma, in relation to disease severity. Haematologica 2005; 90: 706-707.

9. Moreaux J, Hose D, Reme T, Jourdan E, Hundemer M, Legouffe E, et al. CD200 is a new prognostic factor in multiple myeloma. Blood 2006; 108: 4194-4197.

10. Robillard N, Pellat- Deceunynck C & Bataille R. Phenotypic characterization of the human myeloma cell growth fraction. Blood 2005; 105: 4845-4848.

11. Moreau P, Robillard N, Avet-Loiseau H, Pineau D, Morineau N, Milpied N, et al. Patients

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with CD 45 negative multiple myeloma receiving high-dose therapy have a shorter survival than those with CD45 positive multiple myeloma. Hematologica 2004; 89: 547-551.

12. Ocqueteau M, Orfao A, Almeida J, Blade J, Gonzalez M, Gercia- Sanz R, et al. Immuno-phenotypic characterization of plasma cells from monoclonal gammopathy of undetermined significance patients: implications for the differential diagnosis between MGUS and multiple myeloma. American Journal of Pathology 1998; 152: 1655-1665.

13. Rawstron AC, Owen RG, Davies FE, Johnson RJ, Jones RA, Richards SJ, et al. Circulating plasma cells in multiple myeloma: Charac-terization and correlation with disease stage. British Journal of Haematology 1997; 97: 46-55.

14. Terstappen LW, Johnsen S, Segers-Nolten IM & Loken MR. Identification and characteri-zation of plasma cells in normal human bone marrow by high-resolution flow cytometry. Blood 1990; 76: 1739-1747.

15. Perez-Persona E, Vidriales MB, Mateo G, Garcia- Sanz R, Mateos MV, de Coca AG, et al. New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smouldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells. Blood 2007; 110: 2586-2592.

16. San Miguel JF, Gutierrez NC, Mateo G & Orfao A. Conventional diagnostics in multiple myeloma. European Journal of Cancer 2006; 42: 1510-1519.

17. Mateo G, Montalban MA, Vidriales MB, Lahuerta JJ, Mateos MV, Gutierrez N, et al. Prognostic value of immunophenotyping in multiple myeloma: A study by the PETHEMA/ GEM cooperative study groups on patients uniformly treated with high-dose therapy.

Journal of Clinical Oncology 2008; 26: 2737-2744.

18. Mateo G, Castellanos M, Rasillo A, Gutierrez NC, Montalban MA, Martin ML, et al. Genetic abnormalities and patterns of antigenic expression in multiple myeloma. Clinical of Cancer Research 2005; 11: 3661-3667.

19. Lima M, Teixeira Mdos A, Fonseca S, Goncalves C, Guerra M, Queiros ML, et al. Immunophenotypic aberrations, DNA content, and cell cycle analysis of plasma cells in patients with myeloma and monoclonal gammopathies. Blood Cells Molecular Disease 2000; 26: 634-645.

20. Lemoli RM & Fortuna A. C-kit ligand (SCF) in human multiple myeloma cells. Leukemia & Lymphoma 1996; 20: 457-464.

21. Kovarova L & Hajek R. Flow cytometric analysis of plasma cells in multiple myeloma. Klinicka Onkologie 2008; 21(Suppl 1): 249-252.

22. Rawstron AC. Immunophenotyping of plasma cells. Currrent Protocols in Cytometry 2006; Chapter 6: Unit 6, 23.

23. Sahara N, Takeshita A, Shigeno K, Fujisawa S, Takeshita K, Naito K, et al. Clinicopatho- logical and prognostic characteristics of CD56-negative multiple myeloma. British Journal of Haematology 2002; 117: 82-5.

24. Sahara N & Takeshita A. Prognostic significance of surface markers expressed in multiple myeloma: CD56 and other antigens. Leukemia & Lymphoma 2004; 45: 61-65.

25. Kumar S, Rajkumar SV, Kimlinger T, Greipp PR & Witzig TE. CD45 expression by bone marrow plasma cells in multiple myeloma: Clinical and biological correlations. Leukemia 2005; 19: 1466-70.

26. Harada H, Kawano MM, Huang N, Harada Y, Iwato K, Tanabe O, et al. Phenotypic difference of normal plasma cells from mature myeloma cells. Blood 1993; 81: 2658-63.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Physical Fitness of the Elderly People from Home for the Aged (Hninzigone), Yangon

Khin Mi Mi Lay*, Nway Htike Maw, Pyae Phyo Kyaw, Sandar Win, Khin San Lwin, Htet Htet Lwin, Yi Yi Mon, Lei Lei Win Hlaing & Theingi Thwin

Department of Medical Research

A cross-sectional, descriptive study was conducted to assess physical fitness in 145 elderly; 52 men (mean age=81.23±6.36 years) and 93 women (mean age=79.03±5.27 years), from the Home for the Aged (Hninzigone), Yangon by using the Senior Fitness Test. The test consists of six measures of physical fitness: (a) 30-second chair stand test, (b) 30-second arm curl test, (c) chair sit and reach test, (d) back scratch test, (e) 8-foot up and go test and (f) 2-minute step test. Most of elderly women had higher BMI (23.19±6.46 kg/m2 vs. 22.98±4.24 kg/m2, p=0.83) and significantly higher body fat percent (24.23±7.6% vs. 20.11±6.46%, p=0.001) than those of elderly men. Higher BMI of elderly women might be due to more body fat than elderly men. All the elderly had completed the six fitness tests. Elderly men had better performance in chair stand test (strength test) and 2-minute step test (aerobic endurance test) but elderly women had better performance in 8-foot up and go test (dynamic balance). This might be due to the fact that elderly men had more lean body mass than women. Scores from each test were compared to Americans’ norms by subject’s age and gender and described as performance better than the norm, same as the norm, or worse than the norm. Generally, better performance were seen in strength tests (chair stand and arm curl) than cardiovascular tests (2-minute step and 8-foot up and go) or flexibility tests (chair sit and reach and back scratch). The low scores of cardiovascular tests may be attributed to the fact that those types of exercise are not very common in the daily activity of elderly in Myanmar. However, the good result of chair sit and reach test is an essential part of their daily physical activity. In conclusion, this study found that there was a decrease in body fat percent and decrease levels of dynamic balance and flexibility in the aging process.

Key words: Physical fitness, Elderly, Senior Fitness Test Battery

INTRODUCTION

The older adult population of Myanmar has been increased over the last three decades. Percentage of persons older than 60 years was increased from 6.37 in 1980 to 8.77 in 2011.1 With the projected growth in the older adult population, preventing or delaying physical disability in later years has become a national goal. Evidence suggests that physiological decline, especially that associated with physical inactivity is modifiable through proper assessment and activity intervention. However, a major limitation in reducing loss of function in later years is the lack of

suitable assessment tools. Of special concern is the ability to assess underlying physical parameters associated with common activities of daily living.2 Recognizing the need for a tool to evaluate the functional fitness performance of older adults, researchers at California State University, Fullerton, developed and validated a new fitness test battery especially for older adults: the Senior Fitness Test.3

Physical fitness parameters such as strength, flexibility, coordination and endurance can

*To whom correspondence should be addressed. Tel: +95-9402741523 E-mail: khnlay883@gmail.com

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be measured by the Fullerton Functional Fitness Test, invented by Rikli and Jones in the Lifespan Wellness Clinic at California State University in Fullerton. It uses six items to assess these parameters; (a) arm curl test to indirectly assess the upper body strength, (b) 30-second chair stand test to assess lower body strength, (c) back scratch test to assess upper body flexibility, (d) chair sit-and-reach test to assess lower body flexibility, (e) 8-foot up-and-go test to assess the agility/dynamic balance and (f) 6-minute walk trial describes indirectly, the level of aerobic endurance and step-in-place test is performed instead of 6-minute walk trial in case of persons, who use orthopaedic devices during walking, as well as in case of persons with difficulties associated with maintenance of balance. Because of limitations resulting from age and coexisting diseases, it is required that some easy and safe motor patterns are used that should be based on everyday activities. It is safe for the adults, requires minimal equipment and can measure small unit changes. It is suitable even for patients with cardiovascular disease.4

Although many studies had been done on assessment of physical fitness of school children;5 office workers and labourers;6 military personnel, dependents and some civilians,7 but lack of studies on physical fitness of elderly in Myanmar.

The Hninzigone Home for the aged, Yangon, is a non-profit humanitarian non-governmental organization in Myanmar. Its mission is to provide accommodation and care for the helpless and homeless aged senior citizens in the evening years of their lives. Preventing or reducing loss of function in later years depends, in part, on the ability to detect and treat any physical declines that may be precursors to more serious loss of function.8

Early identification of physical decline and appropriate interventions could help to prevent functional impairments, such as in walking and stair climbing that often result

in falls and physical fraility.9 Monitoring the level of functional fitness is especially important for elderly people above 60 years for preventing many diseases, occurrence of immobilization and reduction of mortality rate, thus, the study aimed to assess physical fitness of elderly people from Home for the Aged (Hninzigone), Yangon.

MATERIALS AND METHODS

A cross-sectional, descriptive study was done at the Home for the Aged (Hninzigone), Yangon. A total of 145 elderly men and women (52 men and 93 women) (>65 year of age), who can walk and dress unaided were voluntarily participated.

Exclusion criteria Participant’s bad general feeling Chest pain (discomfort) ECG evidence of ischemia and

arrhythmia Uncontrolled arterial blood pressure

exceeding 160/100 mmHg Musculoskeletal disorder Recommendation of the Medical officer

not to perform the test

Procedure of the study Firstly, primary screening such as blood pressure (BP) measurement and electro-cardiogram (ECG) were done after taking informed consent and elderly were selected according to inclusion and exclusion criteria. Then, filling out proforma, anthro-pometric measurements (height, weight, waist and hip circumferences, skinfold thickness) were done by well-trained technician. BMI, waist-hip ratio and body fat composition were calculated. The testing location was equipped with drugs to deal with emergencies and medical officer standby. Before starting the functional fitness tests, the elderly was asked to perform the tasks as good as possible. Appropriate safety was secured by proper positioning of the devices used during the testing procedure.

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Before and after the tests, arterial blood pressure and heart rate were measured. Performance of each functional fitness test was preceded by a demonstration and the examined elderly could preliminarily check his ability to perform particular tests in order to get familiar with their proper course.

The performance of the tests was started with the arm curl test, and subsequently the back scratch test, the 30-second chair stand test, the chair sit-and-reach test, the 8-foot up-and-go test, and the last, 2-minute walk in place test was performed. It was terminated in case the examined person reports dizziness, nausea, excessive fatigue, pain, or if the examiner notices other alarming symptoms. “Anthropometric measurements”

Waist circumference (WC) Waist circumference was measured by measuring tape with the subject stands with feet 25-30 cm apart and weight evenly distributed. Measurement was taken at the midway between the inferior margin of the last rib and the crest of the ilium in the horizontal plane. The measurer sat by the subject and fit the tape snugly without compressing the soft tissue. The circumference was measured to nearest 0.1 cm.

Hip circumference (HC) This was measured as in WC around the pelvic at the point of the maximal protrusion of the buttocks. Waist-hip ratio

The ratio of WC and HC was calculated. Skinfold thickness

Triceps skinfold thickness (TSF) and biceps skinfold thickness (BSF) of the right arm were measured to the nearest 0.1 mm with a pair of calipers (GIMA, Italy), in duplicate. Body fat percentage was calculated using the equations described by Kwok, Woo and Lau.10

“Tests for functional fitness of the elderly” Each subject was asked to perform the following tests. Arm curl test (To assess upper body strength)

Description Number of bicep curls that can be completed in 30 seconds holding a hand weight of 5 lbs (2.27 kg) for women, 8 lbs (3.63 kg) for men. Risk zone Less than 11 curls using correct form for men and women. 30-second chair stand test (To assess lower body strength) Description Number of full stands that can be completed in 30 seconds with arms folded across chest. Risk zone Less than 8 unassisted stands for men and women. Back scratch test (To assess upper body (shoulder) flexibility) Description With one hand reaching over the shoulder and one up the middle of the back, the number of inches (cm) between extended middle fingers (+ or -). Risk zone Men: Minus (-) 4 inches or more Women: Minus (-) 2 inches or more Chair sit-and-reach test (To assess lower body flexibility)

Description From a sitting position at front of chair, with leg extended and hands reaching toward toes, the number of inches (cm) (+ or -) between extended fingers and tip of toe.

Risk zone Men: Minus (-) 4 inches or more Women: Minus (-) 2 inches or more 8-foot up-and-go test (To assess agility/ dynamic balance)

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Description Number of seconds required to get up from a seated position, walk 8 feet (2.44 m), turn, and return to seated position. Risk zone More than 9 seconds. The 2-minute walk-in-place test (Aerobic endurance test) Description Number of full steps completed in 2 minutes, raising each knee to a point midway between the patella (knee cap) and iliac crest (top hip bone). Score is number of times right knee reaches the required height. Risk zone Less than 65 steps for men and women Statistical analysis Data were analyzed by using SPSS-version 16. Results were expressed as the mean± standard deviation (SD) or standard error (SE). Univariate analysis of variance (ANOVA) was used to determine the differences between age groups. Bonferroni correction was used to determine which of groups were statistically different. Statistical significance was set at p<0.05.

RESULTS

Table 1. General characteristics of the elderly

men and women

Characteristics Men Women p value

Mean age (yr) 81.23±6.36 79.03±5.27* 0.027 Weight (kg) 56.46±11.26 49.88±14.01* 0.004 Height (cm) 156.8±8.27 146.68±5.33* 0.000 BMI (kg/m2) 22.98±4.24 23.19±6.46 0.83 Body fat percent (%) 20.11±6.46 24.23±7.6* 0.001 WC (cm) 85.55±13.77 82.28±8.53 0.08 HC (cm) 93.6±16.28 91.28±8.42 0.26 Waist/hip (cm) 0.93±0.15 0.9±0.07 0.197 Resting HR (beats/min) 86.06±12.03 81.32±9.37** 0.009 Resting SBP (mmHg) 139.25±14.4 142.24±12.84 0.201 Resting DBP (mmHg) 80.29±8.6 80.37± 9.26 0.96 BMI=Body max index, WC=Waist circumference, HC=Hip circumference, HR=Heart rate, SBP=Sys-tolic blood pressure, DBP=Diastolic bold pressure Data were calculated by using Independent samples. “t” test and expressed as mean±SD *means statistically significant (p<0.05) **means statistically significant (p<0.01)

Table 2. General characteristics of elderly men and women according to age groups

Age Number (%)

Standing height (cm)

Body weight (kg)

BMI (kg/m2)

Body fat (%)

70-74 26 (17.93)

150.14 ±5.83

54.5 ±10.79

24.18 ±4.55

24.94 ±6.43

75-79 51 (35.17)

149.99 ±9.66

53.69 ±17.4

23.92 ±7.84

24.19 ±9.2

80-84 40 (27.59)

150.10 ±7.78

51.6 ±11.45

22.8 ±4.12

22.13 ±5.46

85-89 19 (13.1)

151.24 ±8.57

48.21 ±9.92

21.0 ±3.66

19.41 ±6.29

90 9 (6.21)

151.56 ±5.96

48.89 ±6.85

21.33 ±3.20

18.1 ±5.09

Total 145 150.31 ±8.13

52.24 ±13.43

23.11 ±5.75

22.75 ±7.45

One way ANOVA test (Bonferroni correction) Data were expressed as mean±SD. Table 3. Comparison of functional fitness tests’

scores between elderly men and women Tests Men Women p value Chair stand (No. of stands)

14.5±0.92 11.6 ±0.51** 0.003

Arm curl (No. of reps)

19.19±0.67 18.54±0.37 0.35

2-minute step (No. of steps)

69.37±2.82 58.22±2.56** 0.006

Chair sit-and-reach (Inches +/-)

-0.87±1.35 1.99±1.38 0.18

Back scratch (Inches +/-)

-10.66±2.03 -9.84±1.37 0.73

8- foot up-and-go (Seconds)

8.82±0.42 10.43±0.31** 0.002

Independent paired “t’ test Data were expressed as mean±SE. **means statistically significant (p<0.01).

Table 4. Physical fitness of elderly men and women in comparison with Americans’ norms3

DISCUSSION

The level of physical activity is often used as a parameter for monitoring and evaluation of public health. This monitoring is especially important for the elderly to prevent many diseases, occurrence of immobilization and reduction of mortality rate.11 In this study, although the elderly

Test Norm (Percent)

Worse than Same as Better than Men Women Men Women Men Women

Chair stand 19.23 23.66 48.08 52.69 32.69 23.66 Arm curl 3.85 4.3 46.15 26.88 50 68.82 2-minute step 53.85 46.24 42.31 52.69 3.85 1.08 Chair sit-&-reach 25 20.43 30.77 31.18 44.23 48.39 Back scratch 55.77 56.99 13.46 16.13 0.77 26.88 8-foot up-&-go 9.62 83.87 30.77 16.13 9.62 0

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men and women had no significant difference in body mass index (BMI), elderly men had significantly higher height and weight than elderly women. Conversely, body fat percentage was significantly higher in elderly women than that of the elderly men. Higher BMI of elderly women might be due to more body fat than elderly men. Resting systolic blood pressure (SBP) and diastolic blood pressure (DBP) had no significance difference in terms of gender Table 1.

In this study, the average BMI of all the elderly (with the mean age of 79.82± 5.76 years) was 23.11±5.75 kg/m2 and the highest BMI value was seen in 70-74 year age group (24.18±4.55 kg/m2). This finding was agreed with the finding of Milanovic, et al. 201211 in which physical fitness of men older than 60 years was studied and found higher BMI values in the age group of 70-74. Lower BMI values were seen in the age group of 85 years and above in the present study. In contrast to the finding, Perissinotto, et al. (2002)12 found lower BMI values between the ages of 65-75 than in the period between 75-80. In this study, the higher the age, the lower the BMI and body fat percent were seen. This finding was agreed with the previous Myanmar study.13 Reducing body fat could lead to better physical fitness and working capacity. However, this finding was not agreed with the concept that age-related changes in body composition were decreases in fat free mass and increases in fat mass. Milanovic, et al. (2012)11 also stated that aging is associated with a higher percentage of body fat and body fat distribution. Distribution of lower-body subcutaneous adipose tissue in the abdominal and visceral part is the most common with the elderly people. In this study, all the elderly had completed the six fitness tests. Elderly men had better performance in chair stand test (strength test) and 2-minute step test (aerobic endurance test). This might be due to the fact that elderly men had more lean body

mass than women. However, elderly women had better performance in 8-foot up-and-go test (dynamic balance). There was no statistically significant difference (p>0.05) in arm curl test, back scratch test and 2-minute step test in all age groups. The subjects aged 70-74 significantly differed (p<0.05) in the chair stand test and 8-foot up-and-go test compared to 80-84 years of age and they also significantly differed (p<0.05) in the chair sit-and-reach test in compared to subjects aged 85-89 years of age. The results showed statistically significant decrease of muscle strength, dynamic balance and flexibility in elderly older than 80 years. Elderly men and women are less physically active with aging process which could be reflected on their muscular strength and dynamic balance. Decrease in muscle strength during the aging process is the result of significant loss of muscle mass, which may cause the decrease in physical activity14 but impaired dynamic balance may also increase the risk of falls and injuries in older people.

Scores from each test were compared to Americans’ norms by subject’s age and gender and described as performance better than the norm, same as the norm, or worse than the norm. Generally, performance was better in strength tests (chair stand and arm curl) than cardiovascular tests (2-minute step and 8-foot up-and-go) or flexibility tests (chair sit-and-reach and back scratch). The low scores of cardiovascular tests may be attributed to the fact that those types of exercise are not very common in the daily activity of elderly in Myanmar. However, the good results of chair sit-and-reach test is essential part of their daily physical activity. The reduction of muscle function should be attributed to a combination of factors such as aging and physical inactivity.15, 16

The reduction of the number of muscle fibers and reduction in activation of motor units during the aging process14 might decrease the muscle strength, flexibility and endurance in elderly people. These changes

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could lead to a greater risk of cardiovascular and respiratory diseases. Other researchers found that appropriate level of training can maintain muscle strength and endurance.17, 18 Thus, physical activity inter-vention and monitoring of physical fitness should be done regularly in elderly people.

REFERENCES

1. Country profile, Health in Myanmar, 2012. 2. Rikli RE & Jones CJ. Assessing physical

performance in independent older adults: Issues and guidelines. Journal of Aging and Physical Activity 1997; 5: 244-261.

3. Rikli R & Jones CJ. Measuring functional fitness of older adults. Journal of Active Aging March-April 2002; 24-30.

4. Kirschke AR, Kocur P, Dylewicz P, et al. The Fullerton Fitness Test as an index of fitness in the elderly. Medical Rehabilitation 2006; 10(2): 9-6.

5. U Maung Gale. Report on the dietary and nutrition surveys. Rangoon Government Printing and Stationery, Burma, 1948.

6. Postmus S. Final report on nutrition in Burma. WHO Project in Burma, 26.

7. United States. Interdepartmental committee on nutrition and national defense (1963). Report on Nutrition survey. Oct-Dec, 1961 in Union of Burma, Washington DC, 1957.

8. Evans WJ. Keys to successful aging. Paper presented at the International Conference on Aging and Physical activity, Colorado Springs, 1995.

9. Alliance for aging research. Independence for older Americans: An investment for our nation’s future. Washington DC, 1999.

10. Kwok T, Woo J & Lau E. Prediction of body fat by anthropometry in older Chinese people. Obesity Research 2001; 9(2): 97-101.

11. Milanović Z, Pantelić S, Trajković N & Sporiš G. Basic anthropometric and body composition characteristics in elderly population: A Systematic Review. Facta Universitatis: Series Physical Education and Sport 2011; 9(2): 173-182.

12. Perissinotto E, Pisent C, Sergi G & Grigoletto F. Anthropometric measurements in the elderly, age and gender differences. British Journal of Nutrition 2002; 87(2): 177-86.

13. Ye Tint Lwin, Zaw Myint, Mi Mi Nwe, Mya Mya Win, Ni Ni Than, et al. Body composition of Myanmar elderly people from home for the aged (Hninzigone), Yangon. Myanmar Health Sciences Research Journal 2014; 16(1): 1-5.

14. Milanović Z, Pantelić S & Jorgic B. Changes in physical fitness of men older than 60 years: A pilot study. Sport Logia 2012; 8(1): 43-49.

15. Shephard J. Aging, physical activity, and health. Champaign, IL: Human Kinetics Publishers, Inc: USA, 1997.

16. Spirdusso WW. Physical dimension of aging. Champaign, IL: Human Kinetics Publishers, Inc: 1995.

17. Jozsi AC, Campbell WW, Joseph L, Davey SL & Evans WJ. Changes in power with resistance training in older and younger men and women. The Journals of Gerontology: Series A 1999; 54(11): M591-M596.

18. Toraman N, Ayceman N, & Yaman H. Effects of six weeks of detraining on retention of functional fitness of old people after nine weeks of multi component training. British Journal of Sports Medicine 2005; 39(8): 565-568.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018 Genotypic Analysis of Epstein-Barr Virus (EBV) in Nasopharyngeal Carcinoma Patients

Moh Moh Htun1*, Chaw Su Hlaing2, Myat Mon Oo1, Kay Thi Aye1,

Aung Zaw Latt1, Soe Aung3, Hlaing Myat Thu1, Khin Pyone Kyi3 & Kyaw Zin Thant1

1Department of Medical Research

2Department of Health Services 3Myanmar Medical Association

Nasopharyngeal carcinoma (NPC) is endemic in certain populations, 0.6% of all cancers in the world. It occurs at high incidence in Southeast Asia, Southern China and North Africa. In Myanmar, the prevalence of NPC is gradually rising yearly and it is one of the common head and neck cancer in Yangon General Hospital (YGH). Epstein-Barr virus (EBV) is a member of the Herpesviridae family. It is a well-known causative agent in NPC and mainly infect in lymphocytes and epithelial cells. The polymerase chain reaction (PCR) was used to study DNA extracted from the blood samples of 35 histologically confirmed NPC patients. The commonest age group was 51-60 years in both gender and male was more common than female (1.5:1). The most common histological type was poorly differentiated squamous cell carcinoma (SCC) (45.7%) and other histological types were non-keratinized carcinoma (20%), undifferentiated anaplastic carcinoma (17.2%), moderately differentiated SCC (11.4%) and well differentiated SCC (5.7%) according to World Health Organization (WHO) classification. Primers were directed to conserved regions of the EBV genome encoding Epstein-Barr nuclear antigen 1(EBNA1) region. Specific EBV amplification (EBV-DNA positive) was found in 5 samples of NPC patients (14.3%) at 262 bp. The purified PCR products were carried out to do genetic sequencing by using ABI genetic analyzer. These isolates were found to be EB virus (Herpesviridae genotype 4) and firstly detected in blood samples of nasopharyngeal carcinoma patients in Myanmar. The new Myanmar EBV sequences were analyzed with a group of 14 previously published EBV strain sequences including 8 from China, 5 from Australia and one from Japan within 2006-2016. A phylogenetic tree was generated and the new Myanmar EBV strains were recorded that was different from other isolates these countries. Cancer treatment for early stage of NPC is good response but 70-80% of NPC is found in advanced or metastatic state. EBV DNA may be currently related biomarker in NPC which allows to one of the indicators for early diagnosis, better prognosis, treatment response and recurrent of disease during cancer therapy.

Key words: Nasopharyngeal carcinoma (NPC), Epstein-Barr virus (EBV), Genotypic analysis

INTRODUCTION

Global cancer rates could further increase by 50% to 15 million new cases in the year 2020, according to the World Cancer Report from the World Health Organization in 2003.1 Non-communicable diseases (NCDs) are the leading causes of death in the world and nearly 80% of NCD deaths occur in low- and middle-income countries.

It comprises of mainly cardiovascular diseases, cancers, diabetes and chronic lung diseases.2 Nasopharyngeal cancer (NPC) is an uncommon cancer with approximately 80,000 new cases reported per year and accounting 0.7% of all cancers. In endemic ___________________________________ *To whom correspondence should be addressed. Tel: + 95-95070214 E-mail: mohmoh.htunn@gmail.com

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areas including Southern China (Hong Kong) and Southeast Asia, the annual age- standardized incidence rates are as high as 20-30 cases per 100,000 populations in men and 8-15 cases per 100,000 populations in women.3 In Myanmar, the prevalence of NPC is 2.04% of total admission to Oto-rhinolaryngology Head and Neck Surgery Specialist Hospital, Yangon and 7.12% of total head and neck cancers in Yangon (2008-2009).4 NPC is the second most common head and neck cancer in Yangon General Hospital (YGH) and is gradually rising the incidence yearly. According to 52 NPC cases in 2008 and 76 NPC cases in 2011, males are more common than females (2.5:1) in previous study.5

There are three common histological types according to the World Health Organization (WHO) such as Type (I) squamous cell carcinoma, Type 2a (II) keratinizing undifferentiated carcinoma, Type 2b (III) non-keratinizing differentiated and undiffer-entiated carcinoma. There were three histological grades in squamous cell carcinoma (SCC) types of NPC such as well, moderate and poorly differentiated SCC. Among them, NPC subtype (III) is the commonest form of NPC in endemic areas. Type (I) NPC is associated with Epstein-Barr virus (EBV) and sensitive to chemotherapy and radiotherapy. Type (III) NPC is also related to regulation of gene expression in DNA packaging or modulation of gene activities. Epstein-Barr virus (EBV) is a member of the Herpesviridae family and Gammaher-pesvirinae subfamily. EB viral particle has a diameter of 120-180 nm and EBV genome is a linear, double stranded 172 kb DNA molecules.6

It can mainly infect in lymphocytes and epithelial cells. Infectious mononucleosis and oral hairy leukoplakia (OHL) are caused by EBV. There are two genotypes such as EBV-1 and EBV-2 distinguished by divergent gene sequences encoding the EBNA-2, 3A, 3B, and 3C proteins.7

The primary infection of EB virus occurs during childhood with replication of the virus in the oropharyngeal linning epithelial cells followed by a latent infection of B lymphocytes. Latent EBV infection was recognized in neoplastic cells of almost all cases of NPC. In some areas of Asia, 80% of children are infected with EBV by 6 years of age, 100% have sero-converted by 10 years of age.8

EBV DNA was detected in plasma/serum of 98 of 167 (58.7%) of NPC patients prior to treatment in Chulalongkorn University Hospital, Bangkok Thailand.9 Common clinical presentations are unilateral and bilateral mass in neck, nasal obstruction with instead of bloody drainage (epistaxis) and decreased hearing.10 The three major etiologic factors related to NPC are genetic predisposition, dietary factors including salt-cured fish and meat, foods containing aerosolizes carcinogenic nitrosamine and EB viral infection.11

Alcohol drinking, shrimp paste consumption and infrequent vegetables eating were found to be significantly associated to the risk of NPC in previous study from Myanmar.12 Cancer treatment for early stage of nasopharyngeal carcinoma is good response but 70-80% of NPC is found in locally advanced or metastatic state. EB virus is currently related biomarkers in NPC that can be allowed for better prognosis, treatment response and recurrent of disease during cancer therapy.13

Genotypic analysis of EB virus in nasopharyngeal carcinoma patients in Myanmar was main objective of this study because the number of the NPC cases is rising gradually mainly in young adults that was mainly associated with EBV infection. Moreover, association of different histo-logical types of NPC and genotypic characteristic of EB virus was analysed in present study. This study is provided for the strong evidence supporting the etiologic role of EBV infection in nasopharyngeal carcinoma in Myanmar.

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MATERIALS AND METHODS

A cross-sectional, descriptive laboratory- based study was carried out the total 35 histologically confirmed NPC patients attending at Clinical Unit of Ear, Nose, Throat (ENT) and Head and Neck Specialist Hospital, Yangon during 2014-2016. Blood samples of NPC patients were collected from Clinical Unit of Ear, Nose, Throat and Head and Neck Specialist Hospital, Yangon. All clinical and laboratory data were collected in proforma.

DNA extraction and molecular detection of EBV was done in Pathology Research Division and EBV DNA sequencing was carried out in Advanced Molecular Research Centre in Department of Medical Research. Thirty-five histologically con-firmed new NPC cases with the age of 15-75 years and both genders were recruited. Five millilitres of whole blood with ethylene diamine tetra-acetic acid (EDTA) contained tube were collected from these subjects. All serum samples were kept in a -20ºC freezer before DNA extraction after separation of plasma/ serum by centrifugation at 3000 rpm. Detection of EBV DNA was done by polymerase chain reaction (PCR). DNA extraction and PCR reaction

DNA was extracted from serum or plasma of each sample by using QIAmp Blood Kit (Qiagen, Germany) according to manu-facturer’s protocol and amplified by using EBNA-1 primer set. Genomic DNA (0.1-0.5 µg) was added to 25 µl of PCR mix. The reaction mix contained a final concentration of 250 µM dNTP, 1.5 mM MgCL2, 0.1 µM each primers (EBNA-1F and EBNA-1 R), and 2.5 U of Taq polymerase. Primers were directed to conserve Epstein- Barr nuclear antigen (EBNA-1). After being denatured at 94ºC for 5 minutes, samples were subjected to 40 cycles of amplification (30 seconds at 94ºC, 30 seconds at 55ºC and 5 minutes at 72ºC).

Identification of viral sequence PCR products were visualized with UV light as a single band by staining with ethidium bromide after 1.5% gel electrophoresis. The PCR products were purified to remove primers and excess nucleotides by using Montage’s purification method. Two micro-litres of purified product were added to a master-mix containing 3.5 µl of 5 X sequencing buffer, 0.5 µl of (Big-dye V 3.1, Applied Biosystems), 2.4 µl of sterile DDW and 1.6 µl (1 µlM concentration) of each of forward and reverse primers of EBNA gene. Amplification was done under the following conditions: one minute of activation at 96ºC followed by 25 cycles of 10 seconds denaturation at 96ºC, 5 seconds of annealing at 50ºC and 4 minutes of extension at 60ºC. After purification of cycle sequencing product by ethanol, this product was carried out to DNA sequencing by using Applied Biosystems 3500 XL Genetic Analyzer, Hitachi. Phylogenetic analysis

Sequence quality was checked by BioEdit software v 7.0.5 and manual correction was done. Two sequences of EBNA-1 (262nt) were compared with that of other sequences of EBV available from sequence numbers of GenBank (KX950746 and KX950747 EBNA1). Sequence of primers used for EBNA -1 gene

Gene Primer name Nucleic sequences Amplicon

size EBNA-1 Forward

primer TGAATACCACCAAGAGGTG 262 bp

Backward primer

AGTTCCTTCGTCGGTAGTC

Then, nucleotide and amino acid of all sequences were aligned by using the Cluster W 1.6 method of MEGA software version 6.0.6. Phylogenetic tree was generated by the neighbor-joining method.

Ethical consideration This study was approved by the Ethics Review Committee of the Department of Medical Research, Ministry of Health and Sports.

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RESULTS

In this study, 35 blood samples were collected from 15-75 years with both genders, 21 males (60%) and 14 females (40%) of histologically proven nasopharyn-geal carcinoma patients who were attending in Clinical Unit of Ear, Nose, Throat and Head and Neck Specialist Hospital, Yangon during 2014-2015. The mean age of both genders was 47.9±14.9 years. The age of youngest NPC patient was 15 years (male) and the oldest NPC patient was 74 years (male). The commonest age group of NPC cases was 51-60 years (51.4%) including 11 males and 7 females (Table 1). Table 1. Age and sex distributions of naso-

pharyngeal carcinoma

Age (Year) Male Female Total cases (%) <20 2 2 4(11.4) 21-30 1 1 2(5.7) 31-40 1 1 2(5.7) 41-50 3 2 5(14.3) 51-60 11 7 18(51.4) >60 3 1 4(11.4) Total 21 14 35(100.0)

Among the 35 cases of NPC, the commonest histological type was Kerati-nized poorly differentiated squamous cell carcinoma, 45.7% (16/35 cases) and the lowest type was Keratinized well differen-tiated squamous cell carcinoma 5.7% (2/35 cases) (Table 2). Table 2. Proportion of EBV DNA in different

histological types and grades of NPC according to WHO classification

The commonest clinical staging was stage I (T1N0M0) NPC in 11/35 patients (31.4%) and the lowest stages was Stage 0 (lymph node is not detected) in 4/35(11.4%) in this study (Table 3).

Table 3. Clinical staging (TNM staging) of nasopharyngeal carcinoma (NPC) according to WHO classification14

Clinical stages T N M No. of cases

(%) EBV positive

(%) 0 Tis N0 M0 4(11.4) 0 I T1 N0 M0 11(31.4) 1(9) II T1 N1 M0 3(17.1) 0 T2 N0 M0 2(2.9) 1(50) T2 N1 M0 4(20) 1(25)

III T2 N2 M0 3(11.4) 0 T2 N2 M0 2(5.7) 0 T3 N0 M0 1(8.6) 0 T3 N1 M0 1(2.9) 0 T3 N2 M0 0(0) 0

IVA T4 N0 M0 1(8.6) 1(100) T4 N1 M0 1(2.9) 1(100) T4 N2 M0 1(2.9) 0

IVB T any N3 M0 1(2.9) 0 IVC T any N any M1 0(0) 0 Total 35 5

T=Tumor, N=Lymph node, M=Metastasis

Of 35 NPC patients, 5 patients were Epstein-Barr virus (EBV) DNA positive (14.3%) and 30 patients (85.7%) were EBV-DNA negative by amplification of polymerase chain reaction (PCR) with specific primer set of EBNA-1 gene (Fig. 1 & Fig. 2).

Fig.1. Detection of EBV-DNA by using PCR with specific EBNA-1 gene

EBV was detected in only one case of stage I NPC and two cases of clinical staging II and IVA NPC. This DNA was found in 16-year-old female, a case of 48 years old male, 2 males and 1 female case in the age range

Histological types and grades

Total cases (%)

EBV-DNA positive

(%)

EBV-DNA negative

(%)

Keratinized well differentiated SCC

2(5.7) 0(0) 2(6.7)

Keratinized moderately differentiated SCC

4(11.4) 0(0) 4(13.3)

Keratinized poorly differentiated SCC

16(45.7) 4(80) 12(40)

Non-keratinizing differentiated

7(20.0) 0(0) 7(23.3)

Non-keratinizing Un-differentiated

(Anaplastic type)

6(17.1) 1(20) 5(16.7)

Total 35(100.0) 5(100) 30(100)

100 bp maker

EBV-DNA bandat 262 bp

EBV-DNA bandat 262 bp

EBV-DNA Band at 262 bp

EBV-DNA Band at 262 bp

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Fig. 2. Phylogenetic relationship of Epstein-Barr virus (Herpes virus genotype -4) done with EBVNA-1 gene (262 nt) analysis by Cluster W 1.6 method of MEGA software version 6.0.6. Phylogenetic tree was generated by the neighbor-joining method

of (51-60) years. EBV-DNA positivity was 4/5 cases (80%) in keratinized poorly differentiated NPC and 1/5 cases (20%) of non-keratinized undifferentiated NPC. EBV-DNA negative was found in non-keratinized differentiated type, keratinized well differentiated SCC and moderately differentiated SCC. EBV-DNA positive was detected in 2/5 cases (40%) of clinical stage I, 2/5 (20%) of Stage II and 1/5 cases (20%) of stage IV. EBV-DNA was not detected in clinical stage 0(0/4) and stage III (0/5) in this study. Specific EBV amplification (EBV-DNA positive) was found in 5 samples of NPC patients (14.3%) at 262 bp. The purified PCR products were carried out to do genetic sequencing by using ABI genetic analyzer. These isolates were found to be EB virus (Herpesviridae genotype 4) and firstly detected in blood samples of nasopharyn-geal carcinoma patients in Myanmar.

The new Myanmar EBV sequences were analyzed with a group of 14 previously published EBV strain sequences including 8 from China, 5 from Australia and 1 from Japan within 2006-2016. A phylo-genetic tree was generated and the new Myanmar EBV strains were recorded that was different from other isolates these countries.

DISCUSSION

In this study, the commonest age group is 51-60 years in both gender and male was more common than female (1.5:1). The most common histological type was poorly differentiated squamous cell carcinoma (SCC) (45.7%). In this study, the common clinical stages were Stage I (31.4%) and stage 0(11.4%) and EBV-DNA was detected in stage I 1/11cases (9%), stage II 2/6 cases (33.3%) and Stage IVA of NPC 2/2 cases (100%), respectively. Therefore, EBV may be enhanced to progress of tumor growth.

Histologically, 4 cases of poorly differen-tiated type and only one case of non- keratinized undifferentiated anaplastic type were found EBV-DNA. In other study found that EB virus is commonly associated to non-keratinized lympho proliferative type of NPC. In Myanmar, the most common histological type is squamous cell carci-noma according to previous results. Therefore, 80% of EBV-DNA positive were found in poorly differentiated SCC type in this study. EBV sequence was identified in 100% of undifferentiated NPC, 50% of moderately differentiated type and 60% of keratinized NPC in a study of China.15

EBV has not been detected in normal nasopharyngeal epithelial cells but EBV-

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DNA was detected in NPC patients and suspected NPC (carcinoma in situ) in other previous research findings.16 Epstein-Barr virus is a ubiquitous herpes virus, latently infecting in all populations but primary mostly occurs in childhood with asympto-matic and acute infection in young adults, however, causes a lymphoproliferative disease called infectious mononucleosis. It also related to development of nasopharyn-geal carcinoma and Burkitt’s lymphoma.

It can persist for as long as ten years. There were two major types of EB virus, type1 (A) is much more B lymphocyte transformation than type 2 (B). Type 1 EBV is more common than type 2 EBV in NPC patients among Chinese and Japanese populations.17 Type A is predominant in populations of Southern China, Japan, Tunisian, Slovenia and North America whereas type B has been found mainly in Alaska.18

In this study, primers were directed to conserved regions of the EBV genome encoding Epstein-Barr nuclear antigen 1(EBNA1) because this type is more commonly occur in many countries. EBV type 1 was firstly detected in 5 cases out of 35 nasopharyngeal carcinoma patients by using polymerase chain reaction and identified by genetic sequences of this virus by genetic sequencer. The new Myanmar EBV sequences were analysed with a group of 14 previously published EBV strain sequences including 8 from China, 5 from Australia and one from Japan within 2006-2016. A phylogenetic tree was generated and the new Myanmar EBV strains were recorded that was different from other isolates of these countries. Molecular detection of serum or plasma EBV-DNA could be diagnosed for early detection of NPC that was highly sensitive and specific method.

In other countries, screening of EBV-DNA could be done in patients with serially increased anti- EBV with positive family history of NPC.19 EBV is associated with the development of NPC and EBV-DNA detection is potential for early diagnosis,

monitoring and prognosis of NPC. It acts as a biomarker for better prognosis, treatment response and recurrent of disease during cancer therapy.

REFERENCES

1. World Health Organization (WHO, 2003): The major findings of the World Cancer Report [Internet]. 2009 [cited 2009 Oct 22]. Available from: http://www.who.int/mediacenter/ news/ releases/2003/pr27/en/

2. World Health Organization (WHO, 2010). 3. GLOBOCAN 2012: Estimated cancer incidence,

Mortality and Prevalence Worldwide, 2012. Nasopharyngeal carcinoma: Union for International Cancer Control, 2014 Review of Cancer Medicines on the WHO list of Essential Medicines. UICC EML Review- NPC 2014. [Internet]. 2015 [cited 2015 August 31]. Available from: http://www.who. int/selection_ medicines/committees/expert/20/appl

4. Myat Thiri. A histopathological study of malignant nasopharyngeal tumors at Eye Nose Throat Hospital, Yangon. [MMedSc thesis]. Institute of Medicine 1: Yangon; 2005.

5. Wai Chan JY & Sze Wong ST. The role of plasma Epstein Barr virus DNA in nasopharyn-geal carcinoma. Journal of Molecular Biomarkers and Diagnosis 2013. [Internet]. Available from: http:// dx.doi.org/ 10.4172/ 2155-9929 S5-004

6. Robaina TF, Valladares CP, Tavares DS, et al. Polymerase chain reaction genotyping of Epstein-Barr virus in scraping samples of the tongue lateral border in HIV-1 sero-positive patients. Journal of Microbes & Their Vectors Causing Human Infections 2008; 103(4): 326-31.

7. Ahmed HG, Suliman RSAG, et al. Molecular screening for Epstein Barr virus (EBV) among Sudanese patients with nasopharyngeal carci-noma (NPC). Infected Agent Cancer 2015; 10: 6.

8. Shotelersuk K, Khorprasert C, Sakdikul S, et.al. Epstein Barr virus DNA in serum/plasma as a tumor marker for Nasopharyngeal cancer. Clinical Research Journal 2000; 6(3): 1046-1051.

9. Kreimer AR, Clifford GM, Boyle P & Franceschi S. Human papilloma virus types in head and neck squamous cell carcinomas Worldwide: A system review. Journal of Cancer Epidemiology, Biomarkers and Prevention 2005; 14(2): 467-475.

10. Clifton P & Titcomb Jr. High incidence of nasopharyngeal carcinoma in Asia. Journal of Insurance Medicine 2001; 33: 235-238.

11. Sai San Win. A case control study on epidemiology of nasopharyngeal carcinoma.

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[MMedSc thesis]. Institute of Medicine 1 Yangon; 1997.

12. Chien YC, Chen JY, Liu MY, et al. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. New England Journal of Medicine 2001; 345(26): 1877-1882.

13. Li YH, Shao JY, Zhao MQ, et al. Quantitative analysis of plasma Epstein-Barr virus DNA for monitoring of recurrence and metastasis in nasopharyngeal carcinoma patients after radiotherapy. Ai Zheng 2003; 22: 645-648.

14. Stevenson MM. Nasopharyngeal cancer staging (WHO criteria), Drug and Diseases: Oncology [Internet]. 2016 [updated 2016 Feb; cited 2017 June 17]. Available from: http://www. emedicine. medscape.com

15. Wai Chan JY & Sze Wong ST. The role of plasma Epstein-Barr virus DNA in naso- pharyngeal carcinoma. Journal of Molecular Biomarkers and Diagnosis 2013; 1-5.

16. Niedobitek G. Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma. Journal of Molecular Pathology 2000; 53(5): 248-254.

17. Tamura S, Kunimoto M, Tabata T & Yoshie O. Genotypic analysis of Epstein-Barr virus associated with Nasopharyngeal Carcinoma of Japanese patients. Japanese Journal of Cancer Research 1993; 84(3): 246-249.

18. Cui Y, Wang Y, Liu X, Chao Y, Xing X, Zhao C & Bing Luo CL. Genotypic analysis of Epstein-Barr virus isolates associated with nasopharyngeal carcinoma in northern China. Intervirology 2011; 54: 131-138.

19. Liao LJ & Lai MS. Epstein-Barr virus serology in the detection and screening of nasopharyngeal carcinoma [internet]. 2012 [update 2012 Feb 15]. Available from: https://www. intechopen.com/ books/ carcinogenesisdiagnosis- and- molecular-targeted-treatment-fornasopharyngeal-carcinoma /epstein-barr-virus-serology-in-the-detection-and-screening-of-nasopharyngeal-carcinoma

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Plasma Malondialdehyde Level and Vibration Perception Threshold in Non-exposed Subjects and Lead-exposed Battery Workers

Thura Tun Oo1*, Zaw Linn Thein2, Zarli Thant2 & Ohnmar2

1Physiology Research Division

Department of Medical Research 2University of Medicine 1(Yangon)

Lead is toxic to multiple organ systems and oxidative stress is one of the key mechanisms in lead toxicity. Long-term exposure can result in lead neuropathy, typically motor neuropathy. Peripheral sensory neuropathy caused by lead exposure is still controversial. The aim of this study was to determine and compare plasma malondialdehyde (MDA) level and vibration perception threshold (VPT) between non-exposed subjects and lead-exposed battery workers. This case-control analytical study included 28 non-exposed subjects and 28 lead-exposed battery workers of small-scale battery work-places in Insein and North Okkalapa townships. Plasma malondialdehyde level was determined by colorimetric method. The function of large myelinated peripheral sensory nerve fibers was determined by vibrometer and described as vibration perception threshold (VPT). The mean blood lead level of the lead-exposed battery workers (4.25±3.87 µg/dl) was signi-ficantly higher (p=0.007) than that of the non-exposed subjects (2.14± 1.02 µg/dl). The mean plasma MDA level of lead-exposed battery workers was significantly (p<0.001) higher than that of the non-exposed subjects and their plasma MDA levels were 2.08±0.94 µmol/l and 0.9±0.43 µmol/l, respectively. Both mean values of VPT (hand and foot) in the lead-exposed battery workers were significantly higher than that of non-exposed subjects (p=0.002). There was no significant correlation between plasma MDA level and VPT measurements in the lead-exposed battery workers. Therefore, lead-induced lipid peroxidation and early abnormality in peripheral sensory nerve function could occur in the lead-exposed battery workers even at the low blood lead level but there was no evidence in relationship between lead-induced lipid peroxidation and peripheral sensory nerve impairment in lead-exposed battery workers.

Key words: Lead, Oxidative stress, Plasma malondialdehyde, Vibration perception threshold

INTRODUCTION

Lead is a toxic metal and its widespread use has caused extensive environmental conta-mination and lead exposure is estimated to account for 0.6% of the global burden of disease, with the highest burden in developing regions.1 In developed countries, due to better identification, monitoring, and improvement in industrial safety methods, occupational lead exposure has been signi-ficantly reduced. However, in developing countries, lead toxicity is a persistent health problem for occupational workers and lead-

acid battery manufacturing plants are one of the leading sources of occupational lead poisoning.2

Lead potentially induces oxidative stress and lipid peroxidation in animal studies. Yiin and Lin found that blood lead concentration was positively associated with lipid peroxidation in workers exposed to lead.3 Agency for Toxic Substances and Disease Registry (ATSDR) indicated that _____________________________________________________

*To whom correspondence should be addressed. Tel: + 95-95084347 E-mail: drthuratunoo.neurophysiologist@gmail.com

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lead toxicity can affect every organ system and the nervous system is the most sensitive target of lead exposure.4 Traditionally, the neuro-muscular disorder associated with lead poisoning has been purely motor neuropathy.5 Peripheral sensory neuropathy caused by lead exposure is still contro-versial. The main pathological change in the peripheral nerve fibres consists of segmental demyelination and may be associated with pronounced slowing of nerve conduction velocity.6 Sata, et al. suggested that fast nerve fibres which conduct vibration sense were sensitive to chronic exposure to lead.7

This study was undertaken to determine and compare plasma malondialdehyde (MDA) level and vibration perception threshold (VPT) between non-exposed subjects and lead-exposed battery workers and to find out the relationship between plasma MDA level and VPT of lead-exposed battery workers.

MATERIALS AND METHODS

This is a case-control analytical, one year study from April 2015 to February 2016. In this study, male participants were only recruited to avoid gender difference that was one of the confounding factors in determining the peripheral sensory neuro-pathy. Twenty-eight male workers from small-scale battery workplaces in Insein and North Okkalapa townships and another twenty-eight non-exposed male subjects from University of Medicine 1 (Yangon) were recruited. The age range of the partici-pants was 20-45 years. Written informed consent was obtained. Two milliliter of blood were withdrawn from ante-cubital vein under aseptic con-dition and collected in a test tube containing anticoagulant (EDTA) for plasma MDA assay. After centrifuge, plasma was kept in a plain test tube and plasma MDA was determined within 6 hours from time of sample collection. After the blood samples were collected, the subjects were asked to take sitting rest for 5 minutes and they were explained about the procedure for vibration perception threshold. The sites used in the

measurements were the left and right index finger, the left and right big toes. The Yes/No method was used to find out whether the subject perceived the vibration sense or not. The vibration was increased gradually from the least minimum voltage and the transition from no vibration perception to the onset of perceiving vibration, i.e. when the subject sensed the vibration by the Yes was taken as vibration perception threshold (VPT). The test was repeated three times for each subject and the average was taken for analysis. The vibration perception threshold (VPT) was expressed in volts (V).8

Data were presented as mean±SD. Data analysis was done by using the Statistical Package for Social Sciences (SPSS) soft-ware version 16. This study was done according to Guideline of Board of Studies Physiology, University of Medicine 1, (Yangon).

RESULTS The blood lead levels (BLL) were found to be significantly higher in the lead- exposed group compared to the control group (4.25±3.87 µg/dl vs. 2.14±1.02 µg/dl, p=0.007). Figure 1 shows that plasma MDA level in the lead-exposed group was significantly higher (2.08±0.94 µmol/l) when compared to the control group (0.9±0.43 µmol/l). Vibration perception threshold levels were significantly higher in lead-exposed workers than in controls (Fig. 2 & Fig. 3). Table 1. Relationship between plasma malon-

dialdehyde (MDA) level and vibra-tion perception threshold (VPT) in the lead-exposed battery workers (n=28)

Correlation parameters Pearson’s correlation ‘r’ p

MDA vs. VPT (hand) 0.018 0.928 MDA vs. VPT (foot) 0.094 0.635

The strength of the correlation between plasma MDA level and VPT of the lead-exposed battery workers are shown in Table 1.

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Fig. 1. Comparison of plasma malondialdehyde (MDA) level between the non-exposed subjects and lead-exposed battery workers (Solid line ( ) indicates mean of different groups)

Fig. 2. Comparison of vibration perception threshold (VPT) (hand) between the non-exposed subjects and lead-exposed battery workers (Solid line ( ) indi-cates mean of different groups)

Fig. 3. Comparison of vibration perception threshold (VPT) (foot) between the non-exposed subjects and lead- exposed battery workers (Solid line

( ) indicates mean of different groups)

DISCUSSION

Several lines of evidence have demonstrated that lead induces oxidative damage by inducing the generation of reactive oxygen species and by reducing the antioxidant cell defense systems.9 Free radicals activity has been implicated in the pathogenesis of a variety of human diseases and the analysis of the data showed that oxidative stress was quite clear in lead-exposed workers (Fig. 1), as noticed by increased plasma MDA levels, which is in agreement with other studies.10, 11

According to the study of Nielsen and co-workers, the estimated reference range of plasma MDA level for male is from 0.39 to 1.53 µmol/l.11 In this study, it was observed that plasma MDA level of the non-exposed subjects was within normal range, whereas only one subject of the non-

Non-exposed

subjects (n=28)

Lead-exposed battery workers

(n=28)

23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

p=0.002

4.93±2.62 V 8.36±4.81 V

VPT

foot

(V)

VPT

hand

(V)

15 14

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Non-exposed subjects (n=28)

Lead-exposed battery workers

(n=28)

p=0.002

2.66±0.71 V 4.20±2.29 V

4.5 4

3.5 3

2.5 2

1.5

1

0.5

0

p<0.001

0.9±0.43 µmol/L 2.08±0.94 µmol/L

Non-exposed subjects (n=28)

Lead-exposed battery workers

(n=28)

Pla

sma

MD

A le

vel (

µmol

/l)

74

exposed group had higher MDA level than 1.53 µmol/l. Among the lead-exposed battery workers, 19 out of 28 subjects had plasma MDA levels higher than the upper limit of the estimated reference range of plasma MDA (1.53 µmol/l) and it indicated that higher lead exposure could generate more reactive oxygen species which lead to an increase in lipid peroxidation. Most studies have reported that lead exposure causes oxidative stress by increasing lipid peroxidation.4, 9, 10, 12 Although the findings of the this study are consistent with those results, the parti-cipants had lower blood lead level. Meanwhile, according to an invited critical review by Ahamed and Siddiqui, low level of lead exposure also induces oxidative stress.13 The classic description of lead neuropathy is that of a motor neuropathy, which typically presents as wrist drop.14 More recently, investigators demonstrated that, in the development of lead neuropathy, sensory nerve fibers are affected earlier than motor nerve fibers.14-16 Few studies have focused on peripheral nerve toxicity of lead with VPT changes.15, 16

In this study, as the participants’ age group criterion was 20-45 years, the VPT cut-point based on VPT age-specific reference value for diagnosis of neuropathy was 15V.5 Therefore, VPT ≤15 V indicated no neuro-pathy for our participants’ age group. Although the mean VPT (hand) and mean VPT (foot) in both groups were within normal limit, the lead-exposed battery workers showed significantly higher VPT (hand) and VPT (foot) values than the non-exposed subjects. The most likely explanation for this finding is that sensory nerve conduction was slowed in battery workers and it might be due to myelin loss or axonal degeneration.15 Whatever the underlying nerve pathology, there may seem to be a minimal conduction defect in the lead-exposed workers. The evidence we have obtained indicates that exposure to lead may give rise to peripheral sensory nerve

fiber damage even at the low level of lead exposure.

These findings indicate no correlation between lipid peroxidation parameter and vibration perception threshold. There was limited evidence on effect of lead-induced lipid peroxidation on VPT measurements. Lead-induced neurotoxicity was found to be associated with lead-induced production of ROS which caused an increase in lipid peroxidation and the effects were con-centration dependent.3

Lack of the correlation between the plasma MDA level and VPT in the lead-exposed battery workers might probably be due to either low lead level exposure or smaller sample size. It could be speculated that low lead level exposure might produce inadequate amount of ROS formation to initiate significant effect of lipid peroxi-dation on the large myelinated peripheral sensory fibers in the lead-exposed battery workers. Therefore, a large scale study with high-lead exposed subjects is recommended to confirm the relationship between lipid peroxidation and neuronal impairment. Moreover, this study suggests that lead-exposed battery workers should have their peripheral nerve function examined carefully, including fast sensory nerve fibres. The VPT screening test is non-invasive, painless, and easy to perform in the field, and it has the potential to be developed for occupational physicians to screen for lead-induced peripheral sensory neuropathy at the workplace.

ACKNOWLEDGEMENT We are particularly grateful to Professor Thet Khaing Win, Former Rector, University of Medicine 1, Yangon, for his kind permission and encouragement to conduct this study. We would like to express our gratitude to Dr. Kyaw Zin Thant, Director-General, Chairperson of External Grant Committee, Department of Medical Research, for supporting financially in part for this study. We wish to

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express our deepest thanks to all the participants from battery shops and the staffs from University of Medicine 1, (Yangon) who kindly consented to take part in this study.

REFERENCES

1. World Health Organization. Exposure to lead: A major public health concern. Preventing disease through healthy environment, Geneva, 2010.

2. Khan DA, Qayyum S, Saleem S & Khan FA. Lead-induced oxidative stress adversely affects health of the occupational workers. Toxicology and Industrial Health 2008; 24(9): 611-618.

3. Yiin SJ & Lin TH. Lipid peroxidation in workers exposed to lead. Archives of Environ-mental Health 1994; 49(4): 256-259.

4. Agency for Toxic Substances and Disease Registry. Toxicological profile for lead. US Department of Health and Human Services, Public Health Service, Atlanta, 2010.

5. Maffei L, Premrou V, Roldan P, Copetti M, Pellegrini F, Rossi MC & Vespasiani G. Vibration perception threshold in the screening of sensorimotor distal symmetric polyneuro-pathy: The need of more accurate age-specific reference values. Journal of Diabetes Science and Technology 2014; 8(3): 621-622.

6. Windebank AJ. Textbook of Metal Neuropathy. In: Peripheral neuropathy Dyck PJ, Thomas PK & Griffin JW (eds). 3rd ed, WB Sanders, Philadelphia, 1993: 1549-1570.

7. Sata F, Araki S, Murata K, FujimuraY & Uchida E. Are faster or slower large myelinated nerve fibers more sensitive to chronic lead exposure?: A study of the distribution of conduction veloci-ties. Environmental Research 1993; 62(9): 333-338.

8. Operation User Manual of Biothesiometer. Diabetik Foot Care India Pvt Limited, 2014.

9. Van Dam PS, Van Asbeck BS, Erkelens DW, Man JJM, Gispen WH & Bxavenboe PB. The role of oxidative stress in neuropathy and other diabetic complications. Diabetes/Metabolism Reviews 1995; 11(3): 181-192.

10. Singh Z, Chadha P & Sharma S. Evaluation of oxidative stress and genotoxicity in battery manufacturing workers occupationally exposed to lead. Toxicology International 2013; 20(1): 95-100.

11. Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR & Grandjean P. Plasma malondialdehyde as biomarker for oxidative stress: Reference interval and effects of lifestyle factors. Clinical Chemistry 1997; 43(7): 1209-1214.

12. Patil AJ, Bhagwat VR, Patil JA, Dongre NN, Ambekar JG, Jailkhani R, et al. Effect of lead exposure on the activity of superoxide dismutase and catalase in battery manufacturing workers of Western Maharashtra (India) with reference to heme biosynthesis. International Journal of Environmental Research and Public Health 2006; 3(4): 329-337.

13. Ahamed M & Siddiqui MK. Low level lead exposure and oxidative stress: Current opinions. Clinica Chimica Acta 2007; 383:57-64.

14. Rubens O, Logina I, Kravale I, Eglite M & Donaghy M. Peripheral neuropathy in chronic occupational inorganic lead exposure: A clinical and electrophysiological study. Journal of Neurology, Neurosurgery and Psychiatry 2001; 71(2): 200-204.

15. Kovala T, Matikainen E, Mannelin T, Erkkila J, Riihimaki V, Hanninen H & Aitio A. Effects of low level exposure to lead on neurophysio-logical functions among lead battery workers. Occupational and Environmental Medicine 1997; 54(7): 487-493.

16. Chuang HY, Schwartz J, Tsai SY, Lee MLT, Wang JD & Hu H. Vibration perception thresholds in workers with long term exposure to lead. Occupional Environmental Medicine 2000; 57(9): 588-594.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

Are They Disclosed?: Situation of HIV Status Disclosure among Adolescents in Four Townships of Myanmar

Kyaw Min Htut*, Myo Myo Mon, Lwin Lwin Ni, Aung Soe Min & Ni Ni Htay Aung

Department of Medical Research

Disclosing HIV status to vertically HIV infected adolescents is crucial for preventing further transmission of HIV, maintaining drug adherence, and preventing possible unsafe reproductive behavior. Little is known about the situation of HIV status disclosure among these adolescents. A cross-sectional, exploratory mixed method design was conducted in 2015 with the objective of identifying disclosure related information among HIV infected adolescents. Total of 66 adolescents were interviewed by using a structured questionnaire. Focus group discussions (FGD) and in-depth interviews (IDIs) were conducted with the guardians, providers and focal persons. Age of the adolescents ranged from 10-16 years (12.5±2.0). About 44% of adolescents were double orphans and 82% were currently attending school. Nearly 82% knew their HIV status in which over 42% were disclosed by health staffs. During FGDs, about half of the guardians stated that they have not disclosed HIV status to their children properly. They just let them know that they had an illness which needed daily medication for their survival. Less than half of the children were discussed about HIV with their guardians including transmission of HIV (16.7%), HIV virus (40.9%) and treatment (19.7%). Regarding reproductive health (RH), only few adolescents received information about sexually transmitted diseases (18.5%), puberty changes (7.7%), pregnancy (6.2%) and reproductive organs (4.6%). Most guardians revealed that they rarely discussed about RH with their children although they know that it is important for their future reproductive life. Proper and comprehensive disclosure counselling is needed for vertically HIV infected adolescents.

Key words: Adolescents, HIV, Status disclosure, Counselling, Myanmar

INTRODUCTION

Globally, an estimated number of people currently living with HIV are about 35.3 million. Additionally, there were 1.6 million deaths of HIV around the world in 2012. One of the adverse health consequences of HIV on children is mother-to-child transmission of the virus.1

Specifically, 1,000 new cases of HIV were detected every day in the children under 15 years of age predominantly due to vertical transmission.2 In low- and middle-income countries, about 260,000 new infections among children occurred in 2012.3 According to the estimation of UNAIDS in 2012, there were 2.1 million adolescents aged between 10 to 19 years

living with HIV all over the world.1 According to the WHO guideline, all people living with HIV should be on ART regardless of clinical stage and CD4 count.4

Along with the increasing coverage of ART, children with vertically acquired HIV infection are surviving and reaching adolescence.1 Physical, mental, psycho-logical and social changes occurring among adolescents (10-19 years) that lead to physical and sexual maturity. These situations make many opportunities for development, but at the same time, also pose risks to their health.5 ____________________________________ *To whom correspondence should be addressed. Tel: +95-9595143248 E-mail: 84kyawminhtut@gmail.com

77

Besides usual developmental changes, adolescents living with HIV are experiencing the social, economic, mental and develop-mental consequences of life-long HIV.6

Among these consequences, issues around disclosure are challenging for both children and their guardians.6-8 Adolescents living with HIV in Asia Pacific region learn about their HIV status for the first time at different ages and from a range of sources. Disclosure should start as early as possible since it can empower children to take care of their health. However, negative psycho-logical effects would be resulted if they learned their HIV positive status during middle adolescence.6

In Myanmar, National AIDS Program (NAP) estimated that adult HIV prevalence is 0.59% in 2014.9 According to the results of sentinel surveillance in 2011, percent of pregnant women who are HIV infected was 0.9%. Moreover, percent of infants born to HIV infected mothers who are infected was 13%.10 Disclosing HIV status to vertically HIV infected adolescents is crucial for preventing further transmission of HIV, maintaining drug adherence, and preventing possible unsafe reproductive behavior. However, little is known about the situation of HIV status disclosure among these adolescents in Myanmar. Therefore, the aim of the study was to assess HIV-disclosure status amongst adolescents living with HIV in Myanmar.

MATERIALS AND METHODS

Study design and setting

A cross-sectional, exploratory mixed method study was conducted at four townships of Myanmar in 2015. These four townships were situated in central part of Myanmar and all had similar socio-demographic background.

Study population Adolescents aged between 10 to 16 years who were vertically HIV infected from their parents were included in the study. Vertical

transmission was confirmed since all these children got HIV diagnosis since they were very young. Confirmation about the transmission of HIV was also done with their parents/guardians. Sample size was calculated based on the assumption of proportion of adolescents who have received comprehensive HIV status disclosure as 20% (p=0.2), precision of 0.1(d=0.1) and at 95% confidence level (z=1.96) resulting in a sample size of 61, by using the formula “n=z2 pq/d2”.

Data collection

A structured- questionnaire was drafted after reviewing the literature and it was pre-tested in a non-study township in Yangon. A pre-tested and modified structured questionnaire and guidelines for focus group discussions (FGDs) were used by trained interviewers and moderators, respectively. Adolescents were recruited through a network of people living with HIV (PLHIV) after coordination meeting at each township.

Firstly, a list of eligible adolescents was prepared with the help of adult PLHIV which was followed by random sampling. Fifteen to 17 vertically HIV infected adoles-cents were recruited from each township to get the required sample size. Face-to-face interviews were carried out with the adolescents by trained interviewers and four FGDs were done with their guardians by the investigators. Guardians consisted of fathers, mothers, grandparents, aunts/uncles and elder siblings. Six in-depth interview (IDIs) were conducted with service providers and focal persons from PLHIV network.

Variables

Variables were age, sex, parental status (both parents alive/ paternal orphan/ maternal orphan/ double orphan), schooling (never attended school/ out of school/ in school), disclosure of HIV (disclosed/ not disclosed), age at knowing HIV status, receiving HIV information (yes/ no/ don’t know), communication between parents and

78

adolescents about HIV transmission, virus and treatment (yes/ no), communication between parents and adolescents about RH information such as STI, puberty, pregnancy and reproductive organs (yes/ no).

Data management and analysis

Data entry and analysis were done by EpiData version 3.1 and SPSS version 16.0 after checking and coding the questionnaire. Range and consistency checks were done. Descriptive statistics were shown by mean/ median as appropriate for continuous variables and frequency distribution for categorical variables. Transcripts were prepared for each FGD session which was followed by manual coding and thematic analysis. Finally, quantitative and qualitative information were triangulated. Ethical consideration

Informed consent was taken from the guardians and informed assent was taken from the adolescents after thorough explanation about the study. Confidentiality and anonymity were ensured. Privacy was strictly ensured in such a way that all the interviews were taken place at the private place as agree by the interviewees. All the ethical guideline required for conducting the research with the children were strictly followed. The proposal was approved by the Ethics Review Committee of Department of Medical Research.

RESULTS

Background characteristics Total of 66 adolescents were included in the study. As shown in Table 1, proportion of male exceeded female and their age ranged from 10-16 years with the mean age of 12.5±2.0. About 44% of adolescents were double orphans (both parents passed away) whereas nearly one-fourth (24.2%) had both parents alive. Nearly 82% were currently attending school and 20.4 % were at the level of grade 4. Over 16% of the children were dropped out from the school and out of

school duration ranged from 1-7 years (mean: 3.3±2.4).

Table 1. Background characteristics of the adolescents infected with HIV from four selected townships, 2015 (n=66)

Characteristics Number Percent Sex Male 37 56.1 Female 29 43.9 Age Range 10-16 Mean±SD 12.48±2.04

Parental condition Both parents alive 16 24.2 Paternal orphan 15 22.7 Maternal orphan 6 9.1 Double orphan

(both parents passed away) 29 43.9

Schooling Never attended school 1 1.5 Out of school 11 16.7 In school 54 81.8 Table 2. Information related to HIV status dis-

closure among adolescents Characteristics Number Percent Disclose about HIV (n=66)

Disclosed 54 81.8 Not disclosed 12 18.2

Age at knowing HIV status (n=54) Range 5-16 Mean±SD 10.15±2.77

Received HIV (n=54) Yes 44 81.5 No 8 14.8 Don’t know 2 3.7

Person disclosed (n=54) Either parent 19 35.2 Family member 8 14.8 Health staff 24 44.4 Others (Neighbours, Friends) 3 5.6

Reaction on knowing HIV status* (n=54) Shocking 4 7.4 Crying 4 7.4 No expression 48 88.9

Parent/guardian communicate adolescent about HIV (n=66) Yes 37 56.1 No 29 43.9

*Multiple response

Of all adolescents, 81.8% (54/66) knew their HIV status in which over 44.4% (24/54) of the children were disclosed by health staffs. Their mean age at the time of knowing HIV status was 10.2±2.8 years. About two-third of them reported that they had ever received counselling whereas 33% had not received any type of counselling. Nearly 90% of adolescents did not express their emotional responses when they were disclosed (Table 2).

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Status of disclosure, counselling and communication with guardians among adolescents according to their parental condition was mentioned in Table 3. Disclosure status and receiving HIV counselling were not statistically different among children with both parents alive and single/ double orphans. Significantly higher proportion of adolescents having both parents received communication about HIV in comparing to orphans (81.2% vs. 66.7% vs. 34.5%, p<0.01). Table 3. Status of disclosure, counselling and communication with guardians among adolescents according to their parental condition

Characteristic

Parental condition Both

parents alive n(%)

Paternal/ Maternal

orphan n(%) Double orphan n(%)

Disclose about HIV (n=66) Disclosed 15(93.8) 15(71.4) 24(82.8) Not disclosed 1(6.2) 6(28.6) 5(17.2)

Receive HIV (n=54) Yes 2(13.3) 12(80.0) 21(87.5) No 2(13.3) 3(20.0) 3(12.5) Don’t know - - -

Parent/guardian communicate adolescent about HIV (n=66) Yes 13(81.2) 14(66.7) 10(34.5) No 3(18.8) 7(33.3) 19(65.5)

Table 4. Responses of the participants during FGD

During FGDs, about half of the guardians admitted that they did not disclose HIV status to their children properly. They just let them know that they had an illness which needed daily medication for their survival. Most of the adolescents gradually recognized their HIV status without proper counselling before disclosure. Some of the responses to the focus group discussions are shown in Table 4.

Fig. 1. Information on discussion about HIV and reproductive health information with the adolescents as reported by the guardians

Figure 1 depicts the information on discussion about HIV and reproductive health information with the guardians. Less than half of the adolescents discussed about HIV such as transmission of HIV (16.7%), HIV virus (40.9%) and treatment of HIV (19.7%). Very few adolescents had ever received RH information necessary for them. In particular, 18.5%, 7.7%, 6.2%, and 4.6% of adolescents received discussion about sexually transmitted diseases, puberty changes, pregnancy and reproductive organs, respectively from their parents/ guardians.

DISCUSSION

Current study aimed to explore the situation of HIV status disclosure among vertically infected adolescents. Of all adolescents, majority knew their HIV status and most of them awared their status gradually without receiving proper disclosure counselling.

A = Transmission D = Sexually transmitted disease B = Virus E = Puberty C= Treatment F = Pregnancy G= Reproductive organs

Responses during FGD Opinion on disclosure

by grandparent “… She doesn’t understand why she has to take medicine every day. We don’t try to explain her. Just let her know that she has to take those pills if she doesn’t want to die like her parents” (55 years, grandmother)

Opinion on disclosure by service provider

“… It’s difficult to disclose the children if they have little knowledge on HIV. So, we need to raise their awareness first …” (Public service provider)

Opinion on disclosure by adopted parent

“…We have to tell him since he has to go to clinic and don’t want to miss the school. We told him “you have to take the drugs till you die and it would be good for his health” (53 years, adopted father)

Opinion on disclosure by parent

“… We just tell my child as he had to take medicine for TB … till now, he doesn’t know that he had HIV” (48 years, mother)

Opinion on disclosure by adult PLHA

“Most parents don’t allow disclosing directly to their children. We’ve to try hard to counsel them only when they are transferred to adult side …” (35 years, PLHA Network)

Per

cent

age

of re

spon

dent

s

16.7

40.9

19.7 18.4

7.7 6.2 4.6

HIV RHA B C D E F G

80

Mean age when they knew their status was around 10 years.

Previous studies have documented the disclosure status of children and adolescents.6-7, 11 In Nigeria, the mean age at disclosure was just over 10 years and about one third of the children were informed their status.7 Another study in rural Uganda also recorded that most children were informed their status between the age of 5 to 9 with the mean age at 7 years.6 Similar age at disclosure was detected in present study, however, higher proportion of adolescents has known their status in comparing to other studies. In a study in Tanzania, among 4-17 years perinatally HIV infected children and adolescent, about 32% of care givers reported that they have disclosed the children’s HIV sero-positive status.11 The discrepancy might be due to the difference in age group of this study population in which only adolescents over 10 years were included in this study whereas other studies included children over 6 years of age.

Adolescents living with HIV in Asia and the Pacific region gain knowledge of their status for the first time at different ages and sources. How and when adolescents receive information about their status plays an important role in how they understand what living with HIV means for their health and well-being.1 Surprisingly, over 90% of the children from the present study did not express anything at the first time they knew about their HIV status. Children’s age, maturity and background knowledge about HIV may contribute their responses towards knowing their status. Likewise, in a study among adolescents aged 9 to 16 years in United States, 70% of them had disclosed and did not shown any psychological consequence after the disclosure.12

Communication between caregivers and service providers is essential for smooth and successful disclosure of HIV status to the children. In addition, getting child consent is crucial in disclosing HIV status to children and adolescents according to the United

Nations Convention on the Rights of the Child (CRC). Adolescents’ right to privacy is protected in Article 16 of the CRC. It was mentioned that information on the HIV status of children may not be disclosed to third parties, including parents, without the child’s consent.10

One of the important issues surrounding disclosure is counselling where improper post-test counselling and support may worsen their emotional status.13 One third of the adolescents from current study had never received any type of counselling. In a study in Nigeria, 11% of HIV disclosure were performed by health staff including counsellor while much higher proportion of adolescents (42%) in the current study were informed by health staff.6 On the other hand, about one-third of disclosure was done by the caregivers in Tanzania.11 Adherence to take medicine for HIV is one of the important reasons for disclosure in the current study as reported by the caregivers and similar reason was found in other study.6

Regarding reproductive health information, very few adolescents were ever discussed with their guardians in this study. In contrast, previous study in Myanmar documented that 87% of the parents communicated on RH issues at least one time with their children.14 The difference might be due to the fact that many adolescents in present study were double orphans who are staying in extended family members and they may rarely get chance to discuss about RH with their guardians.

Previous study has highlighted the attitudes and behaviours of caregivers regarding disclosure where 42% of caregivers told their children as they were suffering from a disease other than HIV, with malaria being the most common disease.15 Similarly, some children from the present study were told that they had to take medicine for TB.

There were certain limitations in current study. Firstly, findings were solely based on

81

self-reported answers from children and their caregivers. Verification could not be done whether these reported behaviors were really practiced by the participants. Another limitation was about the disclosure process. Although 82% of the adolescents were disclosed, the process of disclosure could not be clarified in this study.

Conclusion

Comprehensive disclosure counselling is very limited and many guardians do not want to disclose their children’s HIV status in this study. About one-third of the adolescents have not received any type of counselling. Additionally, parents/ guardians of over half of them have never discussed with their children about HIV related information. More importantly, many adolescents have never received RH information though it is critical for them to have RH knowledge during the adolescent period. Recommendation

Strengthening of proper and comprehensive disclosure counselling is suggested for vertically HIV infected adolescents. RH information should be provided to all infected adolescents since very few of them had ever received RH information. Awareness raising activities should be carried out for guardians on advantages of disclosure counselling.

ACKNOWLEDGEMENT

The authors would like to express the great thanks to our Director-General and Ethics Review Committee, Department of Medical Research for approval. The authors are also grateful to authorized persons from National AIDS Program and respondents for participation in this study.

REFERENCES

1. UNAIDS. Report on the AIDS epidemic [Internet]. 2013 [cited 2015 June 23]. Available from: http://www.unaids.org/sites/default/ files/

en / media/ unaids/ content-assets / documents/ epidemiology/ 2013/ gr2013/ UNAIDS _ Global_ Report_2013_en.pdf

2. World Health Organization, Guidance for HIV testing and counselling and care for adolescents living with HIV: Recommendations for a public health approach and considerations for policy-makers and managers. WHO Geneva, 2013.

3. Joint United Nations Programme on HIV/AIDS (UNAIDS), Global Report: UNAIDS report on the global AIDS epidemic 2013, UNAIDS, Geneva, 2013.

4. World Health Organization, Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection recommendations for a public health approach, 2nd ed, WHO Geneva, 2016.

5. World Health Organization. Adolescent development. [Internet]. [cited 2015 June 15]. Available from: http:// www. who. int/ maternal_child adolescent/ topics/ adoles-cence/development/en

6. Chandler C & Ngoksin A. Lost in transitions: Current issues faced by adolescents living with HIV in Asia Pacific, Bangkok: UNICEF 2013.

7. Lorenz R, Grant E, Muyindike W, Maling S, Card C, Henry C, et al. Caregiver’s attitudes towards HIV testing and disclosure of HIV status to at risk children in rural Uganda. PLoS ONE 11(2): e0148950.

8. Odiachi A, Abegunde D. Prevalence and predictor of paediatric disclosure among HIV-infected Nigerian children on treatment. AIDS Care 2016; 28(8): 1046-51.

9. National AIDS Program, Ministry of Health and Sports. National Strategic Plan on HIV and AIDS, Myanmar, 2016-2020.

10. Myo Myo Mon, Saw Saw, Yin Thet Nu Oo, Khin Ohnmar San, Wai Wai Myint, San San Aye, et al. Threat of HIV/AIDS in children: Social, education and health consequences among HIV orphans and vulnerable children in Myanmar. WHO South-East Asia Journal Public Health 2013; 2(1): 41.

11. Nzota MS, Matovu JKB, Draper HR, Kisa R & Kiwanuka SN. Determinants and processes of HIV status disclosure to HIV-infected children aged 4 to 17 years receiving HIV care services at Baylor College of Medicine Children’s Foundation Tanzania, Centre of Excellence (COE) in Mbeya: A cross-sectional study. BMC Paediatrics 2015; 15:18

12. Santamaria EK, Dolezal C, Marhefka SL, Hoffman S, Ahmed Y, Elkington K, et al. Psychosocial implications of HIV serostatus disclosure to youth with perinatally acquired

82

HIV. AIDS Patient Care and STDS 2011; 25(4): 257-264.

13. UNICEF, Mapping of social protection measures for children affected by HIV and AIDS in Asia and the Pacific: A report by the Economist Intelligence Unit of the Economist, UNICEF East Asia and Pacific Regional Office (EAPRO), 2012.

14. The Global Fund to Fight AIDS, Tuberculosis and Malaria. Orphans and vulnerable children, 2011.

15. Ko Ko Zaw, Yin Thet Nu Oo, Kyu Kyu Than & Thae Maung Maung. Reproductive health communication between parents and adolescents in North Okkalapa Township. Myanmar Health Sciences Research Journal 2009; 21(1): 50-55.

16. National AIDS Programme, Global AIDS Response Progress Report Myanmar 2014 [Internet]. 2014 [cited 2015 June 15]. Available from: http:// www. MMR_narrative_report_ 2014.pdf

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

SHORT REPORT

Applications of Alumina Nanoparticles from Coal Fly Ash

Myat Myat Thaw & Khine Thin Thin Aye

Department of Chemistry, University of Yangon Key words: Coal fly ash, Power plant, Sensor

In this research, coal fly ash samples collected from Tigypt Power Plant located in Pinlaung Township, Taunggyi District in Shan State, Myanmar were studied. The power plant is the only operating coal-fired power station in Myanmar. Power plant is a complex of structures, machinery, and associated equipment for generating electric energy from another source of energy and is also called generating station, power station. Alumina nanoparticles were obtained at 1000°C for 3 hours. Prepared alumina nanoparticles from coal fly ash were used to treat wastewater samples collected from textile workshop in Inle Lake, Southern Shan State. Fly ashes (FAs) are the major combustion residues (normally 60-88%) produced during the burning of pulverized coal in thermo-electric power stations (TPSs) and collected by the cleaning equipments of fuel emis-sions (commonly electrostatic precipi-tators). Ash which does not rise is termed bottom ash.1 Fly ash is generally captured by electrostatic precipitators. There is wide range of variation in the principal consti-tuents - silica (25-60%), alumina (10-30%) and ferric oxide (5-25%). When the sum of these three principal constituents is 70% or more and reactive calcium oxide is less than 10%, technically the fly ash is considered as siliceous fly ash or class F fly ash. If the sum of these three constituent is equal to or more than 50% and reactive calcium oxide is not less than 10%, fly ash will be considered as calcareous, also called as class C fly ash.2 Four classes of nanoscale materials, being evaluated as functional

materials for water purification include (i) dendrimers (ii) metal-containing nano-particles (iii) zeolites, and (iv) carbonaceous nanomaterials. These have a broad range of physicochemical properties that make them particular attractive as separation and reactive media for water purification.3 The aim was to present the application of alumina nanoparticles from coal fly ash. Objectives were to prepare alumina nano-particles from coal fly ash by using alkaline leaching sintering method, to characterize alumina nanoparticles by using XRD (X-ray Diffraction technique), FTIR (Fourier Transform Infrared Spectrometer), SEM (Scanning Electron Microscope) and FE SEM techniques (Field Emission Scanning Electron Microscope) and to carry out some applications (adsorption properties, gas sensor applications and antimicrobial activity) of prepared alumina nanoparticles.

Fifty milliliters of waste water samples were taken out and centrifuged for 15 minutes and alumina nanoparticles from coal fly ash were added and stirred for 1 hour at room temperature. The concentrations of Cu, Zn and Cd in supernatant solutions were measured by atomic absorption spectrophotometer (AAS). Alumina nano-particles 0.5 g were mixed with 5% poly-vinyl acetate binder well and pasted on the interdigitated electrodes (IDEs). Alumina coated electrodes were dried at 150°C for 2 hours. ___________________________________ *To whom correspondence should be addressed. Tel: +95-95189779 E-mail: dr.myat.myat.thaw@gmail.com

84

Copper, zinc and cadmium contents in waste water samples were found to be 0.12, 0.15 and 0.17, ppm, respectively, before treatment. After treatment with alumina nanoparticles for 2 hours, copper, zinc and cadmium contents were found to be 0.060, 0.009 and 0.0054 ppm, respectively. It was found that the removal percentages of copper, zinc and cadmium were 95.50%, 94% and 67.70%, respectively. Among these three metal ions, the highest removal percent was found in copper.

In this work, effect of pH and time of coal fly ash on metal removal were carried out. Prepared alumina nanoparticles were used for the removal of Cu (II), Zn (II) and Cd (II) ions from aqueous solutions at different pH values and temperatures. By the increase of pH the removal efficiency of alumina nanoparticles from coal fly ash increased for elimination of Zn (II) ion. The removal percentages of Zn (II) at pH 3 were found less effective than at pH 5 and 7. Among the three cations, the removal percentage of Cu (II) ion was found to be the highest at pH 7. It was found that the maximum adsorption of Cu (II) were observed, and followed by Zn (II)) and Cd (II) metal cations in aqueous solution are found in their hydrated forms. It was observed that the smaller hydrated diameter of Cu (II), the more adsorption properties pronounced and better than those of Zn (II) and Cd (II). Therefore, it was found that the low adsorption of Zn (II) and Cd (II) ions was observed compared with adsorption of Cu (II).

Gas sensor applications of alumina nano-particles were investigated by using ammonia gas to observe the response of gas in sensing properties. The gas sensor testing system was designed to test sensor by a static environment method and fixed volume (1.18 L). The changes of resistance were measured by using multimeter of type U 1232 A True RMS with Keysight Handheld Meter logger Software. In addition, it was

found that Al2O3 sensor can detect the much smaller concentration of ammonia gas. According to the results, Al2O3 sensor is more suitable for ammonia gas. Gas sensing properties of Al2O3 sensor were tested with ammonia gas by using 8.47ppm, 16.94 ppm and 32.45 ppm, respectively. According to experiment, the resistance of alumina sensor decreased when contacted with air ammonia gas mixture. The response and recovery times were about 220 s, 130 s for 8.47 ppm, 455 s and 200 s for 16.94 ppm and 560s and 630s for 32.45 ppm, respectively. Alumina nanoparticles showed remarkable anti-microbial activity against both gram-positive and gram-negative bacteria.5 The inhibition zones of alumina nanoparticles against tested microbes show Bacillus subtilis (40 mm), Staphylococcus aureus (35 mm), Pseudomonas aeruginosa (30mm), B. pumilius (40 mm), Candida albicans (50 mm) and E. coli (33 mm), respectively. It was observed that the maximum anti-microbial activity of alumina nanoparticles was found on Candida albicans and its maximum inhibition zone diameter was shown to be 50 mm.

REFERENCES

1. Goswami AK, Kulkarni SJ, Dharmadhikari SK,

& Patil PE. Fly Ash as Low Cost Adsorbent to Remove Dyes. Journal of Scientific Research and Management 2013; 2(5): 842-845.

2. Kumar V & Jha CN. Fly Ash for High Value Added Applications. Jamshedpur 1991; 6: 23-31.

3. Veeradate P, Voranuch T, Piyapang A & Pichet L. Preparation and Characterization of Alumina Nanoparticles in Deionized Water Using Laser Ablation Technique. Journal of Nanomaterial 2012; 20: 12-17.

4. Hongbin T, Jianhua Z & Haiwa B. Continuous Alumina Gel Fibers by Sol-Gel Method Using Glycolic Acid, Aluminum Nitrate and Polyvinyl pyrolidone. Ceramic-Silikaly 2011; 3: 276-279.

5. Amitava M, Mohamned DI, Prathna, TC, & Chandrasekaran, N. Antimicrobial activity of alumina oxide nanoparticles for potential clinical applications, School of Bio Science & Technology, Centre for Nanobiotechnology 2011: 245-251.

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Myanmar Health Sciences Research Journal, Vol. 30, No. 1, 2018

SHORT REPORT

Iodine Deficiency in Pregnant Women Living in the Coastal Area of Mon State

Theingi Thwin*, Moh Moh Hlaing, Mya Ohnmar, Sandar Tun, Thidar Khine, Wah Wah Win, Su Su Hlaing & Hla Phyo Lin

Department of Medical Research

Key words: Iodine deficiency, Pregnant women, Coastal area

Iodine requirements sharply increase during pregnancy because of the transfer of iodine and thyroid hormone to the fetus, an increased needs for maternal thyroid hormone and maternal renal iodine clearance.1 Thus, recommended iodine intakes during pregnancy are 250 g/day compared with 150 g/day for non-pregnant women and the corresponding median urinary iodine concentration (UI) indicating optimal iodine nutrition increases from 100-199 g/l in non-pregnant women to 150-250 g/l during pregnancy.2

Adequate iodine nutrition in pregnant women residing in coastal area where solar salt is available, is crucially important to prevent miscarriages, still birth, low birth weight babies as well as inevitable cretinism. In Mon State, there are coastal fishing and related industries such as production of dried fish, fish sauce and solar salt (unfortified salt).

Measurements of urinary iodine levels are often used for assessment of iodine deficiency disorders in populations because of accessibility of urine samples in community-based survey. Therefore, urinary iodine concentration can be used as an indicator for sufficiency of iodine nutrition status of pregnant women living in certain pocket areas like Panga Village and Kalokepi Village in Thanbyuzayat Township, Mon State.

A cross-sectional, descriptive study was carried out among the 144 pregnant women

living in above mentioned villages. All pregnant women who were in generally good health, without visible goitre enlarge-ment or other thyroid disorders or history of iodine supplements were included in the study. In the pilot survey, pie sorting was conducted to identify commonly consumed food among pregnant women. Based on these findings, food frequency question-naires for consumption pattern of iodine rich food were constructed. The studied pregnant women were interviewed by using the structured questionnaires including back-ground, parity, consumption patterns of salts and frequency of iodine rich food. Three each of iodized salt and non-iodized salt samples from local markets were collected for determination of iodine content by the iodometric titration method.3 Casual urine samples were collected for urinary iodine determination based on Sandell-Kolthoff reaction4 indicating changes in colour due to the presence of iodine. In this study, 16.7% of study population used non-iodized salt for consumption. Majority of pregnant women (74.3%) did not consume seaweed (wet or dried) although they are rich in iodine content. Among sea foods, marine fish was the most common food which was consumed by 36.1% of study women. Mean iodine content of iodized salt and non-iodized salt were 20.6±9.2 ppm and 5.1±1.2 ppm, respectively. ____________________________________ *To whom correspondence should be addressed. Tel: +95-95021540, 95136703 E-mail: ttgthwin16@gmail.com

86

Although iodine concentration in salt at the point of production should be within the range of 20-40 ppm,5 the lower limit could be considered these conditions such as salt with poor quality, long termed exposure to moisture, light, heat and contaminants. Proper packing, transport and storage should be carried out to maintain the desirable iodine content in salt as it may loss up to 50%.

It was found that the median urinary iodine concentration was 105 µg/l (5 to 295 µg/l). Out of 144 pregnant women, 65(45.1%), 41(28.5%) and 38(26.4%) pregnant women had urinary iodine level with less than 100 µg/l, 100-149 µg/l and 150 µg/l and above, respectively. It showed insufficient iodine nutrition in pregnant women. Table 1. Urinary iodine concentration of pregnant

women by types of salt consumed

Types of salt consumed

Urinary iodine concentration p value Median

(µg/l) <150 µg/l*

No.(%) >=150 µg/l

No.(%) Iodized salt Non-iodized salt

105.5 102.5

86(71.7) 20(83.3)

34(28.3) 4(16.7)

0.336

* 150 µg/l was used as cut-off point because during pregnancy, median urine iodine concentration between 150-249 µg/l defines a population which has no iodine deficiency.2

Table 1 shows the urinary iodine concen-trations in pregnant women who consumed iodized salt and non-iodized salt were not significantly different where 150 µg/l was used as a cut-off. Only 16.7% of pregnant women consumed non-iodized salt and 19.4% used it for making dried fish. Majorities of food producers were not willing to use iodized salt in processing due to the appearance of unfavourable colours in their products. Majority of pregnant women (74.3%) did not consume seaweed (wet or dried) although they are rich in iodine content. Therefore, the contributing factors for insufficiency of iodine nutrition in these pregnant women were low iodine content in iodized salt, inadequate consumption of iodine rich food and easily availability of non-iodized salt in the villages. Iodine contents of sea water fish and prawn were

three to seven times higher than those of fresh water fish.6 The sources of iodine from food are iodized salt and sea food in Myanmar.

Therefore, adequate consumption of seafood which are abundant in these areas, should be encouraged during pregnancy. Health education should be given to pregnant women to take iodine rich food and iodized salt for attaining sufficient iodine nutrition which is essential for growth and develop-ment of baby and themselves. Iodized salt should be used in production of dried fish and fish sauce. Universal salt iodization should be covered up to small scale factories especially in coastal area where solar salt are abundant.

Sustainable monitoring of iodine content in salt at retailed shops/ household levels, quality control procedures of salt iodization and urinary iodine concentration among pregnant women should be enhanced. Furthermore, information on standard operating procedures of salt iodization and the role of iodine in health should be disseminated to iodized salt manufacturers.

REFERENCES

1. Zimmermann MB, Jooste PL & Pandav C. Iodine

deficiency disorders. Lancet 2008; 372: 1251-2. 2. WHO, ICCIDD, UNICEF. Assessment of the

iodine deficiency disorders and monitoring their elimination, 3rd ed. Geneva: WHO, 2007.

3. Hetzel BS, Dunn JT & Stanbury JB. The prevention and control of iodine deficiency disorders. Amsterdam: Elsevier; 1987.

4. ICCIDD/WHO/UNICEF. A practical guide to the correction of Iodine Deficiency. In: Technical mamual no. 3: International council for control of iodine deficiency disorders, Dunn JT and Vander HF, Netherlands, 1990; 1-62.

5. WHO/ICCIDD/UNICEF. Recommended iodine levels in salt and guidelines for monitoring their adequacy and effectiveness [Internet]. [cited 2017 December 14]. Available from: http://www. who. int/nutrition/publications/micronutrients/iodine-deficiency

6. Thiri Myint Oo. Comparison of iodine content in fresh and sea water fish and crustaceans. [MSc, thesis]. University of Yangon; 1996.

Websites of the Department of Medical Research

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www.myanmarhsrj.com (Myanmar Health Sciences Research Journal Website)

www.mhrr-mohs.com (Myanmar Health Research Registry Website)

"Achieving a Healthier Nation through

Application of Research Findings "

DEPARTMENT OF MEDICAL RESEARCH

No.5, ZIWAKA ROAD, DAGON TOWNSHIP, YANGON 111-91, MYANMAR

Tel: 951-375447 | 951-375457, 951-375459 Fax: 951-2515L4E-mail I M HSRladm in@ myanmarhsrj.com Website: www.rnyanmarhsrj.com

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