Comparison between Polymerase Chain Reaction, Mayer‘s Haematoxylin and Diff- Quick stains in the diagnosis of Madurella mycetomatis in Sinnar State, Sudan (2014-2015) Nuha Yousif Ali Yousif (B.Sc in Haematology and Histopathology, University of Shandi 2000) A Dissertation Submitted to University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science in Histopathology and Cytology Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences University of Gezira August 2015 1 Comparison between Polymerase Chain Reaction, Mayer‘s Haematoxylin and Diff- Quick stains in the diagnosis of Madurella mycetomatis in Sinnar State, Sudan (2014-2015) Nuha Yousif Ali Yousif Supervision Committee: Name Position Signature Dr. Rania Mahgoub Sied Ahmed Supervisor …………….……………. Dr. Nagwa Adam Mahmoud Co Supervisor ………………..………… Date 19: /8/ 2015 2 Comparison between Polymerase Chain Reaction, Mayer‘s Haematoxylin and Diff- Quick stains in the diagnosis of Madurella mycetomatis in Sinnar State, Sudan (2014-2015) Nuha Yousif Ali Yousif Examination Committee: Name Position Signature Dr. Rania Mahgoub Sied Ahmed Chairman …………….……………. Dr. Ali Sied Ahmed Mohammed Ali External examiner ………………..………… Dr. Bakri Yousif Mohamed Nour Internal examiner …………………………. Date: 19 /8/ 2015 3 Authorization I authorized that my dissertation “Comparison Between Polymerase Chain Reaction, Mayer’s Haematoxylin and Diff- Quick Stains in the Diagnosis of Madurella Mycetomatis in Sinnar State-Sudan” submitted by me, under the supervision of Dr. Rania Mahgoub Siad Ahmed and Dr. Nagwa Adam Mahmoud for the partial fulfillment for the award of Master degree in Medical Laboratory Sciences in Histopathology and Cytology. University of Gezira Faculty of Medical Laboratory Sciences Department of Histopathology and Cytology; Wad-Medani, Sudan. Name and Signature of Candidate: Nuha Yousif Ali Yousif ……………………………. 4 Dedication To my parents; thanks for support; Thanks for giving me a chance to prove and improve myself through all my walks of life. To my brother; thanks for believing me, for allowing me to further studies. To my daughter AMNA and my son AHMED. To my friends and all people support me. 5 Acknowledgements I should like to thanks and gratitude to all of helped me in completing this dissertation and provided me with information and special thanks and appreciation to my supervisors Dr Rania Mahgoub, University of Gezira, Faculty of Medical Laboratory Sciences, Department of Histopathology. Also, I’d like to thank Dr Nagwa Adam. University of Khartoum , Faculty of Medical Laboratory Sciences, department of microbiology and head manager of molecular biology lab of Mycetoma Research Center. who followed and supported me in all stages of this dissertation. Also, thanks to Uataz Mohammed Ibrahim. Sinnar research Center of malaria And all people supported me in this dissertation. Special thanks to Ustaz Eglal ELamin. Ustaz. Mubarak Elshafia who helped me in conducting statistical analysis of the research. 6 Comparison between Polymerase Chain Reaction, Mayer‘s Haematoxylin and Diff- Quick stains in the diagnosis of Madurella mycetomatis in Sinnar State, Sudan (2014-2015) Nuha Yousif Ali Yousif Abstract Mycetoma is a chronic, specific, granulomatous, progressive inflammatory disease; it usually involves the subcutaneous tissue after a traumatic inoculation of the causative organism. Mycetoma may be caused by true fungi or by higher bacteria. In Sudan; Madura is spread mainly in agricultural and grazing areas like Gezira and sinnar states, the adults being the mostly affected because they work on fields so they are most susceptible to infection by the causative agents. This study aimed to compare between polymerase chain reaction, Mayer’s Haematoxylin and Diff- Quick Stains in the diagnosis of madurella mycetomatis to detect sensitivity and specificity in order to evaluate the reliability of these tests. 50 samples of fine needle Aspiration Cytology were collected randomly from infected site clinically diagnosed with madurella mycetomatis in sinnar state. These samples were diagnosed using Mayer’s Haematoxilin and Diff- Quick stains of cytology and polymerase chain reaction (PCR) techniques. The results showed that polymerase chain reaction (PCR) was more accurate. The positivity of the infection using Polymerase Chain Reaction techniques was 87% and only 13% of the total cases gave negative results. Whereas the positivity of the infection using Mayer‘s Haematoxylin and Diff- Quick stains of cytology was only 26% and the remaining 74% gave negative results. According to the Receiver Operating Curve (Rinvolved in assessing the specificity and sensitivity of the three methods; there is a difference between the three methods. PCR technique showed 93.3% sensitivity and 100% specificity. Whereas H&E and RALL stains of cytology showed 32.1% sensitivity and 100% specificity. The study recommended to use PCR in the diagnosis of Madurella Mycetomatis and local health care facilities and health education must be sufficient and adequate in areas where mycetoma is endemic, because the morbidity is massive and enormous. And it has many clinical and socio-economic impacts on patients, families and the community. 7 مقارنة بين تقنية التفاعل السلسلي للخميرة وصبغتى الهيماتوكسلين ايوسين والديف كويك تشخيص النبت في وﻻية سنار, السودان )2015-2014( نهى يوسف على يوسف ملخص الدراسة مرض النبت الفطرى هو من اﻷمراض المزمنة و ذو خاصية حبيبية إلتهابية مستفحله يبدأ من اﻷنسجة تحت الجلد و يمتد إلى الجلد و العضﻻت و العظام. تسببه ميكروبات فطرية و بكتيرية دقيقة تنتقل من التربة إلى جسم اﻹنسان عن طريق اﻷدوات التي تسبب جرحا في أماكن اﻹصابة. يعد داء المايستوما أو النبت من اﻷمراض الشائعة في السودان و تعتبر وﻻيتي الجزيرة وسنار أكثر تأثرا به. ينتشر بكثافة في أواسط المزارعين و الرعاة و الشرائح الضعيفة إقتصاديا حيث أنهم أكثر عرضة لمسبب المرض, كما أن فئات الشباب هم اﻷكثر عرضة لﻹصابة بهذا المرض, تكمن خطورة المرض في أنه قد يؤدي إلي بتر العضو المصاب هذا بجانب التكلفة العﻻجية العالية. هذه الدراسة تهدف إلى مقارنة تقنية التفاعل السلسلي للخميرة وصبغتى الهيماتوكسلين ايوسين والرال في تشخيص المادورا. تم جمع 50 عينة خزعه باﻻبر الناعمه عشوائيا من مرضى من مناطق ينتشر فيها المرض في وﻻية سنار. هذه العينات تم تشخيصها خلويا باستخدام صبغتي الهيماتوكسلين إيوسين والرال, كما تم تشخيصها أيضا بإستخدام تقنية التفاعل السلسلي للخميرة. أعطى التفاعل السلسلي للخميرة %87 نتيجة إيجابية و 13 % نتيجة سلبية. أما التشخيص باستخدام التقنيات الخلوية أعطى 26% نتيجة إيجابية و 74% نتيجة سلبية. بعد إجراء التحاليل اﻹحصائية وضحت النتائج أن الحساسية و التخصصية لتقنية التشخيص الخلوي هي 32.1 % و 100% على التوالي, و الحساسية و التخصصية عند إستخدام التفاعل السلسلي للخميرة هي 93.3% و 100% على التوالي. من هذه النتائج توصلت الدراسة إلى أن تقنية التفاعل السلسلي للخميرة هي اﻷكتر حساسية و تخصصية في تشخيص مرض المادورا مقارنة بتقنية التشخيص الخلوي. أوصت هذه الدراسة بإستخدام تقنية التفاعل السلسلي للخميرة في تشخيص مرض النبت الفطرى , و أوصت بضرورة التثقيف الصحي الكامل لﻷشخاص الذين يسكنون بتلك المناطق التي ينتشر فيها المرض و يشكل خطرا كبيرا و ذلك نسبة ﻹمراضيته العنيفة مما قد يؤدي في نهاية اﻷمر إلى بتر العضو المصاب, وكما أن تأثيره اﻹقتصادي و المادي عالي جدا يؤثر إجتماعيا و ماديا على حياة الفرد و اﻷسرة و المجتمع ككل. 8 Table of contents Title Page Supervision Committee I Examining Committee II Authorization III Dedication IV Acknowledgements V Abstract in English VI Abstract in Arabic VII Table of content VIII List of tables X List of figures XI List of abbreviation XII Chapter one Introduction Introduction 1 Rationale 2 objectives 3 Chapter two Literature review Historical background 3 Classification 3 Microscopic structure 4 The causative organism 5 Clinical presentation 5 Site of mycetoma 6 Diagnosis of mycetoma 7 Radiology 7 Ultrasonic imaging 7 Culture 8 Histological technique 8 Serodiagnosis 8 9 Fine needle aspiration 9 Molecular diagnosis 10 Chapter three: Material and methods Material and methods 11 Study area and study population 11 Study design 11 Sample size 11 Data collection 11 Materials and equipment 11 FNA sampling and Collection 13 Methods 13 H&E Stain 13 DNA extraction 13 PCR procedure 14 Chapter four: Result and Discussion Results and discussion 15 Conclusion 28 Recommendation 28 References 29 Appendix 32 10 List of tables Serial NO. Title Page 1. Distribution of Madurella by PCR according age group, gender ,occupation and 21 race 2. Distribution of Madurella by H&E and Diff- Quick stains according age group, 24 gender ,occupation and race 11 List of Figures Serial NO. Title Page 4.1 Disease frequency among different age groups 15 4.2 Disease frequency according to the gender 16 4,3 Disease frequency in different organ 17 4.4 Disease frequency among the different Occupation 18 4.5 Disease frequency according Race of patients. 19 4.6 PCR result 20 4.7 Positivity of M. Mycetomatis using PCR 20 4.8 H&E stain result 22 4.9 Diff- Quick stain result 22 4.10 Positivity of M. Mycetomatis using H&E and Diff Quick 23 4.11 Specificity and sensitivity of H&E 25 4.12 Specificity and sensitivity of Diff Quick 26 4.13 Specificity and sensitivity of PCR 26 12 List of abbreviations ID………………………………………….. Iimmunodiffusion CIE…………………………………………… Counter immune-electrophoresis ELISA…………………………………………… Enzyme linked immunosorbant assay FNA…………………………………………… Fine needle aspiration RCA…………………………………………… Rolling circle amplification PCR…………………………………………… Polymerase chain reaction H&E…………………………………………… Haematoxylin and Eosin ROC……………………………………………. Receiver Operating Curve MRC…………………………………………… Mycetoma Research Center 13 CHAPTER ONE INTRODUCTION