Comparison Between Polymerase Chain Reaction, Mayer's

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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

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

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

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 …………………………….

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.

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.

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.

مقارنة بين تقنية التفاعل السلسلي للخميرة وصبغتى الهيماتوكسلين ايوسين والديف كويك تشخيص النبت في والية سنار, السودان )2015-2014(

نهى يوسف على يوسف

ملخص الدراسة مرض النبت الفطرى هو من األمراض المزمنة و ذو خاصية حبيبية إلتهابية مستفحله يبدأ من األنسجة تحت الجلد و يمتد إلى الجلد و العضالت و العظام. تسببه ميكروبات فطرية و بكتيرية دقيقة تنتقل من التربة إلى جسم اإلنسان عن طريق األدوات التي تسبب جرحا في أماكن اإلصابة. يعد داء المايستوما أو النبت من األمراض الشائعة في السودان و تعتبر واليتي 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 األكثر عرضة لإلصابة بهذا المرض, تكمن خطورة المرض في أنه قد يؤدي إلي بتر العضو المصاب هذا بجانب التكلفة العالجية العالية. هذه الدراسة تهدف إلى مقارنة تقنية التفاعل السلسلي للخميرة وصبغتى الهيماتوكسلين ايوسين والرال في تشخيص المادورا. تم جمع 50 عينة خزعه باالبر الناعمه عشوائيا من مرضى من مناطق ينتشر فيها المرض في والية 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 اإلحصائية 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 األكتر 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 لألشخاص الذين يسكنون بتلك المناطق التي ينتشر فيها المرض و يشكل خطرا كبيرا و ذلك نسبة إلمراضيته العنيفة مما قد يؤدي في نهاية األمر إلى بتر العضو المصاب, وكما أن تأثيره اإلقتصادي و المادي عالي جدا يؤثر إجتماعيا و ماديا على حياة الفرد و األسرة و المجتمع ككل.

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

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

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

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

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

CHAPTER ONE INTRODUCTION

1.1 General introduction: 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 and hence it is usually classified into eumycetoma and actinomycetoma respectively (Fahal et.al, 1992). Tumefaction and formation of sinus tracts characterize mycetoma. The sinuses usually discharge purulent and seropurulent exudate containing grains. It may spread to involve the skin and the deep structures resulting in destruction, deformity and loss of function very occasionally it could be fatal (Abbott et.al, 1956; Mahgoub et.al, 1973). The true incidence and the geographical distribution of mycetoma throughout the world is not exactly known due to the nature of the disease which is usually painless, slowly progressive which may lead to the late presentation of the majority of patients. Mycetoma has a worldwide distribution but this is extremely uneven. It is endemic in tropical and subtropical regions. The African continent seems to have highest prevalence (Gonzalez et.al, 1975). It prevails in what is known as the mycetoma belt stretching between the latitudes of 15 south and 30 north (Magana et.al, 1984). The belt includes Sudan, Somalia, Senegal, India, Yemen, Mexico, Venezuela, Columbia, Argentina and others. The geographical distribution of the individual mycetoma organism shows considerable variations, which can be convincingly explained on an environmental basis. Areas where mycetoma prevails are relatively arid zones with a short rainy season with a relative humidity. In sudan Madura is widely spread mainly in agricultural and andgrazing areas like Gezira and sinnar states with adults being the mostly affected because they work on fields so they are most susceptible to infection by the causative agents as a result of that , it is one of the most economically dangerous diseases ,moreover ,the treatment is expensive also, it often and by amputation of the affected organ so all these are seriousness factors of the infection, and what ensures the seriousness of the infection is that the number of patients is high however it decrease every years for instance , according to soba center of Mycetoma, which is a national center, the number of cases in 2013 is 4800 decreases in very good and remarkable way to approximately 1500 in 2014 ,60 %of them is between 15---35 years old under poverty line, but that doesn’t mean it not a serious number because this is only the registered number of cases who came to the center. In Gazira state alone the number of

14 patients is about 748 from thousands of patients considered to be in 2013 that make Gezira state nearly most affected area in world followed by sinnar state (sudaress.com, 2015).

Justifications and objectives

1.2 Justifications:  The morbidity caused by mycetoma is massive and enormous. It has many clinical and socio-economic impacts on patients, families and the community. In areas where mycetoma is endemic, local health care facilities and health education are usually insufficient and inadequate. Also it is a common medical and health problem in sinnar state.  More sensitive and specific diagnostic tools are needed for accurate detection of Mycetoma.

1.3 Objectives:

1.3.1 General objective:

 To compare between polymerase chain reaction,Mayer’s Heamatoxiline and Diff- Quick stains in the diagnosis of Madurella Mycetomatis.

1.3.2 Specific objectives:

 To Diagnosis of Mycetoma specimens using H&E stains.  To Diagnosis of Mycetoma specimens using RALL 555 stain.  To Diagnosis of Mycetoma specimens using molecular technique (PCR).  To compare between three methods to identify the accurate method to be used in the diagnosis of Mycetoma.  To assess the sensitivity and specificity of the three methods.  To find out the most affected age group, Gender and occupation by infection.

CHAPTER TWO

LITERATURE REVIEW

2.1 Historical Background: Mycetoma was probably known by the ancient Indian writers as padavalmita. The earliest medical description of the mycetoma appears to be in the doctoral thesis of the German physicion and traveler Engelbert Kaempfer (651-1716) he was reported some cases in india, Ceylon, Japan. Century later mycetoma recorded by French missionaries in india 1812 .Billingall in 1855 described the microscopic details of the disease. Van Dyke Carter (1860) after isolation of the fungus, stressed the fungal parasitic origin of the disease and he was the first who coined the term of the mycetoma he also described two varianies of mycetoma . the melanoid type with black granules. And the ochriod one with white granules, in 1874, he produced a monograph on mycetoma and fungal disease of india. in 1881, Dymock reported for the first time on local recurrence after surgery in a patient with yellow mycetoma. Boyce and Surveryor (1894) studied in details the morphology of the grain and their chemical reaction to different solvents and they were able to describe the host tissue reaction to the organism. in sudan the disease has been known before the advent of modern medicine , by its present common name (Nebit) meaning growth. The first documented report of case of mycetoma in sudan was published by Balfour in 1904 he noted that the disease was common amongst Northern Sudanese. In 1972 El Hassan Mahgoup confirmed beyond doubt the spread of mycetoma through the lymphatics. Mycetoma Research Centre in 1992 was established at Soba University of Khartoum, Sudan. The centre is committed to the maintenance of the highest standards of mycetoma patients (Ahmed et.al, 2006).

2.2 Classification of mycetoma: The species of madurella .mycetomatis related to Genus madurella, Familly of Incetaesedis in order Sordariales class Sordariomycetes, phylum Ascomycota klingdom of Fungi. Before the introduction of molecular methods for phylogenetic analysis, taxonomists considered fungi to be members of the plant kingdom because of similarities in life style: both fungi and plants are mainly immobile, and have similarities in general morphology and growth habitat. Like plants, fungi often grow in soil, and in the case of mushrooms form conspicuous fruit bodies, which

16 sometimes resemble plants such as mosses. The fungi are now considered a separate kingdom, distinct from both plants and animals, from which they appear to have diverged around one billion years ago some morphological, biochemical, and genetic features are shared with other organisms (Harris et.al, 2008; Bruns et.al, 2006). A fungus is any member of a large group of eukaryotic organisms that include microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, protists, and bacteria. One major difference is that fungal cells have cell walls that contain chitin, unlike the cell walls of plants and some protists, which contain cellulose, and unlike the cell walls of bacteria (Baldauf et.al, 1993). Like other eukaryotes, fungal cells contain membrane-boundnuclei with chromosomes that contain DNA with noncoding regions called introns and coding regions called exons. Fungi have membrane-bound cytoplasmic organelles such as mitochondria, sterol-containing membranes, and ribosomes of the 80S type They have a characteristic range of soluble carbohydrates and storage compounds, including sugar alcohols (e.g., mannitol), disaccharides, (e.g., trehalose), and polysaccharides (e.g., glycogen, which is also found in animals the cells of most fungi grow as tubular, elongated, and thread-like (filamentous) structures and are called hyphae, which may contain multiple nuclei and extend at their tips. Each tip contains a set of aggregated vesicles—cellular structures consisting of proteins, lipids, and other organic molecules—called Spitzenkörper (Bowman et.al, 2006).

2.3 Microscopic structures: Most fungi grow as hyphae, which are cylindrical, thread-like structures 2–10 µm in diameter and up to several centimeters in length. Hyphae grow at their tips (apices); new hyphae are typically formed by emergence of new tips along existing hyphae by a process called branching, or occasionally growing hyphal tips fork, giving rise to two parallel-growing hyphae. the combination of apical growth and branching/forking leads to the development of a mycelium, an interconnected network of hyphae. Hyphae can be either septate or coenocytic. Septate hyphae are divided into compartments separated by cross walls (internal cell walls, called septa that are formed at right angles to the cell wall giving the hypha its shape), with each compartment containing one or more nuclei; coenocytic hyphae are not compartmentalized (Ferguson et.al, 2003).

2.4 The Causative Organisms OF Mycetoma: A variety of eumycetes and actinomycetes are incriminated in the causation of mycetoma. Not less than 18 organisms have been reported from different parts of the world. The organisms are usually present in the soil in the form of grains but grains are not capable of inducing infection neither in man or animals, this support by a study on molecular identification of M. mycetomatis in experimental animals the finding support the presence of intermediate host but that is not known, it may be thorn, plant or animal. The disease is not contagious from person to person (Ahmed et.al, 2006). The incubation period in mycetoma in unknown due to the difficulty in establishing the time of initial infection .in experimental animals the information of granuloma was noted after three weeks from the inoculation of the organism ( Ahmed et.al, 2004).

2.5 Clinical Presentation: Male predominance is a constant finding in mycetoma with a sex ratio of 3.7:1. This is commonly attributed to the greater risk of exposure to organisms in the soil during the outdoor activities. No age is exempted but mycetoma commonly affects adults between 20-40 years of age and these are the earning members of the society especially in under developed countries. However, in endemic regions children and elderly people may also be affected (Mahgoub et.al, 1973; Magana et.al, 1984). Mycetoma is seen more conventionally in cultivators, field laborers and in herdsmen who come in contact with the land in endemic areas people of other occupations are affected ( Ahmed et.al, 2004 ; Ahmed et.al, 2006). The rate of progress is more rapid with actinomycetoma than with eumycetoma. In eumycetoma, the lesion grows slowly with clear defined margins and remains encapsulated for a long period, whereas, in actinomycetoma the lesion is more inflammatory, more destructive and invades the bone at an earlier period (Magana et.al, 1984). The characteristic triad, of a subcutaneous mass, sinus and the presence of discharge containing grains are pathognomic of mycetoma. It presents as a slowly progressive painless subcutaneous swelling at the site of previous trauma. The swelling is usually firm and rounded but it may be soft, lobulated, and rarely cystic and it is often mobile. Multiple secondary nodules then evolve as well, the nodules may suppurate and drain through multiple sinus tracts and these sinuses may close transiently after discharge during the active phase of the disease. Fresh adjacent sinuses may open while some of the old ones may heal completely. They are connected with each other,

18 with deep sterile abscesses and with the skin surface. The discharge is usually serous, serosanguinous or purulent. During the active phase of the disease the sinuses discharge grains (fungal colonies) the colour of which depends on the causative organism. The grains can be black, yellow, white or red and they are of variable size and consistency. The black grains are usually due to M. mycetomatis, the red ones are due to A. pelletierii and the white grains can be due to A. madurae (Ahmed et.al, 1992). Mycetoma is usually painless in nature, it was suggested that the mycetoma produces substances that have an anaesthetic action (Mahgoub et.al, 1973). At a late stage of the disease the pain may become negligible due to nerve damage by the tense fibrous tissue reaction, endarteritis obliterans or poor vascularisation of the nerve. Pain may be produced by the expansion of the bone with the mycetoma granuloma and grains or it may be due to secondary bacterial infection (Magana et.al, 1984). In the majority of patients the regional lymph nodes are small and shotty. An enlarged regional lymph node is not uncommon and this may be due to secondary bacterial infection, genuine lymphatic spread of mycetoma or it may be due to immune complex deposition as part of a local immune response to mycetoma infection (Gumaa et.al, 1983). The infection remains localised and the constitutional disturbances are rare but when they do occur they are generally due to secondary bacterial infection of the open sinus tracts, fistula formation in some patients or generalisedimmuno-suppression. Cachexia and anaemia may be seen in late mycetoma. This is often due to malnutrition, sepsis and mental depression. Mycetoma can produce many disabilities, distortion and deformity. It can be fatal especially with cranial mycetoma (Fahal et.al, 1994 ; Fahal et.al, 1996 ; Fahal et.al, 1998).

2.6 Site of Mycetoma: The commonest site for mycetoma is the foot (79.2%), most of the lesions is seen on the dorsal aspect of the forefoot and for unexplainable reasons the left foot is affected more. The hand ranks as the second commonest site (6.6%), the right hand is more affected. This may imply a traumatic basis of the infection in this site. In endemic areas other parts of the body may be involved but less frequently and these include the knee, arm, leg, head and neck, thigh and the perineum. Rare sites such as the chest and abdominal walls, fascial bones, mandible, paranasal sinuses, eyelid, vulva, orbit, scrotum and surgical incisions may be affected (Fahal et.al, 2006).

2.7 Diagnosis of Mycetoma:

2.7.1 Radiology:

A series of radiological changes are seen in mycetoma. This is due to the fact that all mycetoma agents are osteophilic and it may be due to the effect of the granuloma on both the affected bone and its blood supply. In the early stage, there is a soft tissue granuloma, which is shown as a dense shadow or as scattered multiple soft tissue shadows. Calcification and obliteration of the fascial planes may sometimes be seen as the disease progresses, the cortex may be compressed from outside by the granuloma leading to bone scalloping, this is followed by a variable amount of periosteal reaction. Periosteal new bone spicules are laid down at right angle to the cortex to create a sun-ray appearance and Codman triangle, an appearance that may be undistinguishable from that due to osteogenic sarcoma. Late in the disease, there may be multiple punched out cavities through the normal density of the bone. These cavities are large in size, few in number with well-defined margins in eumycetoma. Whereas, the bone cavities in actinomycetoma are usually smaller in size, numerous and have no definite margins. The cavities are produced by the replacement of the osseous tissue by the grains. Their size is due to the size of the grains of the causative organism. The cavities are usually filled with solid masses of grains and fibrous tissue, which provides bone support. This may explain the rarity of pathological fractures in mycetoma. The bony changes in the skull are unique: they are purely sclerotic with dense bone formation and loss of trabeculation (Fahal et.al, 1992). Osteoporosis at and distal to the affected part is well observed in mycetoma and this may be due to disuse atrophy or due to the compression of the bone and its blood supply by the mycetoma granuloma. Chemotherapy causes radiological improvement consisting of remoulding, absorption of the sclerotic bone and reappearance of the normal trabecular pattern

(Mahgoub et.al, 1973).

2.7.2 Ultrasonic imaging of Mycetoma: The mycetoma grains, its capsule and the accompanying inflammatory granuloma have characteristic ultrasonic appearances. Ultrasound imaging can differentiate between eumycetoma and actinomycetoma and between mycetoma and other non-mycetoma lesions. In eumycetoma lesions, the grains produce numerous sharp bright hyperreflective echoes, which are consistent with the black grains. The grain cement substance is most probably the origin of

20 these sharp echoes. Also there are multiple thick walled cavities with absent acoustic enhancement. In actinomycetoma lesion the findings are similar but the grains are less distinct. This may due to their smaller size and consistency, individual embedding of the grains or the absence of the cement substances in few of them. The ultrasonic diagnosis of mycetoma is more precise and accurate in lesions with no sinuses. The size and extent of the lesion can be accurately determined ultrasonically and this is useful in planning surgical incisions and procedures (Fahal et.al, 1997).

2.7.3 Culture: A large variety of microorganisms are capable of producing mycetoma. They can be identified by their textural description, morphological and biological activities in pure culture. The biological activity may include acid fastness, optimal temperature, proteolytic activity, utilization of sugars and nitrogenous compounds. The grains are the source of the culture and they should be alive and free of contaminants. Many culture media are used e.g. Sabouraud, blood agar and Malt extract agar. The culture technique is cumbersome, time consuming and chance contamination may give a false positive result. It also requires experience to identify the causative organisms (Mahgoub,et al 1985).

2.7.4 The histological technique: Stained sections usually show the grain morphology and the tissue reaction to the organisms. The technique is attractive in that it requires neither aseptic procedure nor the rigid time schedule required for culture, however it lacks the precision of culture (Elhasan et.al, 1994).

2.7.5 Serodiagnosis in mycetoma: In the absence of the classical triad of mycetoma, the demonstration of significant antibodies titers against the causative organism may be of diagnostic value. Serodiagnosis is of a great help in identification and classification of the various organisms, which is an essential prerequisite for medical treatment, and is mandatory for the follow-up of these patients. It has many advantages over the culture and histopathological techniques, as both require surgical biopsy, which may enhance the spread of the organism. The common serodiagnostic tests for mycetoma are the immunodiffusion (ID) and counter-immuno-electrophoresis (CIE) (Gumaa et.al, 1975).

The (ID) test is not sensitive enough and unless antigens are quite pure, it can be negative in early cases. (CIE) test remains reliable, simple, economical, rapid and a sensitive test. It is useful for routine diagnosis of early cases of mycetoma and for checking the sera that are weakly positive by (ID) test. It distinguishes between eumycetoma and actinomycetoma and between Actinomycetes themselves, but cross reactivity between A. madurae and A. pelletierii is quite common. However it is time consuming and the preparation of the antigens take a considerable time. Recently the indirect fluorescent antibody test was used in diagnosing mycetoma and was found to be specific and sensitive (Joshi et.al, 1988).

Enzyme linked immunosorbent assay (ELISA) was used in the diagnosis of mycetoma. It appeared to be a sensitive test for the detection of antibodies, however the high sensitivity of the test makes the cross reactivity unavoidable. ELISA may be a useful tool in community studies as sero-epidemiological survey could give valuable information on the distribution and prevalence of exposure to mycetoma (Taha et.al, 1983).

2.7.6 Fine needle Aspiration Cytology of Mycetoma:

Fine needle aspiration (FNA) of Mycetoma was described for the first time by El hag, Fahal and Gasim in a paper published in (Acta Cytologica 1996; 40:461-464). Which were aspirated with 23 gauge needle attached to 10 ml syringe.

Mycetoma can be accurately diagnosed by Fine Needle Aspiration (FNA) cytology. Mycetoma lesion has a distinct appearance in a cytology smear characterised by the presence of polymorphous inflammatory cells consisting of an admixture of neutrophils, lymphocytes, plasma cells, histiocytes, macrophages and foreign body giant cells and grains. In sections, the grain is closely surrounded by and occasionally infiltrated by neutrophils causing its fragmentation. Outside the neutrophil zone, monocytic cells and giant cells are seen. This is surrounded by granulation tissue rich in fibroblasts and blood vessels. FNA allows morphological identification of mycetoma and its classification into eumycetoma and actinomycetoma, this is important as the treatment depends mainly on the aetiological agents. The technique is simple, cheap, rapid, and sensitive and can be tolerated by patients. It can be used not only in routine diagnosis but can be used as an effective mean in collection of material for culture and immunological studies. Due to the simplicity of the technique it can be

22 used in epidemiological survey of mycetoma and for detection of early cases in which the radiological and serological techniques may not be helpful (EL Hag et.al, 1996).

2.7.7 Molecular diagnosis: Accurate identification of mycetoma causative agent is a priority for treatment. However current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigation. A rapid, simple and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas (Abdalla et.al, 1999). Comparative studies of nucleotide sequences of the rRNA genes provide a means for analysis phylogenetic relationship over a wide range of taxonomic level and to assist in the development of identification assays for fungal (White et.al, 1996). The small-subunit ribosomal rRNA sequences evolve relatively slowly and are useful for studying distantly related organism. rDNA sequences have been utilized for many investigators for the determination of species identity for multitude of yeasts and fungi (Abdalla et.al, 1999). Rolling Circle Amplification (RCA) uses species-specific padlock probes and isothermal DNA amplification. The test were based on ITS sequences and developed for falciformisporasenegalensis, F.tompkinsii, Madurellafahalii, M.mycetomatis, M.pseudomycetoa and others. With the isothermal RCA assay, 62 isolates were successfully identified with 100 % specificity and no cross reactivity or false results. The main advantage of this technique is the low-cost, high specificity, and simplicity. In addition, it is highly reproducible and can be performed within a single day (Sara et.al, 2014).

CHAPTER THREE

MATERIALS AND METHODS

3.1 Study area and study population:

Sennar State is part of the Blue Nile region in South-East Sudan and is delimited by Al-Gazira State in the North, The Blue Nile in the South, Algadarif State and the Sudanese Ethiopian borders in the East and the White Nile State in the West. The area approximately is 40,680 kilometers square with population 1,400,000 persons. The state contains seven localities and Singa city is the capital of the state (sudan.gov.sd, 2012). This study was conducted in Sinnar state. Volunteer patients with Madurlla Mycetmatis were participated in this study.

3.2 Definition of study:

Prospective and cross-sectional study in diagnosis of M. Mycetomatis using H&E, Quick and molecular technique (PCR). The study was conducted during the period from October 2014 to August 2015.

3.3 Sample size:

The study includes 50 fine needle Aspiration samples clinically diagnosed Madurella Mycetomatis were collected from patients.

3.4 Data collection and analysis:

Data was collected by using a questionnaire. A sufficient copy of the questionnaire was produced. Questionnaires were then filled by the investigator during each time when sample collected. Completed questionnaires from selected study areas (50 FNAC) were collected. Data was then analyzed and tabulated using statistical package for social sciences (IBM SPSS) program version 20, T test, a crosstabs and correlation were performed.

3.5 Samples preparation:

 Syringes 10 ml.  Slides.  Cotton.

 Disinfectant, Alcohol.  Containers.  Cover glasses.  DPX.  H&E stain.  Diff Quick stain.  PCR machine TCHNE 500 USA , NO S 5.525.300.

3.5.1 Requirements of DNA Extraction:

Items Quantity

5 Mortor 500 ml CTAB Buffer 1 ml Proteinase K 500 ml Chloroform : isoamylalcohol 500ml Isopropanol

1000 ml 70 % ethanol 500 ml TE buffer

3.5.2 PCR mixture:

ITEM

PCR master mix Primer 10 pmol

Primer 10 pmol

DW Ladder 1 kb no , bio 33025 (Bioline) 3.6 Sample Collection:

The needle used is a disposable one with an outer diameter varying from 0.6 to 1.2 mm (23 to 18 gauge). The length of the needle is 20 cm depending on the location of the lesion. The syringe varies 20 ml. The sample aspirated, smeared directly on glass slide. (FNAC) stained by cytological stain H&E, RALL and other part of fluid tested by PCR.

3.7 Methods: 3.7.1 Cytology methods: 3.7.1.1 H&E stain procedure:

 Bring section to DW.  Stain with the alum heamatoxylin for 7 min.  Rinse in running tap water.  Differentiate with 0.3% acid alcohol for 2 seconds.  Rinse in running tap water.  Stain with eosin for 2 min.  Dehydrate, clear and mount.  Appearance of mycetoma by H&E stain.

3.7.1.2 Procedure of Diff Quick:

 air dry smear.  Rinse in methanol 5 times.  Rinse in Eosin 5 times.  Rinse in Giemsa stain 5 times.  Allow to dry.  Appearance of mycetoma byDiff Quick stain.

3.7.2 Molecular methods:

3.7.2.1 DNA extraction:

DNA will be extract from 50 different samples

 about 10 mg fugal were crushed in mortor and add 490 ml CTAB buffer (2% CTAB , 100 MmtRIS HCL , 20 mM EDTA , 1,4 M Nacl)  About 10 µll of proteinase K were added and the mixure were mixed in MoBio vortex some minutes.  The tubes were incubated at 60 C for 60 minutes and re-mixed again with MoBio vortex to insure mycelia distruption.  Five hundred microliter of chloroform : isoamylalcohol (24:1) were added followed by shaking for two minutattes +50 µl Na acetate.  Tubes were spin in 14.000 rpm microfuge for 10 minutes and the upper layer was collected in new sterile tube with 0.55 ice cold iso-propanol shaking and respine.  Finally, the pellets were washed with 70 % ethanol, left for air dry and re-suspended in 100µl TE buffer.

3.7.2.2 PCR procedure:

 Prepare a PCR mixture ( 20 µl ) containing 2 µl DNA,1 µl primer (f:10 pmol\20 µl 20 pmol primer 26.1A(5,AATGAGTTGGGTTTAACGG-3.) and 21 pmol primer .28-3A ( 5-TCCCGGTAGTGTAGTGTCCCT- 3~) 16 µl DW.  Perform the PCR amplification in thermocycler using a cycle program with 40 cycles of 94 c for 2 min denaturation 94oC for 20 sec Annealing 65 oC Extension 72oC 20 sec Final Extension 72oC 30 sec.  Separate the PCR products on 1% agarose gel in electrophoreses machine.  Reading of the result.

CHAPTER FOUR

RESULTS AND DISCUSSION

This study was carried out on 50 patients of different ages, gender, race and occupation to estimate Madurella mycetomatis spread in sinnar state. It aimed to assess the role of cytology in the diagnosis of Madurella Mycetomatis to detect sensitivity and specificity in order to evaluate the reliability of this test as a diagnostic tool for the disease.

4.1 Disease frequency in different age groups:

The age range of the patients is 8-47 years with mean of 24.4 ± 8.84 years. 5 (10%) of them are less than 15 years, 32 (64%) are between 15--30 years and 13 (26%) are more than 30 years (figure 4.1).

Age frequences

less than 15 10% more than 30 26%

15-30 64%

Figure (4.1): Disease frequency among different age groups.

This figure shows that the age group of cases from 15 years to 30 years is more susceptible to infection by Madurella mycetomatis than the others.

4.2 Disease frequency according to gender:

29 (58%) are male and 21 (42%) are female (figure 4.2). 47 (94%) of them from Eastern countryside of Sinnar state, 3 (6%) from Western countryside of Sinnar state.

Gender frequences

Female 42% Male 58%

Figure (4.2): Disease frequency according to the gender.

This figure shows that males are more susceptible to infection by Madurella Mycetomatis than females.

4.3 Disease frequency in different infection sites:

According to the infection site; 41 (82%) of cases were infected at their feet, 6 (12%) are infected at their hands, 2 (4%) at their hands and feet together and 1 (2%) at other sites (figure 4.3).

Site of infection foot and hand other 4% 2%

hand 12%

foot 82%

Figure (4.3): Disease frequency in different organs.

This figure shows that the foot is more susceptible to infection by Madurella Mycetomatis than the other sites

4.4 Disease frequency in different occupation groups:

23 (46%) are farmers, 16 (32 %) are housewives, 6(12%) are students and 5(10%) have another occupations (figure 4.4).

Occupation others 2%

housewife 35% farmers 50%

students 13%

Figure (4.4): Disease frequency among different occupations.

This figure shows that farmers are more susceptible to infection by Madurella Mycetomatis than other people.

4.5 Disease frequency according to the race:

17 (34%) patients belong to Gawasma tribe, 18 (36%) to Nefedia, 15 (30%) to another tribes (figure 4.5).

Race frequences

Others 30% Gawasma 34%

Nefedia 36%

Figure (4.5): Disease frequency according to the race of the patients.

A study was done by Ahmed Hassan Fahal in Mycetoma Research Centre (MRC) showed that Mycetoma commonly affects young adults males mostly in developing countries and people of low socioeconomic status and manual workers such as agriculturalists, labourers and herdsmen are the worst affected. Also he showed that Mycetoma in general involves those parts of the body that come in contact with soil during daily activities. The foot is the most affected site and this is seen in 70% of patients (Fahal,et,al 2011); these results agree with my results.

4.6 Diagnosis of Mycetoma using PCR:

Figure 4.6: PCR result.

From 32 patients whom diagnosed clinically with Madurella Mycetomatis; there are 28 (87%) cases give positive results and only 4 (13%) cases give a negative results using a PCR technique (figure 4.7).

PCR results

Negative (4) 13%

Positive (28) 87%

Figure (4.7): positivity of Madurella Mycetomatis using PCR technique.

Table (4.1): Positive and negative results using PCR among different age, gender, race, occupation and infection sites: Variables Total No. +ve results -ve result P. value Less than 15 y 3 3 0 Age 15-30 y 21 18 3 0.78 More than 30 8 7 1 M 20 18 2 Gender 0.58 F 12 10 2 Foot 29 25 4 Site of Hand 2 2 0 infection Foot and hand 0 0 0 Other 1 1 0 farmers 17 16 1 housewives 9 8 1 Occupation 0.04 students 5 4 1 others 1 0 1 Gawasma 14 13 1 Race Nefedia 9 8 1 0.56 others 9 7 2

32 samples were tested for Madurella by PCR technique as showed in Table 1. The positivity of Madurella by PCR among age groups showed 3 cases in the age group of less than 15 years; 18 cases in the age group 15-30 and 7 cases in the age group of more than 30 years. There are 18 positive samples among male group, 10 positive samples in female group. There are 16 positive cases among farmers group, 8 cases positive among housewives group, 4 are students while the positivity according the race is as the following: 13 from Gawasma, 8 from Nefedia and 7 from the other tribes.

4.7 Diagnosis of Mycetoma using H&E and Diff-Quick stain:

Figure (4.8): H&E stain result

Figure (4.9): Diff- Quick stain result

From 50 patients whom diagnosed clinically with Madurella Mycetomatis; there are 13 (26%) cases give positive results and 37 (74%) cases give negative results using H&E and Diff- Quick stains (figure 4.7).

H&E and Quick results

Positive (13) 26%

Negative (37) 74%

Figure (4.10): positivity of Madurella Mycetomatis using H&E and Quick stains.

Table (4.2): Positive and negative results using H&E and Diff-Quick stains among different age, gender, race, occupation and infection sites: Variables Total +ve results - ve result P. value Less than 15 y 5 2 3 Age 15-30 y 32 8 24 0.74 More than 30 13 3 10 M 29 8 21 Gender 0.76 F 21 5 16 Foot 41 12 29 Hand 6 1 5 Site of infection 0.67 Foot & hand 2 0 0 Other 1 0 0 Farmers 23 9 14 Housewives 16 3 13 Occupation 0.21 Students 6 1 5 Others 5 0 0 Gawasma 17 5 12 Race Nefedia 18 6 12 0.21 Others 15 2 13

This table shows that there is no difference between the cytological test results using H&E and Diff- Quick stains. According to the age range there are 2 positive cases in the group of less than 15 years, 8 positive cases in 15-30 years group and only 3 positive cases in the group of more than 30 years. 8 of the positive cases are male and 5 positive cases are female. From the all positive cases there are 12 patients infected at their feet and only one patient infected at his hand. According to the occupation there are 9 positive cases from the farmers group, 3 positive cases from the housewives group and one positive case from the students group. Depending on the race difference; 5 positive cases belong to the Gawasma tribe, 6 positive cases belong to the Nefedia tribe and only 2 positive cases from another tribes.

4.8 Receiver Operating Characteristics (ROC) curve to PCR test: ROC curve was used to measure specificity and sensitivity of the test compared to a gold standard method. In this study, we considered that PCR is the gold standard method and we used it to detect the sensitivity and specificity of the cytological methods ( H&E and RALL 555). The curve shows that specificity of H&E stain was 100% and the sensitivity was 32.1%, while the specificity of RALL stain was 100% but the sensitivity was 32.1%.

The sensitivity of PCR is 93.3% and the specificity is 100% and Area under the ROC curve (AUC) is 0.97, whereas the sensitivity and specificity of cytological technique using H&E and RALL 555 stains are 32%, 100% respectively and Area under the ROC curve (AUC) is 0.66 (figure 4.8, 4.9, 4.10).

H & E 100

Sensitivity

Sensitivity: 32.1 20 Specificity: 100.0 Criterion: ≤1

0 0 20 40 60 80 100 100-Specificity

Figure (4.11): Specificity and sensitivity of cytological test using H&E stain.

RALL 100

Sensitivity

Sensitivity: 32.1 20 Specificity: 100.0 Criterion: ≤1

0 0 20 40 60 80 100 100-Specificity

Figure (4.12): Specificity and sensitivity of cytological test using RALL stain.

PCR 100

Sensitivity: 93.3 Specificity: 100.0 80 Criterion: ≤1

Sensitivity

0 0 20 40 60 80 100 100-Specificity

Figure (4.13): Specificity and sensitivity of PCR.

A study was done by El Hag in Mycetoma Research Centre (MRC) – Khartoum university- Khartoum- Sudan demonstrated that Mycetoma can be accurately diagnosed by FNAC which is sensitive technique (El Hag, 1996); these results disagree with my results.

CHAPTER FIVE CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion:

PCR was the most accurate, sensitive and specific of the three methods .

Madurella Mycetomatis commonly affects young adult’s male ( 15—30) and people of low socioeconomic status and manual workers such as agriculturalists. The foot is the most affected site.

5.2 Recommendations:

PCR recommended to confirm the diagnosis of Mycetoma.

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.

More studies including a larger population size should be done .

REFERENCES

Abbott PH (1956). Mycetoma in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene. 50: 11-24.

Abdalla ,O,A. Moawia .M.Mukhtar. MarlyKools-Sijmons .Ahmed H, Fahal (1999). Development of a Species –Specific PCR restriction fragment length polymorphism analysis procedure for identification of madurellamycetomatis . Journal of clinical microbiology, Oct. p. 3175-3178.

Ahmed AO. Van de Sande and Fahal AH (2004). In vitro susceptibilities of Madurella Mycetomatis to itraconazole and amphotericin B. Anti Microb Agent Chemother, 48.2742-6

Ahmed Hassan Fahal (2006). Mycetoma Clinicopathologiacl Monograph, first edition. P: 18.

Baldauf SL, Palmer JD (1993). "Animals and fungi are each other's closest relatives: congruent evidence from multiple proteins". Proceedings of the National Academy of Sciences U.S.A. 90 (24): 11558–11562.

Bowman SM, Free SJ. (2006). "The structure and synthesis of the fungal cell wall". BioEssays 28 (8): 799–808.

Bruns T. (2006). "Evolutionary biology: a kingdom revised". Nature 443 (7113): 758–761.

EL Hag IA, Fahal AH, Khalil EAG (1996). Fine needle aspiration cytology of mycetoma, Acta Cytologica. 40(3):461-464

EL Hassan AM, Fahal AH, EL Hag IA, Khalil EAG (1994). The pathology of mycetoma: Light microscopic and ultrastructural features. Sudan Medial Journal. 32: 23

Fahal AH (2011). Mycetoma, Khartoum Medical Journal, vol. 04. No. 01. pp 514-523.

Fahal AH, EL Hassan AM, Abella AO, Sheik HE (1998). Cystic mycetoma: An unusual clinical presentation of Madurellamycetomatis. Transactions of the Royal Society of Tropical Medicine and Hygiene. 92: 66-67.

Fahal AH, EL Sheik H, EL Hassan AM (1996). Pathological fracture in mycetoma. Transactions of Tropical Medicine and Hygiene, 90: 675-676.

Fahal AH, EL Sheikh HA, EL Lider MA, Homeida MA, EL Arabi YE, and Mahgoub ES (1997). Ultrasonic imaging in mycetoma, British Journal of Surgery, 78: 765-766.

Fahal AH, Hassan MA, Mycetoma (1992). British Journal of Surgery. 79: 1138-1141.

Fahal AH, Suliman SH (1994). The clinical presentation of mycetoma. Sudan Medical Journal. 32: 46-65.

Ferguson BA, Dreisbach TA, Parks CG, Filip GM, Schmitt CL. (2003). "Coarse-scale population structure of pathogenic Armillaria species in a mixed-conifer forest in the Blue Mountains of northeast Oregon". Canadian Journal of Forest Research 33 (4): 612–623.

Gonzalez-Ochoa A. Mycetoma in (1975). Clinical Tropical Dermatology. Edited by Canizares O. Oxford, Blackwell Scientific. pp 24-29.

Gumaa SA, Mahgoub ES, EL Sid MA (1986). Mycetoma of the head and neck. American Journal of Tropical Medicine and Hygiene. 35(3): 596-600.

Gumaa SA, Mahgoub ES (1975). Counter-immuno-electrophoresis in the diagnosis of mycetoma and its sensitivity as compared to immuno-diffusion. Sabouraudia. 13: 309-315.

Gumaa SA(1983). The etiology and epidemiology of Mycetom, Sudan Med. J.32:46-66.

Harris SD (2008). "Branching of fungal hyphae: oted , mechanisms and comparison with other branching systems". Mycologia 100 (6): 823–832.

43 http://www.sudan.gov.sd/index.php/en/pages/details/114/Sennar-State#.VUXrJTO8ZPw

Joshi KR, Singhvi S (1988). Serodiagnosis of mycetoma.Transaction of the Royal Society of Tropical Medicine and Hygiene. 82: 334.

Magana M. Mycetoma (1984). International Journal of Dermatology. 23(4): 221-236.

Mahgoub ES, Murray IG, William Heinemann (1973). Mycetoma , Medical Books Ltd. pp. 1- 50.

Mahgoub ES (1985). Mycetoma. Seminars of Dermatology. 4: 230-239.

Sara A.Ahmed , Bert H.G Gerrits van den Ende , Ahmed H.Fahal , Wendy W . J. van de Sande G. S. de Hoog (2014). Rapid Identification of Black Grain Eumycetoma Causative Agents Using Rolling Circle Amplification. PLOS Negl Trop Dis 8(12): 3368.

Taha A (1983). A serological survey of antibodies to Streptomyces somaliensis and Actinomadura madurae in the Sudan using enzyme linked immunosorbent (ELISA). Transaction of the Royal Society of Tropical Medicine and Hygiene. 77(1): 49-50.

WhiteT.J, T bruns,s Lee and J, Taylr (1996). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. www.sudaress.com/akhirlahza/ 147109.

Appendix: Questionnaire

S. No.

Name: Tel:

Age: years.

Gender: ♀ ♂

Residence:

Occupation:

Race :

Site of infection

foot

hand

foot and hand

other

Duration of disease years

Discharge from infection yes No

Color of grains?

PCR result:

Cytology results:

 H&E:

 Rall 555: