Sero-Detection of Chlamydia Trachomatis Antibodies Among Pregnant Women, Wad Medani Maternity Teaching Hospital, Gezira State, Sudan (2017) Mona Osman Ahmed Ali

Sero-detection of Chlamydia trachomatis Antibodies among Pregnant Women, Wad Medani Maternity Teaching Hospital, Gezira State, Sudan (2017) Mona Osman Ahmed Ali

B.Sc. in Medical Microbiology, Faculty of Medicine and Health Sciences,

Shendi University (2011)

A Dissertation

Submitted to University of Gezira in Partial Fulfillment for the Requirements for the Award of the Degree of Master of Laboratory Science

Medical Microbiology

Department of Medical Microbiology

Faculty of Medical Laboratory Sciences

May, 2018

Sero-detection of Chlamydia trachomatis Antibodies among Pregnant Women, Wad Medani Maternity Teaching Hospital, Gezira State, Sudan (2017)

Mona Osman Ahmed Ali

Supervision Committee: Name : Position Signature

Dr: Nadir Musa Khalil Abuzeid Main supervisor ………….

Dr: Adam Dawoud Abakar Co-supervisor ………….

Date: …/…/2018

Sero-detection of Chlamydia trachomatis Antibodies among Pregnant Women, Wad Medani Maternity Teaching Hospital, Gezira State, Sudan (2017)

Mona Osman Ahmed Ali

Examination Committee: Name Position Signature Dr: Nadir Musa Khalil Abuzeid Chairperson …..……. Dr: Wad-Elbehar Hamad Elneel External examiner ...…….... Dr: Sanaa Elfatih Husain Internal examiner …………

Date: …/…/2018

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Declaration

I authorized that my dissertation” Sero-detection of Chlamydia trachomatis Antibodies among Pregnant Women, Wad Medani Maternity Teaching Hospital, Gezira State, Sudan (2017)" submitted by me under the supervision of Dr: Nadir Musa Khalil Abuzeid and Prof. Adam Dawoud Abakar for the partial fulfillment for the award of Master degree in Medical Laboratory Sciences in Medical Microbiology. University of Gezira Faculty of Medical Laboratory Sciences Department of Medical Microbiology; Wad- Medani, Sudan and this is original and it was not submitted in part or in full, in any printed or electronic means, and is not being considered elsewhere for publication or submission awarding degree elsewhere.

Name and Signature of Candidate

Name: Mona Osman Ahmed Ali

Signature: ……………………….…

Place: Wad Madani

Date: …/…/2018

Dedication

To my parents; thanks for supporting me; Thanks for giving me a chance to prove and improve myself through all my path of life.

To my brothers, sisters, husband, and my daughters.

To my friends and all people support me.

And of course, all my colleagues in master program Batch (3).

If I forgot anyone, please don’t ever doubt my dedication and love for ALL.

Acknowledgements

I am grateful to all of helped me in completing this dissertation and provided me with information and special thanks and appreciation to my supervisors: Dr. Nadir Musa Khalil Abuzeid, Assistant Professor and consultant of medical microbiology University of Omdurman Islamic university, Faculty of Medical Laboratory Sciences, Department of Medical microbiology who followed and supported me in all Stages of this dissertation. I deeply thanks to my Co-supervisor Prof. Adam Dawoud Abakar Salim for hir guidance and great help. I do appreciate effort of all Staff working in Medical Microbiology and Post graduate studies departments.

Especial thank to all staff of Wad Madani Obstetrics and Gynecology Hospital For collaboration during data collection.

My particular thanks to my best friends Alaa Faroge , Awad Elymani, Ahmed omer and all staff of the faculty of medical laboratory sciences in Gezira University.

Finally I am grateful to all who helped me and dedicated his time and effort to help me and did not mentioned hear above.

Sero-detection of Chlamydia trachomatis Antibodies among Pregnant Women, Wad Medani Maternity Teaching Hospital, Gezira State, Sudan (2017)

Mona Osman Ahmed Ali

Abstract

Chlamydia trachomatis is an obligate intracellular microorganism; it has ability to form intra cytoplasmic inclusion bodies within infected cells. In pregnant women, colonization of Chlamydia trachomatis in the genital tract during early gestation has been associated with spontaneous preterm birth. This case control hospital based study aims to detect Chlamydia trachomatis antibodies (IgG and IgM) among spontaneous recurrent miscarriage and non- miscarriage women at Wad Medani Maternity Hospital, Gezira State. A total of 90 women with and without history of miscarriage were enrolled in this study. 45(50%) were women with recurrent miscarriage, while the other 45(50 %) were women without history of miscarriage. Five ml of blood specimens were collected from each woman and dispensed into Ethylene Diamine Tetra Acetic acid and plain containers. Serum was collected by centrifugation at 4000 r/min for 10 minutes. Sera were tested for the presence of Chlamydia trachomatis IgG antibodies and Chlamydia trachomatis IgM antibodies using indirect Enzyme Linked Immunosorbent Assay (ELISA) method, Demographic and clinical data were collected by structured questionnaire after a verbal consent. 3/45 (6.7 %) women of study subjects were positive for Chlamydia trachomatis IgG antibodies and 3/45(6.7%) were positive for Chlamydia trachomatis IgM antibodies, while 2(2.2) women of control group were positive for Chlamydia trachomatis IgM antibodies. The study concluded that seroprevalence rate of Chlamydia trachomatis IgG and IgM antibodies are higher among women with recurrent miscarriage than those without history of recurrent miscarriage. The study recommended the screening for pregnant women for Chlamydia trachomatis.

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الكشف المصلي عن االجسام المضادة للمتدثرة الحثرية بين النساء الحوامل, مستشفي ود مدني التعليمي للنساء والتوليد , والية الجزيرة, السودان )2017م( منى عثمان احمد علي

ملخص الدراسة

المتدثرة الحثرية هي من الكائنات الحية الدقيقة التي تعيش داخل الخاليا. لديها القدرة على تشكيل جسيمات داخل الخاليا المصابة. في النساء الحوامل ارتبط وجود المتدثرة الحثرية في الجهاز التناسلي أثناء الحمل بالوالدة المبكرة التلقائية. تهدف دراسة الحالة والشاهد المستندة إلى المستشفي إلى الكشف عن المتدثرة الحثرية بين النساء الالتي أصبن باالجهاض والنساء الالتي لم يصبن باالجهاض التلقائي المتكرر المتردات علي مستشفي ود مدني للنساء والتوليد, والية الجزيرة. شملت الدراسة 90امرأة , 45 )50 ٪( كن لديهم اجهاض ، 45 آخرين )50 ٪( من النساء ليس لديهم تاريخ اإلجهاض. تم جمع خمسة مل من عينات الدم من كل امرأة وتم توزيعها في انابيب اختبار تحتوى علي مضاد التجلط ايثايلين دايمين تترا اثيدك اسيد واخرى ال تحتوي علي مانع تجلط . تم تحضير المصل بواسطة الطرد المركزي عند 4000 لفة / دقيقة لمدة 10 دقائق. تم اختبار األمصال لوجود األجسام المضادة )ج( و األجسام المضادة )م( للمتدثرة الحثرية عن طريق استخدم طريقة الفحص المناعي المرتبط باإلنزيم غير المباشر ، وتم جمع البيانات الديموغرافية والسريرية من خالل استبيان بعد الحصول على الموافقة شفهيا من المستهدفين. من بين 45 امراة لديها اجهاض كانت 3 نساء )6.7%( نتائجهم ايجابية لـالجسام المضادة )ج( للمتدثرة الحثرية. و ثالث نساء )6.7٪( كانت نتائجهم ايجابية لألجسام المضاد )م( للمتدثرة الحثرية. بينما اثنان )4.4%( من النساء الالتي لم يصبن باالجهاض كانت نتائجهم ايجابية لألجسام المضادة ( م( للمتدثرة الحثرية .وخلصت الدراسة الي أن معدل انتشار االجسام المضادة )ج , م( للمتدثرة الحثرية وسط النساء الالتي تعرضن لالجهاض اعلي من معدل االنتشار وسط النساء الالتي ليس لديهن تاريخ إجهاض. اوصت الدراسة بأهمية اجراء فحص روتيني للكشف عن المتدثرة الحثرية لكل النساء الحوامل .

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TABLE OF CONTENTS

Number CONTENTS Page Supervision committee i Examination committee ii Declaration iii Dedication iv Acknowledgement v English abstract vi Arabic abstract vi Table of contents Vii List of tables viii List of abbreviations ix CHAPTER ONE 1.1 Introduction 1 1.2 Justification 2 1.3 Objectives 2 1.3.1 General objective 2 1.3.2 Specific Objectives 2 CHAPTER TWO LITERATURE REVIEW 2.1 Definition of miscarriage 4 2.2 Classification of miscarriage 4 2.3 Causes of miscarriage 4 2.4 Chlamydia Trachomatis 5 2.4.1 Taxonomy 5 2.4.2 General properties 5 2.4.3 Morphology 6 2.4.4 Genomics 7 2.4.5 Life cycle 7 2.4.6 Antigenicity 8 2.4.7 Pathogenesis and Immunity 8 2.4.8 Clinical Syndromes 9 2.4.8.1 Lymphogranuloma Venereum 10 2.4.8.2 Ocular genital Infections 10 2.4.8.3 Urogenital infections 10 2.4.8.4 Trachoma 11

2.4.8.5 Neonatal pneumonia 11 2.4.8.6 Fitz-Hugh-Curtis 12 2.4.9 laboratory Diagnosis 12 2.4.9.1 Specimens 12 2.4.9.2 diagnostic methods 12

2.4.9.2.1 Direct cytological examination 12

2.4.9.2.2 Isolation in cell culture 13

2.4.9.2.3 Antigen Detection 13

2.4.9.2.4 Nucleic Acid Hybridization 13

2.4.9.2.5 Nucleic Acid Amplification Tests 14

2.4.9.2.6 Serodiagnosis 14

2.4.9.3 Antimicrobial susceptibility testing 15

2.4.10 Treatment 15

2.4.11 prevention and control 15

2.5 The Relationship between Chlamydia trachomatis Genital Infection 16 and Spontaneous Abortion 2.6 Previous studies 16

CHAPTER THREE (MATERIALS AND METHODS)

3.1 Study design 18 3.2 Study area 18 3.3 Study population 18 3.4 Sample Size 18 3/5 Study Criteria 18 3.5.1 Inclusion criteria 18 3.5.2 Exclusion criteria 19 3.6 Data collection 19 3.7 Data analysis 19 3.8 Ethical consideration 19 3.9 Laboratory method 19 3.9.1 Collection of blood specimens 19 3.9.2 Sample processing 19 3.9.3 CBCs processing 19 3.9.4 Enzyme linked immunosorbent assay (ELISA) processing 20

3.9.4.1 Principle of Enzyme linked immunosorbent assay (ELISA) for 20 detection of C. trachomatis IgG and IgM antibodies 3.9.4.2 Procedure 20 3.9.4.3 Quality control and calculation of results 21 CHAPTER FOUR 4.1 Results 22 4.2 Discussion 32 CHAPTER FIVE

5.1 Conclusion 35 5.2 Recommendations 35

References 36 Appendix 42

LIST OF TABLE

Table Title Page 4.1 Socio-demogric and clinical characteristic of study population 23 4.2 Serodetection of anti C. trachomatis IgG and IgM according to Miscarriage 24 4.3 Serodetection of anti C. trachomatis IgG and IgM according to rate of miscarriage 24 4.4 Serodetection of anti C. trachomatis IgG and IgM according to trimester: 24 4.5 Serodetection of anti C. trachomatis IgG and IgM according to family history 25 4.6 Serodetection of anti C. trachomatis IgG and IgM according to last miscarriage 25 4.7 Serodetection of anti C. trachomatis IgG and IgM according to menstrual cycle 25 4.8 Serodetection of anti C. trachomatis IgG and IgM according to vaginal bleeding 26 4.9 Serodetection of anti C. trachomatis IgG and IgM according to vaginal disease 26 4.10 Serodetection of anti C. trachomatis IgG and IgM according to smoking 26 4.11 Serodetection of anti C. trachomatis IgG and IgM according to Thyroid 27 4.12 Serodetection of anti C. trachomatis IgG and IgM according to diabetics 27 4.13 Serodetection of anti C. trachomatis IgG and IgM according to hypertension 27 4.14 Serodetection of anti C. trachomatis IgG and IgM according to autoimmuno 28 disease 4.15 Serodetection of anti C. trachomatis IgG and IgM according to anemia type 28 4.16 Serodetection of anti C. trachomatis IgG and IgM according to preeclampsia 28 4.17 Serodetection of anti C. trachomatis IgG and IgM according to vaccine 29 4.18 Serodetection of anti C. trachomatis IgG and IgM according to vaccine type 29 4.19 Serodetection of anti C. trachomatis IgG and IgM according to blood group 30 4.20 Serodetection of anti C. trachomatis IgG and IgM according to age 30 4.21 Serodetection of anti C. trachomatis IgG and IgM according to BMI 31 4.22 Serodetection of anti C. trachomatis IgG and IgM according to education 31 4.23 Serodetection of anti C. trachomatis IgG and IgM according to jobs 31

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LISR OF ABBREVIATIONS

BMI Body Mass Index CDC Centers For Disease Control And Prevention CF Complement Fixation DFA Direct Fluorescent Antibody EB Elementary Body EIA Enzyme Immunoassays Hb Haemoglobin IgG Immunoglobulin type Gamma IgM Immunoglobulin type M IL Interleukin LGV Lymphogranuloma Venereum LPS Lipopolysaccharide MCH Mean Cell Haemoglobin MCHC Mean Cell Haemoglobin Concentration MCV Mean Cell Volume micro-IF Microimmunofluorescence Assay MOMP Major Outer Membrane Protein NAATs Nucleic Acid Amplification Tests PCR Polymerase Chain Reaction PCV Packed Cell Volume PCV Packed Cell Volume PLT Psittacosis Lymphogranuloma Trachoma RB Reticulate Body RBCs Red Blood Cell RDWCV Red Blood Cell Distribution Width RDWSD Red Cell Distribution Width Distribution Histogram RM Recurrent Miscarriage RPL Recurrent Pregnancy Loss rRNA ribosomal RNA SDA Strand Displacement Amplification TMA Transcription-Mediated Amplification TNF Tumor Necrosis WBCs White Blood Cell

CHAPTER ONE 1.1: Introduction: Recurrent miscarriage is an important reproductive health issue, occurring in 2-5% of fertile women. Early pregnancy loss, also known as miscarriage or spontaneous abortion, is the loss of a clinical pregnancy before 20 completed weeks of gestational age (18 weeks after fertilization) or, if gestational age is unknown, the loss of an embryo/fetus of < 400 g (El Hachem et al., 2017). Recurrent miscarriage or habitual abortion is three consecutive pregnancy losses prior to 20 weeks from the last menstrual period (Ford and Schust, 2009). The causes of miscarriage are often unknown. However, in 50% of early miscarriages the fetus exhibits chromosomal aberrations such as a structural alteration or abnormal chromosomal numbers. Several other factors have been associated with increased risk of miscarriage. The age of both parents has a significant role as the risk of an adverse pregnancy outcome is increased if the parents are 35 years old or older and it is 50% higher if the mother is 42 years of age. In addition, factors such as ethnic origin, psychological state of the mother, very low or very high pre-pregnancy BMI, feelings of stress, use of non- steroidal anti-inflammatory drugs, smoking and alcohol consumption have also been associated with significantly higher rates of miscarriage . Moreover, it has been reported that women whose first pregnancy resulted in miscarriage are at a higher risk of the second pregnancy resulting in miscarriage compared with women who had a live birth. Finally, a number of infections have been linked to miscarriage and to other adverse outcomes, such as stillbirth and preterm delivery. Specifically, 15% of early miscarriages and 66% of late miscarriages have been attributed to infections. In a recent study, 78% of 101 tissue samples from miscarriage were infected with bacteria (chorioamnionitis),whereas all the control samples from medically induced abortions were uninfected (Giakoumelou et al., 2017). Chlamydia is obligate intracellular bacteria, which have all the elements of bacteria except a rigid cell wall. Of the three species causing disease in humans, Chlamydia trachomatis (C. trachomatis) is the most common as a major cause of genital infection and conjunctivitis. Chlamydia psittaci are respiratory pathogens (Hogg et al., 2004). C. trachomatis in Pregnancy: Exploring Adverse Outcomes. As intracellular bacteria with an ability to exist in resting and infectious forms within human epithelial host cells, C. trachomatis presents a unique challenge to eradication. This ability to evade host detection and elimination also contributes to its ability to cause adverse outcomes among women. While it is often an asymptomatic infection in women, is also an important cause of

cervicitis, arthritis, and pelvic inflammatory disease(PID), which is an ascending infection of the uterus, fallopian tubes, or neighboring pelvic structures that can vary in presentation as asymptomatic endometritis, salpingitis, tuboovarian abscess, pelvic peritonitis, perihepatitis, or perappendicitis (Adachi et al., 2016). 1.2 Justification:

Miscarriage is one of the most common yet under-studied adverse pregnancy outcomes. Miscarriage occurs in one in five pregnancies and has considerable physiological and psychological implications for the patient. It is also associated with significant health care costs. C. trachomatis infection is one of the most prevalent sexually transmitted diseases worldwide. The incidence of the C. trachomatis infection has dramatically increased during the past 10 years. The World Health Organization estimates that there are 92 million new cases every year, with 10 million of these in Europe. Unfortunately, more than 80% of cases are asymptomatic, particularly among females, and can lead to continued transmission of the infection and chronic infection with a high risk of pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, salpingitis or tubal factor of infertility. Also C. trachomatis is a recognized agent of preterm labour and premature rupture of membranes; women are predisposed to postpartum pelvic inflammatory diseases and the neonatal complications of infant low birth, conjunctivitis and pneumonia(Fiolka et al., 2013).

1.3 Objectives: 1.3.1. General objectives: Sero-detection of C. trachomatis Antibodies IgG and IgM among Spontaneous Recurrent Miscarriage and Non Miscarriage Women in Gezira state

1.3.2. Specific Objectives:

1. To detect (IgG) antibodies to C. trachomatis among women with recurrent miscarriage in Gezira state. 2. To detect (IgM) antibodies to C. trachomatis among women with recurrent miscarriage in Gezira state. 3. To determine association between C. trachomatis and recurrent miscarriage in Gezira state.

4. To determine the associated factors in related with C. trachomatis infection include age, trimester, blood group, occupation, chronic disease and other among women with recurrent miscarriage in Gezira state.

CHAPTER TWO LITERATURE REVIEW 2.1. Definition of miscarriage:

Miscarriage also known as spontaneous abortion or early pregnancy loss is the loss of a clinical pregnancy before 20 completed weeks of gestational age (18 weeks after fertilization) or, if gestational age is unknown, the loss of an embryo/fetus of < 400 g. Ectopic, molar, and biochemical pregnancies are thus not included. It is a relatively common event, occurring in 15%–25% of pregnancies, and increasing in prevalence with maternal age. Indeed, the risk is between 9% and 12% in women aged ≤ 35 years, but increases to 50% in women aged ≥ 40 years (El Hachem et al., 2017). The recurrent pregnancy loss (RPL) also referred to as recurrent miscarriage or habitual abortion, has long been debated and differs among international societies. For the European Society for Human Reproduction and Embryology and the Royal College of Obstetricians and Gynecologists, RPL refers to three consecutive pregnancy losses, including non visualized ones. However, according to the American Society for Reproductive Medicine, it is defined as two or more clinical pregnancy losses (documented by ultrasonography or histopathologic examination), but not necessarily consecutive. Primary RPL refers to multiple losses in a woman with no previous viable infants, whereas secondary RPL refers to multiple losses in a woman who has already had a pregnancy beyond 20 gestational weeks. Tertiary RPL refers to multiple pregnancy losses between normal pregnancies(El Hachem et al., 2017).Recurrent miscarriage is occurring in ~1-2% of fertile women. Following investigation, most cases fail to reveal an identifiable cause and are therefore classified as idiopathic(Langeveld et al., 2018).

2.2 Classification of miscarriage: Miscarriage can be classified as embryonic loss (or early miscarriage) when it occurs before 10 gestational weeks, and fetal loss (or fetal miscarriage) when it occurs after 10 gestational weeks, because factors associated with each may differ (El Hachem et al., 2017). 2.3 Causes of miscarriage: The causes of miscarriage are often unknown. However, in 50% of early miscarriages the fetus exhibits chromosomal aberrations such as a structural alteration or abnormal chromosomal numbers(Diego-Alvarez et al., 2005, Suzumori and Sugiura-Ogasawara, 2010)Several other factors have been associated with increased risk of miscarriage. The age of both parents has a significant role as the risk of an adverse pregnancy outcome is increased

if the parents are 35 years old or older and it is 50% higher if the mother is 42 years of age In addition, factors such as ethnic origin, psychological state of the mother, very low or very high pre-pregnancy BMI, feelings of stress, use of non-steroidal anti-inflammatory drugs, smoking and alcohol consumption have also been associated with significantly higher rates of miscarriage (Toullec et al., 1991).(Barndorff‐Nielsen and Shephard, 2001) Moreover, it has been reported that women whose first pregnancy resulted in miscarriage are at a higher risk of the second pregnancy resulting in miscarriage compared with women who had a live birth (Knopp et al., 2006). Finally, a number of infections have been linked to miscarriage(Bauman, 2004) and to other adverse outcomes, such as stillbirth and preterm delivery(Garland-Thomson, 2002). Specifically, 15% of early miscarriages and 66% of late miscarriages have been attributed to infections(Agarwal et al., 2007), In a recent study, 78% of 101 tissue samples from miscarriage were infected with bacteria (chorioamnionitis), whereas all the control samples from medically induced abortions were uninfected (Cassidy and Allanson, 2010). 2.4. Chlamydia trachomatis: 2.4.1. Taxonomy: Chlamydia has undergone extensive revision recently on the basis of genomic studies of this microorganism (Parija, 2012). Formerly grouped with the Rickettsia , these non-motile obligate parasites of birds and mammals (Dautry‐Varsat et al., 2005). Chlamydia is included in the order Chlamydiales, which contains one family Chlamydiaceae. Previously, the family consisted of a single genus Chlamydia with four species (Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia pecorum)(Mandal et al., 2012).Now the family has been reclassified into two genera: Chlamydia and Chlamydophila, based on differences in phenotype,16S ribosomal ribonucleic acid (rRNA), and 23S rRNA(Tillé and Wilhelm, 2017) The species C. trachomatis is included in the genus Chlamydia, whereas C. psittaci and C. pneumonia are included in the new genus Chlamydophila, classification is based on antigens, morphology of intracellular inclusions and disease patterns .All chlamydiaceae share a common group antigen but may be distinguished by species-specific antigens(Barone and Eisner, 2011).

2.4.2. General properties: Chlamydiae are obligate intracellular parasites of humans and animals with marked affinity for the squamous epithelial cells of the gastrointestinal and respiratory tracts. The chlamydiae were once regarded as viruses because, like viruses, the chlamydiae require biochemical

resources of eukaryotic host cells to fuel their metabolism for growth and replication by providing high-energy compounds such as adenosine triphosphate (Patricia, 2014),and due to their filterability through 0.45 µm filter. A Earlier they were named Psittacosis lymphogranuloma trachoma (PLT) viruses or PLT agents (Mandal et al., 2012). Chlamydia spp. are similar to bacteria in that they have both DNA and RNA, They possess cell wall as that of Gram-negative bacteria, They contain prokaryotic ribosomes They multiply by binary fission, They produce and synthesize their own nucleic acid, lipids, and proteins, They are susceptible to a wide range of antibiotics, such as tetracyclines, erythromycin, macrolides, and rifampicin (Mandal et al., 2012). The chlamydial lipopolysaccharide (LPS), however, has little endotoxic activity. The chlamydiae have a major outer membrane protein (MOMP) that is very diverse. The variation in MOMP in Chlamydia trachomatis is used to separate the species into18 distinct serovars, yet highly conserved in Chlamydia pneumoniae (Tillé and Wilhelm, 2017)Susceptibility to physical and chemical agents: Chlamydiae are heat-labile bacteria and are readily killed within minutes by heating at 56°C. They are susceptible to ethanol, ether, phenol, formalin, iodine, potassium permanganate, sodium hypochlorite, silver nitrite, and chlorite. They remain fully viable for several days at 4°C. Moreover, they can be preserved at -70°C or in liquid nitrogen for a long period. The chlamydiae occur in two morphologically distinct forms: elementary body and reticulate body(Mandal et al., 2012).The elementary body (EB) is a small, extracellular, infective form. It is a round particle measuring 300–400 nm in size. The cell wall possesses a rigid trilaminar structure as seen in the cell walls of Gram-negative bacteria. These bacteria lack peptidoglycan layer found in other bacteria. However, their outer membrane proteins due to the presence of extensive cross-linked protein of the outer membrane confer rigidity to the cell wall. The chlamydiae do not multiply in the EB form, but are infectious. They cause infections by binding to receptors on the epithelial cells and stimulate uptake of the bacteria by infiltration(Mandal et al., 2012). The reticulate body (RB) is a large, noninfectious form of Chlamydia. It measures 500–1000 nm in size. It is metabolically active and replicating form of Chlamydia. The extensive cross-linked proteins that confer rigidity are absent in the RBs. Hence, this form of Chlamydia is osmotically fragile and friable. This form, however, is protected by its intracellular location by (Mandal et al., 2012). 2.4.3. Morphology: C. trachomatis are round cells between 0.3 and 1 µm in diameter depending on the replicative stage (Hogg et al., 2004). C. trachomatis are Gram-negative bacteria. However,

they can be stained better by Giemsa, Castaneda, Machiavello, or Gimenez stains. C. trachomatis like other chlamydiae occurs in two morphologically distinct forms: elementary body and reticulate body. EB is an extracellular infectious particle. It is small and spherical and measures 800–1200 nm in diameter. These inclusion bodies in the infected cells, such as conjunctiva, urethra, and corneal smears can be demonstrated after staining with Giemsa, Castaneda, or Machiavello methods. These inclusion bodies are large-sized particles, which can be easily demonstrated under light microscope. These bodies consist of glycogen matrix, hence are demonstrated on staining with Lugol’s iodine. Reticulate body is metabolically active and replicating form of Chlamydia (Mandal et al., 2012). 2.4.4. Genomics: C. trachomatis has a genome that consists of 1,042,519 nucleotide base pairs and has approximately 894 likely protein coding sequences. C.trachomatis strains have an extra chromosomal plasmid, which was sequenced to be 7493 base pair plasmid, the plasmid of C. trachomatis is favored target DNA-based diagnosis of C. trachomatis because there are approximately 7-10 copies of the plasmid present per chlamydial particle. Some C. trachomatis strains lack these plasmids, and the consequences aid in detection of the plasmid free variant of C. trachomatis strain .Plaque purified C. trachomatis that do not contain the plasmids have usually inclusion morphology, have no glycogen, and show no alteration in antibiotic sensitivity. However, the fact that existence of such strains shows that the plasmid is not essential for C. trachomatis survival (Islam et al., 2012). 2.4.5. Life Cycle: Elementary bodies are the infective form, which initiate the cycle. The infection is initiated by the attachment of the EB to the micro villi of susceptible epithelial cells followed by penetration into the host cell. Inside the host cells, EB remains within the cytoplasmic phagosomes in which EBs begin to multiply. The fusion of EB containing phagosome with cell wall lysosome is prevented; thus intracellular killing of EB is inhibited. This phago- lysosomal fusion is usually prevented in the host cells with the intact outer membrane. Within 6–8 hours after entering the cell, the EBs within the phagosome is transformed to large, metabolically active RBs. These RBs synthesize their own proteins and nucleic acids but lack the ability to produce their own high-energy phosphate compound. The chlamydiae are called energy parasites because of this deficiency. Some strains of Chlamydia also depend on the host for the requirement of their amino acid. The RBs multiply by binary fission, which continues for next 18–24 hours. The developing phagosome with accumulated

reticulated bodies within the host cell is called the inclusion body. The mature inclusion body contains nearly 100–500 EBs, which can be readily demonstrated by various staining procedures. Ultimately, the host cell ruptures, releasing the EBs .In C. trachomatis infection, the release of EBs occurs within 70–96 hours. The release of the host cell is marked by the presence of a scar. The release of EBs in C. psittaci infection occurs within 48 hours by lysis of the host cell leading to severe damage of the infected host cell. Chlamydiae during the active growth express the Chlamydia specific LPSs on the cell surface of the infected host cell. These LPSs expressed on the outer surface of the cell are highly antigenic and induce immunological and inflammatory responses(Mandal et al., 2012). 2.4.6. Antigenicity: Chlamydiae possess three types of major antigens: (a) genus specific antigens, (b) species- specific antigens, and (c) sero type specific antigen. (a) Genus-specific antigen: This is a heat-stable, complement-fixing, and genus-specific antigen. It is an LPS–protein complex resembling the LPS of Gram-negative bacilli. It is present in EBs and RBs. The antigen can be extracted by ether, chloroform, or methanol. The antigen is identified by CFT. (b)Species-specific antigen: This antigen is present at the envelope surface and is species specific. This antigen is present in all the strains of Chlamydia.(c)Serotype-specific antigen: This antigen is present only in a few species of chlamydiae. They are located in the major outer membrane proteins (MOMPs) and are useful for intra species typing of Chlamydia species. Typing of species on the basis of these antigens, Chlamydia species are classified into various serovars and serologic variants. C. trachomatis has been subdivided into three biological variants known as biovars: (a) trachoma biovar causing trachoma and inclusion conjunctivitis (TRIC), (b) lymphogranuloma venereum (LGV) biovar causing LGV, and (c) serovars causing mouse pneumonitis. These biovars on the basis of antigenic differences in the MOMPs have been further classified into 20 serotypes. The trachoma biovar consists of 13 serotypes (A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, Ja and K). The LGV biovar consists of only five serotypes (L1, L2, L2a, L2b, and L3(Mandal et al., 2012). 2.4.7 Pathogenesis and Immunity: Virulence factors: The ability to multiply intracellularly in the infected cell is the key mechanism of virulence of C. trachomatis. The bacteria prevent fusion of phago lysosome with cellular liposomes, thereby preventing intracellular killing of the bacteria by the host cell. C. trachomatis causes disease mainly by (a) direct destruction of infected host cells

during multiplication and (b) inducing inflammatory responses in the host(Mandal et al., 2012).The mechanisms by which C. trachomatis cause inflammation and tissue destruction are not completely understood. The C. trachomatis can infect a variety of different cells, including epithelial cells of the mucosa and blood vessels, smooth muscle cells, and monocytes . The chlamydial EB is phago cytosed into a host cell and resides in a vacuole that fails to fuse with a lysosome, leading to the intracellular persistence of the organism and escape from the host immune response. Chlamydiae are able to either turn on or turn off apoptosis (programmed cell death pathways) in infected host cells. By inducing host cell death, the organism facilitates its transmission to neighboring host cells and down regulating inflammation in the acute disease process, whereas, by inhibiting apoptosis, the organism keeps the host cell alive, allowing for sustained survival in chronic infections. The host’s immune response accounts for the majority of the tissue destruction after infection with C. trachomatis. Infected epithelial cells secrete proinflammatory cytokines including interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6. Quickly upon infection, Neutrophil and Monocyte migrate to the mucosa and eliminate exposed EB. Later, CD4 T helper cells migrate to the site of infection. Responding Neutrophil and T helper cells release cytokines, resulting in the influx of additional immune cells. The importance of multiple, recurrent infections with C. trachomatis are associated with the development of ocular trachoma. Immunity provides little protection from reinfection and appears to be short lived after infection with C. trachomatis(Patricia, 2014).C. trachomatis infections do not reliably result in protection against reinfection although there is evidence that secretory immunoglobulin A may confer at least some partial immunity against genital tract reinfection. Any strain-specific protection that may result is short-lived. Local production of antibody, along with CD4+ lymphocytes of the Th1 type that traffic to the genital mucosa may together play a role in mitigating most acute infections. This would at least partially explain why most untreated chlamydial genital tract infections are persistent, but often subclinical in character(Patricia, 2014). 2.4.8. Clinical Syndromes: C. trachomatis causes a variety of diseases. It is an important cause of genital and ocular infections worldwide. C.trachomatis LGV biovar causes lymphogranuloma venereum (LGV) and ocular LGV. C. trachomatis trachoma biovar causes (a) trachoma, (b) adult inclusion conjunctivitis, (c) neonatal conjunctivitis, (d) infant pneumonia, and (e) urogenital infections(Mandal et al., 2012).

2.4.8.1. Lymphogranuloma Venereum: Lymphogranuloma venereum (LGV) is a sexually transmitted disease rarely identified in North America but relatively frequent in Africa, Asia, and South America. It is reemerging in Europe, especially in homosexual males. C. trachomatis serovars L1, L2, L2b, and L3 are invasive, causing LGV, in contrast to C. trachomatis serovars A through K, which leave the mucosa to spread to the regional lymph nodes. The disease is characterized by a brief appearance of a primary genital lesion at the initial infection site. This lesion is often small and may be unrecognized, especially by female patients. The second stage, acute lymphadenitis, often involves the inguinal lymph nodes, causing them to enlarge and become matted together, forming a large area of groin swelling, or bubo. During this stage, infection may become systemic and cause fever or may spread locally, causing granulomatous proctitis. In a few patients (more females than males), the disease progresses to a chronic third stage, causing the development of genital hyperplasia, rectal fistulas, rectal stricture, draining sinuses, and other manifestations(Mandal et al., 2012). 2.4.8.2. Ocular genital Infections: C. trachomatis can cause acute inclusion conjunctivitis in adults and newborns. The organism is acquired when contaminated genital secretions get into the eyes via fingers or during passage of the neonate through the birth canal. Autoinfection rarely occurs. The organism can also be acquired from swimming pools, from poorly chlorinated hot tubs, or by sharing eye makeup. Inclusion conjunctivitis is associated with swollen eyes and a purulent discharge. In contrast to trachoma, inclusion conjunctivitis does not lead to blindness in adults (or newborns) (Mandal et al., 2012). 2.4.8.3. Urogenital infections: Genital tract infections caused by C. trachomatis have surpassed gonococcal (Neisseria gonorrhoeae) infections as a top cause of sexually transmitted disease in the United States. Similar to gonococci, C. trachomatis causes urethritis, cervicitis, bartholinitis (Bartholin glands or greater vestibular glands), proctitis, salpingitis (infection of the fallopian tubes), epididymitis, and acute urethral syndrome in females. In the United States, 60% of cases of non gonococcal arthritis are caused by chlamydiae. Both chlamydiae and gonococci are major causes of PID, contributing significantly to the rising rate of infertility and ectopic pregnancies in young women. After a single episode of increases dramatically with each additional episode

PID, as many as 10% of women may become infertile because of tubal occlusion. The risk many genital chlamydial infections in both sexes are asymptomatic or not easily recognized by clinical criteria; asymptomatic carriage in both males and females may persist, often for months. As many as 50% of males and 70% to 80% of females identified as having chlamydial genital tract infections have no symptoms. Of significance, these asymptomatic infected individuals serve as a large reservoir to sustain transmission of the organism within a community. When symptomatic, patients with a genital chlamydial infection will have an unusual discharge and pain or a burning sensation, symptoms similar to those for gonorrhea. 2.4.8.4. Trachoma: There are eight different serotypes of Chlamydia trachomatis responsible for inclusion conjunctivitis (D–K) (Fig. 25.1) and another four serotypes responsible for trachoma (A, B, Ba and C), which, globally, is the most important eye infection in the world. Trachoma is manifested by a chronic inflammation of the conjunctiva and remains a major cause of preventable blindness worldwide. The organism is acquired as a result of contact with infected secretions on towels or fingers or by flies. Early symptoms of infection include mild irritation and itching of the eyes and eyelids. There may also be some discharge from the infected eye. The infection progresses slowly with increasing eye pain, blurred vision, and photophobia. Repeated infections result in scarring of the inner eyelid that may then turn the eyelid in toward the eye (entropion). As the inner eyelid continues to turn in, the eyelashes follow (trichiasis), resulting in rubbing and scratching of the cornea. The combined effects of the mechanical damage to the cornea and inflammation result in ulceration, scarring, and loss of vision(Goering et al., 2013) 2.4.8.5. Neonatal pneumonia: Of newborns infected by the mother, 10–20% may develop respiratory tract involvement 2– 12 weeks after birth, culminating in pneumonia. C. trachomatis may be the most common cause of neonatal pneumonia. Affected newborns have striking tachypnea, a characteristic paroxysmal staccato cough, an absence of fever, and Eosinophilia. Consolidation of lungs and hyperinflation can be seen on radiographs. The diagnosis should be suspected if pneumonitis develops in a newborn that has inclusion conjunctivitis and can be established by isolation of C. trachomatis from respiratory secretions. In such neonatal pneumonia, an immunoglobulin M (IgM) antibody titer to C. trachomatis of 1:32 or more is considered diagnostic. Oral erythromycin for 14 days is recommended; systemic erythromycin is effective treatment in severe cases(Jawetz et al., 2013).

2.4.8.6. Fitz-Hugh-Curtis: Fitz-Hugh-Curtis (FHC) syndrome is defined as perihepatitis associated with pelvic inflammatory disease (PID) (Yang et al., 2008), also known as acute perihepatitis (Muschart, 2015). It was discovered in 1930 and is a rare disorder characterized by inflammation of the peritoneum and the tissues surrounding the liver. This syndrome is not well known in the medical community and is often under (Nougon et al., 2015) 2.4.9. Laboratory Diagnosis: C. trachomatis can be diagnosed by cytology, culture, direct detection of antigen or nucleic acid, and serologic testing. The sensitivity of the diagnostic procedures depends on (a) the nature of the disease, (b) site of infection from where the specimen is collected, and (c) the population of the patient examined or newborns) (Mandal et al., 2012) 2.4.9.1. Specimens: Chlamydiae are labile bacteria, and viability can be maintained by keeping specimens cold and minimizing the time between specimen collections and processing in the laboratory. A variety of swab types can be used, but toxicity related to materials in swabs can be problematic. It is useful, therefore, to test swab types for toxicity in cell cultures or interference in non culture assays when proprietary swabs are not provided by the manufacturer, specimens from urethra, cervix, rectum, or pharynx, and conjunctiva are the frequently collected specimens. In addition, other specimens such as, blood, respiratory secretions, sputum, lung, and other tissues are collected and examined. Pus from bubo is also useful for diagnosis of LGV (Mandal et al., 2012), For successful culture of chlamydiae, swabs, scrapings and small tissue samples should be forwarded to the laboratory in a special chlamydial transport medium such as 2SP (0.2 M sucrose-phosphate transport medium containing 10 μg of gentamicin/mL, 25 μg of vancomycin/mL and 25 U of nystatin/mL). Broad-spectrum antibiotics such as tetracyclines, macrolides or penicillin cannot be used in the transport media because they have activity against chlamydiae. Chlamydial specimens should be refrigerated on receipt in the laboratory; if specimens cannot be processed within 24 h after collection, they should be frozen at –70°C(Shepard et al., 2005) 2.4.9.2 Diagnostic methods: 2.4.9.2.1. Direct cytological examination: Cytological examination of cell scrapings from the conjunctiva of newborns or persons with ocular trachoma can be used to detect C. trachomatis inclusions, usually after Giemsa staining. Cytology has also been used to evaluate endocervical and urethral scrapings,

including those obtained for Pap smears. However, this method is insensitive compared with culture character (Patricia, 2014),Examine the smear microscopically using the 40_ and 100_ objectives. Look for epithelial cells that contain inclusion bodies. C. trachomatis inclusion bodies vary in size and shape and lie in the cytoplasm of the host cell often touching and forming a mantle around the cell’s nucleus (chlamydia means mantle)(Cheesbrough, 2006) Appearance in Giemsa stain: Elementary body -- Purple, Reticulate body----Blue, Host cell cytoplasm----- Blue. Appearance in iodine stain ---Brown inclusions in host cell cytoplasm because of glycogen matrix surrounding the particle(Cheesbrough, 2006) 2.4.9.2.2. Isolation in cell culture: Isolation of C. trachomatis in cell cultures is the more specific method for diagnosis of C. trachomatis infection. Clinical specimens are inoculated in different cell lines for isolation of Chlamydia. The sensitivity of the cell cultures for isolation of C. trachomatis is increased by (a) pretreatment with cycloheximide (i.e., a metabolic inhibitor, which inhibits the metabolism of host cells) and (b) use of irradiated cell lines (treated McCoy cells are most commonly used cell lines for isolation of C. trachomatis). C. trachomatis infection in cell culture is demonstrated by the presence of intracellular inclusion bodies. These are detected by the use of iodine stains or fluorescence-conjugated antibodies. The culture methods are difficult and expensive. These are specifically preferred for isolation of C. trachomatis from rectal specimens, because non cultural methods are usually negative. The culture shows a sensitivity of 50–90% and specificity of 99%(Mandal et al., 2012) 2.4.9.2.3 Antigen Detection: Direct fluorescent antibody (DFA) staining methods use fluorescein-isothiocyanate conjugated monoclonal antibodies to either MOMP or LPS of C. trachomatis to detect elementary bodies in smears of clinical material (Figure 43-2). The sensitivity and specificity of DFA are similar to those of culture. Chlamydial antigen can also be detected by enzyme immunoassays (EIA). Numerous U.S. Food and Drug Administration (FDA) – approved kits are commercially available. These assays use polyclonal or monoclonal antibodies that detect chlamydial LPS. These tests are not species-specific for C. trachomatis and may cross react with LPS of other bacterial species present in the vagina or urinary tract and thereby produce a false-positive result(Patricia, 2014) 2.4.9.2.4. Nucleic Acid Hybridization: Nucleic acid hybridization tests for Chlamydia were first available for the clinical microbiology laboratory in the late 1980s. Two hybridization tests were commercially

developed for the detection of Chlamydia, Gen-Probe PACE 2C (Hologic-Gen-Probe, San Diego, CA) and Digene Hybrid Capture II assay (Digene, Silver Spring, MD). However, the PACE 2 test is no longer commercially available. The Digene Hybrid Capture II assay uses a ribonucleic acid (RNA) probe to detect chlamydial deoxyribonucleic acid (DNA) in a sample. The DNA/RNA hybrids are captured using monoclonal antibodies imbedded on the side of the well that recognize the unique structure produced by the DNA/RNA hybrid. A second enzyme labeled anti-DNA/ RNA hybrid antibody binds to captured hybrids, and enzyme activity is measured by chemiluminescence. The assay is species specific for the detection of C. trachomatis(Patricia, 2014) 2.4.9.2.5. Nucleic Acid Amplification Tests: FDA-approved nucleic acid amplification tests (NAATs) for the laboratory Diagnosis of C. trachomatis infection use three different formats: polymerase chain reaction (PCR), strand displacement amplification (SDA), and transcription-mediated amplification (TMA). The first two assay formats amplify DNA sequences present in the cryptic plasmid that is present in 7 to 10 copies in the chlamydial EB, whereas the last format amplifies 23S ribosomal RNA (rRNA) sequences. Studies clearly indicate that NAATs are more sensitive than culture and other non–nucleic acid amplification assays. Because of the increased sensitivity of detection, first-voided urine specimens from symptomatic and asymptomatic males and females are acceptable specimens to detect C. trachomatis, thereby affording a noninvasive means of C. trachomatis testing. NAATs are the preferred methodology for detecting C. trachomatis in most clinical situations because of increased sensitivity, ease of specimen collection, and the availability of automated high volume methods(Patricia, 2014) 2.4.9.2.6. Serodiagnosis: Serologic testing has limited value for diagnosis of urogenital infections in adults. Most adults with chlamydial infection have had a previous exposure to C. trachomatis and are therefore sero positive. Serology can be used to diagnose LGV. Antibodies to a genus- specific antigen can be detected by complement fixation (CF), and a single-point titer greater than 1:64 is indicative of LGV. This test is not useful in diagnosing trachoma, inclusion conjunctivitis, or neonatal infections. The micro immunofluorescence assay (micro-IF), at edious and difficult test, is used for type-specific antibodies of C. trachomatis and can also be used to diagnose LGV. A high titer of IgM (1:32) suggests a recent infection; however, not all patients produce IgM. In contrast to CF, micro-IF may be used to diagnose trachoma and inclusion conjunctivitis using acute and convalescent phase sera. Detection of C.

trachomatis–specific IgM is useful in the diagnosis of neonatal infections. Negative serology can reliably exclude chlamydial infection (Patricia, 2014) 2.4.9.3 Antimicrobial susceptibility testing: Antimicrobial susceptibility testing in chlamydiae is typically performed in cell culture with increasing concentrations of antibiotics. Drug efficacy is then determined by staining cells with fluorescently labelled antichlamydial antibodies and microscopically enumerating the intracellular chlamydial inclusions. Susceptibility testing of chlamydiae is problematic due to the lack of standardized techniques and because of the variability introduced by the type of cell culture system used, different cell types, inoculum size and the timing and duration of antibiotic application to the cell culture. It is unclear whether the end points measured by in vitro susceptibility testing are relevant when applied to a naturally occurring infection with dividing and non dividing bacteria that infect multiple cell types in vivo. Thus, the results of in vitro susceptibility testing may not predict the microbiological efficacy in vivo. For these reasons, susceptibility testing is not routinely performed by clinical laboratories(Schachter et al., 2005) 2.4.10 treatment: Azithromycin is the drug of choice for treatment of genital chlamydial infections. This antibiotic has the advantage of being given in a single dose regimen and has high tolerability and few contraindications. Tetracyclines are usually recommended for treatment of patients with LGV for at least 3 weeks. Children below 9 years, pregnant women, and patients unable to tolerate tetracyclines are treated with a macrolide, such as erythromycin or Azithromycin in combination with sulfisoxazole.Doxycycline for 7 days or fluoroquinolone (e.g., ofloxacin)for 7 days is also effective for treatment of genital and ocular infections. Erythromycin given for 10–14 days is very useful for treatment of conjunctivitis in infants and infant pneumonia. Erythromycin may be administered orally and topically for treatment of ophthalmia neonatorum(Saravanan and Vignesh, 2012) 2.4.11. Prevention and control: Primary prevention starts with changing sexual behaviors that increase the risk of contracting STDs, and do standardized detection and treatment of STDs. The CDC recommends annual screening for chlamydial infection in all sexually active women 24 years and younger and in women older than 24 years who are at risk of STDs. The U.S. Preventive Services Task Force (USPSTF) strongly recommends that all women 25 years and younger receive routine screening for chlamydia. (KARL E. MILLER, 2006)

Because no effective vaccines are available, strategies to prevent chlamydial urogenital infections focus on trying to manifest behavioral changes(Mandal et al., 2012) 2.5. The Relationship between Chlamydia trachomatis Genital Infection and Spontaneous Abortion: In pregnant women, colonization of C. trachomatis in the genital tract during early gestation has been associated with spontaneous preterm birth (PTB), preterm premature rupture of the membranes, prematurity, spontaneous abortion, perinatal morbidity and mortality. In addition, the pregnancy may increase the risk of C. trachomatis colonization due to changes in the host immune responses. The suggested mechanisms by which C. trachomatis can trigger PTB or abortion are invasion of chlamydia into the choriodecidual space and subsequent immune responses and placental inflammation, especially chorioamnionitis. Chorioamnionitis can influence protease release, which leads to the premature rupture of the membranes, activation of arachidonic acid cascade, uterine contractions and preterm delivery or abortion (Ahmadi et al., 2016) 2.6. Previous studies: Study conducted to detect IgG and IgM in serum of pregnant women, and to detect the presence of C .trachomatis antibodies in women with ectopic pregnancy. In Khartoum, Sudan Three hundred blood samples were collected from pregnant women with a gestational age between 25 and 36 weeks. Anti- Chlamydia IgG and anti- Chlamydia IgM were detected using ELISA technique. The result revealed that the rate of anti Chlamydia IgG among pregnant women was 17 (5.6%) and 4 (1.3%) were positive for both anti- Chlamydia IgM and IgG. This study showed a correlation between C. trachomatis positivity and ectopic pregnancy(Mohager et al., 2014).The case-control study was conducted from August 2012 until January 2013.In Sanandaj, Iran to evaluate the frequency of C. trachomatis infection among pregnant women and its association with spontaneous abortion. The Results shows that the total prevalence of C. trachomatis infection was 38(17.43%) in endocervical swabs of women. However, the number of cases with C. trachomatis infections was 25 out of 109(22.9%) in the case group and 13 out of 109(11.9%) in control group, respectively. Association between chlamydia infection and spontaneous abortion was statistically significant (OR=2.198, CI 95%: 1.058-4.56) (Ahmadi et al., 2016).Other study was done at Zürich, Switzerland, to determine the role of Chlamydia trachomatis in miscarriage; we prospectively collected serum, cervicovaginal swab specimens, and placental samples from 386 women with and without miscarriage. Prevalence of immunoglobulin G against C.

trachomatis was higher in the miscarriage group than in the control group (15.2% vs. 7.3%; p = 0.018). Association between C. trachomatis–positive serologic results and miscarriage remained signifi cant after adjustment for age, origin, education, and number of sex partners (odds ratio 2.3, 95% confi dence interval 1.1–4.9)(Baud and Greub, 2011).The case- study was done in Sudanese pregnant women whose attended Khartoum Educational Hospital “Khartoum state, Sudan” during the period from May to August 2012. Thirteen serum samples (14.1%) were sero-positive for anti-chlamydial IgG and 5 samples (5.4%) were reactive for anti-chlamydial IgA. The results revealed statistically a significant relationship between immunoglobulin IgG and previous abortion with P value 0.016, whereas the presence of IgA showed insignificant relations with the previous abortion (P=0.325)(Eltayeb and Kiliçman, 2013).A study on Serologic evidence of previous C. trachomatis infection has been found to be associated with ectopic pregnancy, in Ottawa, Ont. The study result indicates that serologic evidence of C. trachomatis infection is associated with an increased risk for preterm delivery. An odds ratio of 3.96 for preterm birth among the sero positive women suggests that the serologic status may be more important than other known risk factors for preterm birth(Claman et al., 1995).Visnovsky et al performed a study that aimed to determine the role of Chlamydia trachomatis in miscarriage at Slovakia, the women on their study were evaluated according to age and trimester of abortion, they revealed that prevalence of miscarriage was significantly higher in group with positive Chlamydia trachomatis infection than pregnant women (67.3% vs. 36%), they also revealed that the majority of positive women with Chlamydia trachomatis miscarried at first trimester of pregnancy and aged between 17 to 34 years old(Fiolka et al., 2013). Other study was done at Switzerland to investigate the involvement of Chlamydia trachomatis on 195 cases of spontaneous abortions by using formalin fixed placental tissues and examined immunohistochemical, their result showed that all placental sections were negative for Chlamydial antigen, so they concluded that there is no association of Chlamydia with abortion(Hansen et al., 2002).A study on Chlamydial serologic studies and recurrent spontaneous abortions was performed at Edinburgh, Scotland to investigate the association between antibodies to Chlamydia trachomatis and recurrent spontaneous abortions, their result showed that among 26 (24.5%) of women with recurrent spontaneous abortions had antibodies to Chlamydia trachomatis compared with 28 (34.6%) of their partners (p< 0.03), so they concluded that there is no association between antibodies to Chlamydia trachomatis and recurrent spontaneous abortions(Rea et al., 2004).

CHAPTER THREE MATERIALS AND METHODS

3.1 Study design:

Case Control laboratory base study.

3.2 Study area:

This study was conducted in Al-Gezira state (Wad Madani teaching hospital Department of obstetrics and gynecological).

3.3 Study population:

Pregnant women with and without history of miscarriage were included.

3.4 Sample size:

The study included 90 pregnant women, 45 females with at least three or more consecutive recurrent miscarriage (subject) and 50 with no history of miscarriage (controls). Then were collected blood samples for measurement the serum Chlamydia trachomatis antibody level at both groups. According to formula since the global reported-risk of recurrent miscarriage was 1%; by applying the classical equation to estimate the sample size. n=Z σ2 /d) 2

Z= was the value of specified level of significance, in this study the level of significancewas1%whichgivesavalueofZ=0.799σ = the value of S.D was estimated to be 2.5. d = was the difference between the case mean and control mean was estimated not to exceed0.5.Hence, applying these values n= (0.799×2.52 / 0.5)2 = 100 women. For some limitation fund of research 90 women were involved in this research.

3.5. Study Criteria: 3.5.1. Inclusion criteria:  For study group pregnant women with three or more consecutive recurrent miscarriage  For control group pregnant women with no history of miscarriage.

3.5.2 Exclusion criteria:  Pregnant women with history of miscarriage less than three times  Pregnant women who refused to participate. 3.6. Data collection: Data were collected by using a questionnaire and filled by the investigator during each time when blood samples were collected. 3.7. Data analysis:

Data were analyzed and tabulated using statistical package for social sciences (SPSS) program version 21,unpaired sample test, a crosstabs and correlation were performed by using Chi2 test estimated difference by P value and evaluated the Odd ratio with confidence interval range Lower and upper limitation.

3.8. Ethical consideration:

This study was approved by college of Medical Laboratory Science ethical committee, SUST. Permission from hospital was applied and verbal conset was taken from participants involved in the study.

3.9. Laboratory methods:

3.9.1. Collection of blood specimens:

Five ml of blood specimens were collected from each woman and dispensed into EDTA and plain blood container. Serum sample were collected by centrifugation at 4000 r/min for 10 minutes.

3.9.2 Sample processing:

Each blood specimen was centrifuged at 4000 r/min for 10 minutes, the EDTA specimen to obtain the plasma, and plan specimen to obtain serum. Later was gently collected into eppendorf tubes and stored at -20 °C until the serological analysis. 3.9.3. CBCs processing:

Complete blood count calculated by using hematological analyzer (Sysmex – XP 300) Manfacture Company. The three meain physical technology used in it, electrical impedance,

flow cytometer and fluorescent flow cytometer, these are used in combination with chemical reagent that lyes or alter blood cell to extend the measurable parameters. Wide range of test is done by it. 3.9.4 Enzyme linked immunosorbent assay (ELISA) processing: The specimens were analyzed for qualitative detection of Chlamydia trachomatis IgG and IgM antibodies by commercially available enzyme–linked immunosorbent assay ʽ C. trachomatis IgG and IgM ELISAʼ kit (Foresight, Acon laboratories, Inc., 10125 Mesa Rim Road, san Diego, CA 92121, USA). The assays were performed following the instructions of the manufacturer. According to the information included in the kit’s insert, the immunoassay used has 98.0% sensitivity and 98.3% specificity. 3.9.4.1 Principle of Enzyme linked immunosorbent assay (ELISA) for detection of C. trachomatis IgG and IgM antibodies: The Chlamydia trachomatis IgG or IgM EIA test kits was solid phase enzyme immunoassay based on immune capture principle for the qualitative detection of IgG and IgM antibodies to Chlamydia trachomatis in human serum or plasma .the micro well plate is coated with anti – human IgG or IgM antibodies. During testing, the specimen diluent and the specimens were added to the antibody coating micro well plate was incubated. If the specimens contained IgG or IgM antibodies to Chlamydia trachomatis, it was bound to the Chlamydia trachomatis IgG or IgM antibody complexes. After initial incubation, the micro well plate was washed to remove unbound materials. The enzyme – conjugated recombinant Chlamydia trachomatis antigens was bound to the anti – human IgG or IgM antibody- Chlamydia trachomatis IgG and IgM antibody complexes present. Substrate A and substrate B were added and then reagent was incubated to produce a blue color indicating the amount of Chlamydia trachomatis IgG or IgM antibodies present in the specimens. Sulfuric acid solution was added to the micro well plate to stop the reaction producing a color change from blue to yellow. The color intensity, which corresponded to the amount of Chlamydia trachomatis IgG or IgM antibodies presented in the specimen, was measured with micro plate reader at 450/620-650 nm or 450 nm within 30 minutes of adding the stop solution. 3.9.4.2 Procedure: The reagents and specimens were allowed to reach room temperature. 100 μl of negative control duplicated, cut-off calibrator duplicated and positive control duplicated were added to their respective wells, then 100 μl of specimen diluents was added to each well except the previous - 25 - wells and 5 μl of each specimen was added. The plate was covered with the

cover sealer and incubated for 30 minutes at 37° C. At the end of incubation period, each well was washed for 3 min with 300 μl of working wash buffer, 100 μl of conjugate was added to each well. The plate was covered and incubated again, for 30 minutes at 37°C. By the end of the incubation period, each well was washed 3 times with working wash buffer. Finally 100 μl of substrate solutions were added to each well, and then the plate was incubated at 37°C for 15 minutes. At the end of incubation period, the stop solution was added. 3.9.4.3 Quality control and calculation of results: Quality control: Reagents and calibrators were checked for storage, stability and preparation before starting work. For anti-Chlamydia trachomatis IgM: Calibrator 1 absorbance was 0.079.,Positive control absorbance was 0.026., Negative control absorbance was 0.024. 3.9.5. Calculation of results Extinction of the control or patient sample = Ratio Ratio, Extinction of calibrator 2 Interpreting result as follows: Ratio ≤ 0.8: negative. Ratio =1.1: border line. Ratio ≥ 1.1: positive.

CHAPTER FOUR RESULTS AND DISCUSSION 4.1Result: 4.1.1 Obstetrical, socio-demographical and clinical characteristic of the pregnant women:

Prevalence of anti- C.trachomatis IgG antibodies in pregnant women attending Wad Madani teaching hospital Department of obstetrics and gynecological was 3/90(3.3%) women out of 90 women studied were positive for C.trachomatis IgG, while only 5 (5.6 %) were positive C.trachomatis IgM antibodies. C.trachomatis IgG & IgM seropositivity was analyzed with the associated factors in related with C.trachomatis infection include tribes ,education ,jobs , miscarriage , rate , trimester , family history , last miscarriage , menstrual cycle , vaginal bleeding , vaginal disease ,smoking , thyroid , diabetic , hypertension , autoimmune disease , anemia type , preeclampsia , vaccine , type of vaccine, blood group in wife , age , Hb , RBCs , PCV , MCV , MCH , MCHC ,TWBCs , platelate , MPV, PCT, RDWCV , RDWSD ,Neutrophil , lymphocyte , Monocyte, Eosinophil ,Basophil , Biomass index with means and standard deviation and confidence interval 95% range as shown in table (4.1).

Table (4.1): Socio-demographic and clinical characteristic of study population:

No Items Control N=45 Case N=45 P value (95% confidence Mean ± SEM Mean ± SEM interval)

1 Age 26.02 ± 0.8531 30.89 ± 0.9504 0.0003 "-7.409 to -2.324" 2 Biomass index 25.66 ± 0.6089 27.85 ± 0.5751 0.0104 "-3.860 to -0.5250" 3 RBCs 10.65 ± 6.849 3.843 ± 0.1349 0.3235 "-6.836 to 20.44" 4 Hb 10.93 ± 0.2420 10.58 ± 0.3481 0.4187 "-0.4995 to 1.188"

5 TWBCs 9.109 ± 0.4661 7.907 ± 1.214 0.3577 "-1.386 to 3.790" 6 Platelate 251.7 ± 12.61 243.8 ± 14.61 0.6803 "-30.43 to 46.39" 7 PCV 33.94 ± 0.6871 31.84 ± 1.053 0.0984 "-0.4025 to 4.602" 8 MCV 90.72 ± 1.057 84.22 ± 1.010 0.0001 "3.590 to 9.410" 9 MCH 29.00 ± 0.5027 28.11 ± 0.5391 0.2311 "-0.5784 to 2.356" 10 MCHC 31.91 ± 0.3579 33.16 ± 0.3316 0.0125 "-2.216 to -0.2733" 11 MPV 8.687 ± 0.1015 9.593 ± 0.2327 0.0006 "-1.412 to -0.4012" 12 PCT 0.2115 ± 0.01113 0.2579 ± 0.03219 0.1762 "-0.1143 to 0.02136" 13 RDWCV 15.88 ± 0.2821 14.59 ± 0.3397 0.0044 "0.4121 to 2.170" 14 RWDSD 52.48 ± 0.8195 44.98 ± 0.8974 0.0001 "5.078 to 9.917" 15 Neutrophil 65.34 ± 1.864 66.43 ± 1.908 0.6829 "-6.403 to 4.216" 16 Lymphocyte 27.42 ± 1.617 32.19 ± 4.928 0.3599"-15.10 to 5.551" 17 Monocyte 4.627 ± 0.3153 5.324 ± 0.3098 0.1180 "-1.578 to 0.1822" 18 Eosinophil 2.553 ± 0.1767 2.267 ± 0.14 0.2108 "-0.1660 to 0.7394" 19 Basophil 00.00 00.00 Constant 21 Anti Chlamydia IgG "3.611 ± 3.509" "0.1358 ± 0.02323 " 0.3247"-3.510 to 10.46" 22 Anti Chlamydia IgM "0.1387 ± 0.02607 " "0.1582 ± 0.04015 " 0.6839"-0.1149 to 0.07574"

Table (4.2): Serodetection of anti - C. trachomatis IgG and IgM antibodies according to miscarriage:

IgG IgM Positive Borderline P.V Positive Borderline P.V Count Miscarriage C.trachomatis (% of total) C.trachomatis (% of total) (% of total) IgG antibody IgM antibody Freq(%of total) Freq(%of total) Miscarriage 45 3(3.3%) 0(0.0%) 3(3.3%) 0(0.0%) No 45 0(0.0%) 1(1.1%) 2(2.2%) 1(1.1%) 0.132 0.549 miscarriage Total 90 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.3) Serodetection of anti - C. trachomatis IgG and IgM according to rate of miscarriage:

rate Count (% IgG IgM of total) Positive (% Borderline P.V Positive Borderline P.V

of total) (% of total) (% of total) (% of total) Zero 45(50.0%) 0(0.0%) 1(1.1%) 2(2.2%) 1(1.1%) Twice 2(2.2%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) Three 34(37.8%) 3(3.3%) 0(0.0%) 0.417 1(1.1%) 0(0.0%) 0.378 Fourth 9(10.0%) 0(0.0%) 0(0.0%) 2(2.2%) 0(0.0%) Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.5%) 1(1.1%)

Table (4.4): Serodetection of anti - C. trachomatis IgG and IgM according to trimester:

Trimester Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) ›three trimester 45(50.0%) 0(0.0%) 1(1.1%) 2(2.2%) 1(1.1%) First trimester 33(36.7%) 2(2.2%) 0(0.0%) 1(1.1%) 0(0.0%) Second 11(12.2%) 1(1.1%) 0(0.0%) 0.620 2(2.2%) 0(0.0%) 0.557 Third 1(1.1%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.5): Serodetection of anti - C. trachomatis IgG and IgM according to family history: Family history Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No history 74(82.2%) 2(2.2%) 1(1.1%) 4(4.4%) 1(1.1%) Have history 16(17.8%) 1(1.1%) 0(0.0%) 0.698 1(1.1%) 0(0.0%) 0.890 Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.6): Serodetection of anti - C. trachomatis IgG and IgM according to last miscarriage: Last miscarriage Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No miscarriage 42(46.7%) 0(0.0%) 1(1.1%) 2(2.2%) 1(1.1%) One to two year 33(36.7%) 3(3.3%) 0(0.0%) 2(2.2%) 0(0.0%) miscarriage Two to five year 11(12.2%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) miscarriage 0.375 0.578 More than 5 4(4.4%) 0(0.0%) 0(0.0%) 1(1.1%) 0(0.0%) years miscarriage Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.7): Serodetection of anti - C. trachomatis IgG and IgM according to menstrual cycle:

menstrual Count IgG IgM cycle (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) irregular 21(23.3%) 2(2.2%) 1(1.1%) 2(2.2%) 1(1.1%) regular 69(76.7%) 1(1.1%) 0(0.0%) 0.035 3(3.3%) 0(0.0%) 0.120 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.8): Serodetection of anti - C. trachomatis IgG and IgG according to vaginal bleeding:

Vaginal Count IgG IgM bleeding (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) absent 65(72.2%) 2(2.2%) 1(1.1%) 5(5.6%) 0(0.0%) present 25(27.8%) 1(1.1%) 0(0.0%) 0.806 0(0.0%) 1(1.1%) 0.103 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.9): Serodetection of anti - C. trachomatis IgG and IgM according to vaginal disease:

Vaginal Count IgG IgM disease (% of total) Positive Borderline P.V Positive(% Borderline P.V (% of total) (% of total) of total) (% of total) absent 67(74.4%) 2(2.2%) 1(1.1%) 2(2.2%) 1(1.1%) present 23(25.6%) 1(1.1%) 0(0.0%) 0.803 3(3.3%) 0(0.0%) 0.166 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.10): Serodetection of anti - C. trachomatis IgG and IgM according to smoking:

smoking Count IgG IgM (% of total) Positive Borderline Positive Borderline (% of total) (% of total) (% of total) (% of total) nonsmoking 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%) Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.11): Serodetection of anti - C. trachomatis IgG and IgM according to Thyroid:

Thyroid Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No 89(98.9%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Yes 1(1.1%) 0(0.0%) 0(0.0%) 0.977 0(0.0%) 0(0.0%) 0.965 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.12): Serodetection of anti - C. trachomatis IgG and IgM according to diabetics: diabetics Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No 89(98.9%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Yes 1(1.1%) 0(0.0%) 0(0.0%) 0.977 0(0.0%) 0(0.0%) 0.965 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.13): Serodetection of anti - C. trachomatis IgG and IgM according to hypertension: hypertension Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No 88(97.8%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Yes 2(2.2%) 0(0.0%) 0(0.0%) 0.954 0(0.0%) 0(0.0%) 0.930 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.14): Serodetection of anti - C. trachomatis IgG and IgM according to autoimmunodisease:

Auto immune disease Count IgG IgM (% of total) Positive Borderline Positive Borderline (% of total) (% of total) (% of total) (% of total) No 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%) Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.15): Serodetection of anti - C. trachomatis IgG and IgM according to anemia type: Anemia type Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) Normochro 62(68.9%) 1(1.1%) 1(1.1%) 4(4.4%) 0(0.0%) mic Macrocytic 7(7.8%) 0(0.0%) 0(0.0%) 0(0.0%) 1(1.1%) Microcytic 21(23.3%) 2(2.2%) 0(0.0%) 0.444 1(1.1%) 0(0.0%) 0.015 hypo chromic total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.5%) 1(1.1%)

Table (4.16): Serodetection of anti - C. trachomatis IgG and IgM according to preeclampsia: preeclampsia Count (% of IgG IgM total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No 77(85.6%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%) Yes 13(14.4%) 0(0.0%) 0(0.0%) 0.702 0(0.0%) 0(0.0%) 0.581 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.17): Serodetection of anti - C. trachomatis IgG and IgM according to vaccine: Count (% of IgG IgM vaccine total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) No 28(31.1%) 2(2.2%) 1(1.1%) 2(2.2%) 1(1.1%) Yes 62(68.9%) 1(1.1%) 0(0.0%) 0.125 3(3.3%) 0(0.0%) 0.291 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.18): Serodetection of anti - C. trachomatis IgG and IgM according to vaccine type: Vaccine Count IgG IgM type (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) Not used 29(32.2%) 1(1.1%) 1(1.1%) 1(1.1%) 1(1.1%) MMR 9(10.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) vaccine Tetanus 26(28.9%) 2 (2.2% 0(0.0%) 2(2.2%) 0(0.0%)

vaccine 0.560 0.769 All the 26(28.9%) 0(0.0%) 0(0.0%) 2(2.2%) 0(0.0%) vaccine MMR +TT Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.19): Serodetection of anti - C. trachomatis IgG and IgM according to blood group:

Blood Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V group (% of total) (% of total) (% of total) (% of total) O + 39(43.3%) 2 (2.2%) 0(0.0%) 1(1.1%) 1(1.1%)

O - 8(8.9%) 0(0.0%) 1(1.1%) 0(0.0%) 0(0.0%) AB + 7(7.8%) 0(0.0%) 0(0.0%) 1(1.1%) 0(0.0%) AB - 1(1.1%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) A + 23(25.6%) 1(1.1%) 0(0.0%) 0.628 2(2.2%) 0(0.0%) 0.958 A - 5(5.6%) 0(0.0%) 0(0.0%) 1(1.1%) 0(0.0%) B + 6(6.7%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) B - 1(1.1%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.20): Serodetection of anti - C. trachomatis IgG and IgM according to Age:

Count (% IgG IgM Age of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) ≤ 30 years 58(64.4%) 3(3.3%) 1(1.1%) 3(3.3%) 1(1.1%) > 30 years 32(35.6%) 0(0.0%) 0(0.0%) 0.315 2(2.2%) 0(0.0%) 0.742 total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.21): Serodetection of anti - C. trachomatis IgG and IgM according to BMI:

BMI Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total) Abnormal 55(61.1%) 2(2.2%) 1(1.1%) 4(4.4%) 1(1.1%) Normal 35(38.9%) 1(1.1%) 0(0.0%) 0.708 1(1.1%) 0(0.0%) 0.788 Total 90(100.0%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.22): Serodetection of anti - C. trachomatis IgG and IgM antibodies according to Education:

Education Count (% IgG IgM of total) Positive (% of Borderline P.V Positive Borderline P.V total) (% of total) (% of total) (% of total) illiteracy 11(12.2%) 0(0.0%) 1(1.1%) 2(2.2%) 0(0.0%) primary 36(40%) 2(2.2%) 0(0.0%) 3(3.3%) 0(0.0%) H-secondary 27(30%) 1(1.1%) 0(0.0%) 0.192 0(0.0%) 1(1.1%) 0.194 university 16(17.8%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) total 90(100%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

Table (4.23): Serodetection of anti - C. trachomatis IgG and IgM according to jobs:

Jobs Count IgG IgM (% of total) Positive Borderline P.V Positive Borderline P.V (% of total) (% of total) (% of total) (% of total)

Housewife 78(86.7%) 2(2.2%) 0(0.0%) 4(4.4%) 1(1.1%)

Farmer 5(5.6%) 1(1.1%) 1(1.1%) 1(1.1%) 0(0.0%) Nurse 2(2.2%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) Teacher 3(3.3%) 0(0.0%) 0(0.0%) 0.014 0(0.0% 0(0.0%) 0.990 Doctor 1(1.1%) 0(0.0%) 0(0.0%) 0(0.0% 0(0.0%) Engineer 1(1.1%) 0(0.0%) 0(0.0%) 0(0.0% 0(0.0%) Total 90(100%) 3(3.3%) 1(1.1%) 5(5.6%) 1(1.1%)

4.2 DISCUSSION: Chlamydia trachomatis is obligate intracellular microorganism; it has ability to multiply intracellularly in the infected cell. In pregnant women. The suggested mechanisms by which C. trachomatis can trigger preterm birth or abortion are invasion of chlamydia into the choriodecidual space and subsequent immune responses and placental inflammation, especially chorioamnionitis. Chorioamnionitis can influence protease release, which leads to the premature rupture of the membranes, activation of arachidonic acid cascade, uterine contractions and preterm delivery or abortion. Infection with C. trachomatis is usually asymptomatic and it is difficult to diagnose on clinical grounds. In present study, Sero- detection of C. trachomatis antibodies IgG among spontaneous recurrent miscarriage and non miscarriage women in Gezira state was low. Out of 90 blood specimens investigated, the results showed out of the 45 case samples, 3/45(6.7%) tested positive for anti - C.trachomatis IgG antibody, and 42/45(93.3%) tested negative.Out of the 45 control samples, 0/45(0.0%) tested positive for Chlamydia trachomatis IgG, and1/45(2.2) was borderline, this result was closeness to that obtained in Study to Evaluation of a C. trachomatis IgG antibody among pregnant women in Khartoum, Sudan. the results revealed that the IgG seropositivity among pregnant women with previous abortion was (5.6%)(Mohager et al., 2014). In our study the seropositivity of C. trachomatis IgG antibody relative lower than that obtained in Zürich, Switzerland , which investigated Seroprevalence of anti-C. trachomatis IgG ,DNA amplification from products of conception or placenta and Immunohistochemical analysis among women with and without miscarriage. reported (15.2%) were Chlamydia trachomatis -IgG positive (Baud and Greub, 2011). The result also lower than that obtained in Alzaiem Alazhari University, Khartoum Bahri , Sudan ,which investigated Seroprevalence of anti-Chlamydia trachomatis (IgG) and anti- Chlamydia trachomatis (IgA) using ELISA techniques in 92 pregnant women, 13 (14.1%) were positive for anti- Chlamydia trachomatis IgG antibodies and this is not a surprising result because Sudan is a traditional Islamic society and according to the customs and traditions of Islamic societies, free sexuality is prohibited(Eltayeb and Kiliçman, 2013). In our study in the spontaneous recurrent miscarriage women group the seropositivity of C. trachomatis IgG in participants were (6.7%), and in non-Miscarriage women were (0.0%), showed no statistically significant correlation with seropositivity (P=0.132).

Our experimental results of C. trachomatis IgG seropositivity varies according to women education levels, the highest incidence was showed in women with primary school education, showed no statistically significant correlation with seropositivity (P =.192). Importantly the seropositivity of Chlamydia trachomatis IgG according to occupation, the highest incidence showed in Housewife was (2.2%), showed statistically significant correlation with seropositivity (P =0. 014).The present study showed significant relation between seropositivity of C. trachomatis IgG antibody and menstrual cycle with p value (0.035). Regarding last miscarriage, family history, Vaginal bleeding, vaginal disease, smoking, thyroid, diabetic, hypertension, autoimmune diseases, anemia type, preeclampsia, blood group of wife age, and biomassindix all participants showed no statistically significant correlation (P = >0.5).There were several studies conducted in Sudan that reported the prevalence of C. trachomatis. In this study the percentage of C. trachomatis IgM showed, out of the 45 case samples, 3/45(6.7%) tested positive for C. trachomatis IgM, and 42/45(93.3%) tested negative. Out of the 45 control samples, 2/45(4.4%) tested positive for C. trachomatis IgM, and1/45(2.2) was borderline and 42/45(93.3%) tested negative. Since the control samples showed low frequency of IgM, but this result is lower in comparison with study done by Visnovsky et al (2013) who found that percentage was 67.3% and this is not a surprising result because Sudan is a traditional Islamic society and according to the customs and traditions of Islamic societies, free sexuality is prohibited. Another Sudanese study done by Mohammed and Alfadhil (2012) found that the percentage was 22.5%, this result were relatively near to findings in Scotland and Poland (24.5%, 32% respectively) , and slightly increased than two studies done in Switzerland and had the same percentage(15.2%). In our study in the spontaneous recurrent miscarriage women group the seropositivity of C. trachomatis IgM in participants were (6.6%), and in non-Miscarriage women were (4.4%), showed no statistically significant correlation with seropositivity (P=0.549). Our experimental results of C. trachomatis IgM seropositivity varies according to women education levels, the highest incidence was showed in women with primary school education, showed no statistically significant correlation with seropositivity (P=.194).Importantly the seropositivity of C. trachomatis IgM according to occupation, the highest incidence showed in Housewife was (8.8%), showed no statistically significant correlation with seropositivity (P =0. 990). In the present study the maximum seropositivity of C. trachomatis IgM according to the rate of miscarriage in the participants was found in fourth times losses (2.2%) and followed by those of three times losses (1.1%).showed no statistically significant correlation with seropositivity (P =0 .378).Also our results showed

that Chlamydia trachomatis infection is equal among women with miscarrige in second trimester, and first trimester in contrary to studies of Visnovsky et, al ( 2013) and David and Gerub (2011) who found that Chlamydia trachomatis infection is common among women with abortion in first trimester and they recorded that this may be due to cross reaction of heat shock protein type 10 and embryonic protein which is expressed in early pregnancy., showed no statistically significant correlation with seropositivity (P = 0.557). Our findings revealed there are difference in the percentage of infection between women with and without history of abortion 2.2% and 8.8% respectively, this result was in alignment to Sozio and Ness et al (2001), Steven et al (2003) and Eltayib et al (2013) whom revealed its association with recurrent abortion, and they recorded that this may be due to re-activation of latent Chlamydia trachomatis infection. showed no statistically significant correlation with seropositivity (P = 0.890).In the present study in all participants the biomass-index seropositivity of C.trachomatis IgM was found higher in abnormal participant Related to the biomass-index the (case) group was N=45 Mean ± SEM 25.66 ± 0.6089 and (control) group was N=45 Mean ± SEM 27.85 ± 0.5751 Interestingly, considering the menstruation cycle, Regarding smoking, thyroid, diabetic, hypertension, autoimmune diseases, all participants showed no statistically significant seropositivity to be computed due to constant values. Considering the of miscarriage, vaginal bleeding, vaginal diseases, preeclampsia, vaccination, and type of vaccine associated to the seropositivity of Chlamydia trachomatis IgM in participants there was no statistically significant correlation with seropositivity (P > 0.05). Our findings indicated that Chlamydia trachomatis infections higher among younger women within age group (20-28 year) than those within age group (29-40 year) ,showed no statistically significant correlation with seropositivity, (P >0.742). This result was in alignment with all studies that came to our knowledge done by Eltayib et al (2013), Magaly et al (2005), Fadwa et al(2011), Mohammed and Alfadhil(2012), Sozio and Ness et al(2001). And David and Gerup et al (2011) where they reported that the infection is common among younger women and this may be due to the type of epithelium predominant in younger women which considered a target for Chlamydia trachomatis. The variation of results of different researches conducted in different areas is possibly due to difference in tools and techniques used. In addition, it might be due to different social practices that vary from society to society and genetic factors, age, and state of health and environmental factors.

CHAPTER FIVE

CONCLUSION AND RECOMMENDATIONS

5:1 Conclusion:  The current study revealed higher seroprevalence rate of Chlamydia trachomatis IgG and IgM among recurrent miscarriage women than those women without history of recurrent miscarriage.

 The highest seroprevalence of Chlamydia trachomatis IgG and IgM was among those house wives below 30 years old who attended primary school. Low hygiene perception, and low educational leads to increase the susceptibility to Chlamydia trachomatis infection.

 The detection of IgM among women enrolled in this study 5(5.6 %), show statistically significant (P.V=0.014) correlation with jobs.

 When adjusted for menstrual cycle, the association between miscarriage and IgG against C. trachomatis was significant (P.V=0.035).

5.2: Recommendations:

 Pregnancy health care centers should be improved and routine Chlamydia trachomatis screening for each pregnant women must be done with high sensitive and specific approach.  Health educational programs must be improved to facilitate in prevention and control of Chlamydia trachomatis infections.  Further studies in different geographical locations with large numbers of samples and more advanced techniques are required to validate the results of the present study.

REFERANCE:

ADACHI, K., NIELSEN-SAINES, K. & KLAUSNER, J. D. 2016. Chlamydia trachomatis infection in pregnancy: the global challenge of preventing adverse pregnancy and infant outcomes in Sub-Saharan Africa and Asia. BioMed research international, 2016.

AHMADI, A., KHODABANDEHLOO, M., RAMAZANZADEH, R., FARHADIFAR, F., ROSHANI, D., GHADERI ,E. & FARHANGI, N. 2016. The relationship between Chlamydia trachomatis genital infection and spontaneous abortion. Journal of reproduction & infertility, 17, pp110.

BARNDORFF‐NIELSEN, O. E. & SHEPHARD, N. 2001. Non‐Gaussian Ornstein– Uhlenbeck‐based modelsand some of their uses in financial economics. Journal of the Royal Statistical Society: Series B (Statistical Methodology), 63, 167-241.

BARONE, T. & EISNER, E. W. 2011. Arts based research, Sage. BAUD, D. & GREUB, G. 2011. Intracellular bacteria and adverse pregnancy outcomes. Clinical Microbiology and Infection, 17, 1312-1322.

BAUMAN, Z. 2004. Identity: Conversation with Benedetto Vecchi. Polity, Cambridge. CASSIDY, S. B. & ALLANSON, J. E. 2010. Management of genetic syndromes, JohnWiley & Sons.

CHEESBROUGH, M. 2006. District laboratory practice in tropical countries, Cambridge university press.

CLAMAN, P., TOYE, B., PEELING, R. W., JESSAMINE, P. & BELCHER, J. 1995. Serologic evidence of Chlamydia trachomatis infection and risk of preterm birth. CMAJ : Canadian Medical Association Journal, 153, 259.

DAUTRY‐VARSAT, A., SUBTIL, A. & HACKSTADT, T. 2005. Recent insights into the mechanisms of Chlamydia entry. Cellular microbiology, 7, 1714-1722.

DIEGO-ALVAREZ, D., RAMOS-CORRALES, C., GARCIA-HOYOS, M., BUSTAMANTE-ARAGONES, A., CANTALAPIEDRA, D., DIAZ-RECASENS, J., VALLESPIN-GARCIA, E., AYUSO, C. & LORDA-SANCHEZ, I. 2005. Double trisomy in spontaneous miscarriages: cytogenetic and molecular approach. Human Reproduction, 21, 958-966.

EL HACHEM, H., CREPAUX, V ,.MAY-PANLOUP, P., DESCAMPS, P., LEGENDRE, G. & BOUET, P.-E. 2017. Recurrent pregnancy loss: current perspectives. International journal of women's health, 9, 331.

ELTAYEB, H. & KILIÇMAN, A. A note on double laplace transform and telegraphic equations .Abstract and applied analysis, 2013. Hindawi.

FIOLKA, R., ZUBOR, P., JANUSICOVA, V., VISNOVSKY, J., MENDELOVA, A., KAJO, K., LASABOVA, Z., PLANK, L. & DANKO, J. 2013. Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer. Oncology reports, 30, 2878-2886.

FORD, H. B. & SCHUST, D. J. 2009. Recurrent pregnancy loss: etiology, diagnosis, and therapy. Reviews in obstetrics and gynecology, 2, 76.

FRETTS, R. C., SCHMITTDIEL, J., MCLEAN, F. H., USHER, R. H. & GOLDMAN, M. B. 1995. Increased maternal age and the risk of fetal death. New England Journal of Medicine, 333, 953-957.

GARLAND-THOMSON, R. 2002. Integrating disability, transforming feminist theory. NWSA journal, 14, 1-32.

GIAKOUMELOU, S., WHEELHOUSE, N., BROWN, J., WADE, J., SIMITSIDELLIS, I., GIBSON, D., SAUNDERS, P. T., HORNER, P., ENTRICAN, G. & HOWIE, S. E. 2017. Chlamydia trachomatis infection of human endometrial stromal cells induces defective decidualisation and chemokine release. Scientific reports.2001 ,7 , GOERING, R. V., MIMS, C. A., DOCKRELL, H., ZUCKERMAN, M., CHIODINI, P. L. & ROITT, I. 2013. Mims' Medical Microbiology, With STUDENT CONSULT Online Access, 5: Mims' Medical Microbiology, Elsevier Health Sciences. ,HANSEN, A., ODENDAHL, M., REITER

K., JACOBI, A. M., FEIST, E., SCHOLZE, J., BURMESTER, G. R., LIPSKY, P. E. & DÖRNER, T. 2002. Diminished peripheral blood memory B cells and accumulation of memory B cells in the salivary glands of patients with Sjögren's syndrome. .Arthritis & Rheumatology, 46, 2160-2171

HOGG, J. C., CHU, F., UTOKAPARCH, S., WOODS, R., ELLIOTT, W. M., BUZATU, L., CHERNIACK, R. M., ROGERS, R. M., SCIURBA, F. C. & COXSON, H. O. 2004. The nature of small-airway obstruction in chronic obstructive pulmonary .disease. New England Journal of Medicine, 350, 2645-2653

ISLAM, M., TALUKDAR, P. K., HOQUE, A., HUQ, M., NABI, A., AHMED, D., TALUKDER, K., PIETRONI, M., HAYS, J. & CRAVIOTO, A. 2012. Emergence of .multidrug-resistant NDM-1-producing Gram-negative bacteria in Bangladesh .European journal of clinical microbiology & infectious diseases, 31, 2593-2600

JAWETZ, E., MELNICK, J. L. & ADELBERG, E. A. 2013. Medizinische Mikrobiologie, .Springer-Verlag

KNOPP, M. V., BALZER, T., ESSER, M., KASHANIAN, F. K., PAUL, P. & NIENDORF, P. 2006. Assessment of utilization and pharmacovigilance based on spontaneous adverse .H event reporting of gadopentetate dimeglumine as a magnetic resonance contrast agent after .45 million administrations and 15 years of clinical use. Investigative radiology, 41, 491-499

KRAFZIG, D., BANKE, K. & SLAMA, D. 2005. Enterprise SOA: service-oriented .architecture best practices, Prentice Hall Professional

LANGEVELD, B. W., MOL, D., ZAZULA, G. D., GRAVENDEEL, B., EURLINGS, M., MCMICHAEL, C. N., GROENENBERG, D., VAN REENEN, G. B., PALMEIRA, M. & VOGEL, J. 2018. A multidisciplinary study of a Late Pleistocene arctic ground squirrel .(Urocitellus parryii) midden from Yukon, Canada. Quaternary Research, 89, 333-351

MACONOCHIE, N., DOYLE, P., PRIOR, S. & SIMMONS, R. 2007 Risk factors for first trimester miscarriage—results from a UK‐population‐based case– .control study. BJOG: An International Journal of Obstetrics & Gynaecology, 114, 170-186

MANDAL, J., ACHARYA, N. S., BUDDHAPRIYA, D. & PARIJA, S. C. 2012. Antibioticresistance pattern among common bacterial uropathogens with a special reference .to ciprofloxacin resistant Escherichia coli. The Indian journal of medical research, 136, 842

MOHAGER, M. O., MOHAGER, S. O. & KADDAM, L. A. 2014. THE ASSOCIATION BETWEEN SHISTOSOMIASIS AND ENTERIC FEVER IN A SINGLE SCHISTOSOMA ENDEMIC AREA IN SUDAN. International Journal of Pharmaceutical Sciences and .Research, 5, 2181

NOUGON, G., MUSCHART, X., GÉRARD, V., BOULOUFFE, C., JAMART, J., VAN PEE, D. & DE CANNIÈRE, L. 2015. Doesoffering pricing information to resident physicians in the emergency department potentially reduce laboratory and radiology costs? .European Journal of Emergency Medicine, 22, 247-252

PATRICIA, M. T. 2014. Bailey and Scott’s diagnostic microbiology. Mosby Inc., Maryland .Heights

REA, T. D., EISENBERG, M. S., SINIBALDI, G. & WHITE, R. D. 2004. Incidence of .EMS-treated out-of-hospital cardiac arrest in the United States. Resuscitation, 63, 17-24

SARAVANAN, H. S. I. & VIGNESH, R. 2012. Symposium on HIV andInfectious Diseases. .BMC Infectious Diseases, 12, O1

results from a multicenter evaluation of the APTIMA assays for both infections. Sexually transmitted diseases, 32, 725-728. . SHEPARD, C. W., FINELLI, L. & ALTER, M. J. 2005. Global epidemiology of hepatitis .Cvirus infection. The Lancet infectious diseases, 5, 558-567.

SUZUMORI, N. & SUGIURA-OGASAWARA, M. 2010. Genetic factors as a cause of .miscarriage. Current medicinal chemistry, 17, 3431-3437.

TILLÉ, Y. & WILHELM, M. 2017. Probability sampling designs: principles for choice of .design and balancing. Statistical Science, 32, 176-189.

TOULLEC, D., PIANETTI, P., COSTE, H., BELLEVERGUE, P., GRAND-PERRET, T., AJAKANE, M., BAUDET, V., BOISSIN, P., BOURSIER, E. & LORIOLLE, F. 1991. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. .Journal of Biological Chemistry, 266, 15771-15781.

GOERING, R. V., MIMS, C. A., DOCKRELL, H., ZUCKERMAN, M., CHIODINI, P. L. & ROITT, I. 2013. Mims' Medical Microbiology, With STUDENT CONSULT Online .Access, 5: Mims' Medical Microbiology, Elsevier Health Sciences

K., JACOBI, A. M., FEIST, E., SCHOLZE, J., ,HANSEN, A., ODENDAHL, M., REITER BURMESTER, G. R., LIPSKY, P. E. & DÖRNER, T. 2002. Diminished peripheral blood memory B cells and accumulation of memory B cells in the salivary glands of .patients with Sjögren's syndrome. Arthritis & Rheumatology, 46, 2160-2171

JAWETZ, E., MELNICK, J. L. & ADELBERG, E. A. 2013. Medizinische Mikrobiologie, .Springer-Verlag

KNOPP, M. V., BALZER, T., ESSER, M., KASHANIAN, F. K., PAUL, P. & NIENDORF, P. 2006. Assessment of utilization and pharmacovigilance based on spontaneous .H adverse event reporting of gadopentetate dimeglumine as a magnetic resonance contrast agent after 45 million administrations and 15 years of clinical use. .Investigative radiology, 41, 491-499

KRAFZIG, D., BANKE, K. & SLAMA, D. 2005. Enterprise SOA: service-oriented .architecture best practices, Prentice Hall Professional

LANGEVELD, B. W., MOL, D., ZAZULA, G. D., GRAVENDEEL, B., EURLINGS, M., MCMICHAEL, C. N., GROENENBERG, D., VAN REENEN, G. B., PALMEIRA, M. & VOGEL, J. 2018. A multidisciplinary study of a Late Pleistocene arctic ground squirrel (Urocitellus parryii) midden from Yukon, Canada. Quaternary Research, 89, .333-351

MACONOCHIE, N., DOYLE, P., PRIOR, S. & SIMMONS, R. 2Risk factors for first trimester miscarriage—results from a UK‐population‐based case–control study. .BJOG: An International Journal of Obstetrics & Gynaecology, 114, 170-186

MANDAL, J., ACHARYA, N. S., BUDDHAPRIYA, D. & PARIJA, S. C. 2012. Antibioticresistance pattern among common bacterial uropathogens with a special reference to ciprofloxacin resistant Escherichia coli. The Indian journal of medical .research, 136, 842

NOUGON, G., MUSCHART, X., GÉRARD, V., BOULOUFFE, C., JAMART, J., VAN PEE, D. & DE CANNIÈRE, L. 2015. Doesoffering pricing information to resident physicians in the emergency department potentially reduce laboratory and radiology .costs? European Journal of Emergency Medicine, 22, 247-252

PATRICIA, M. T. 2014. Bailey and Scott’s diagnostic microbiology. Mosby Inc., Maryland .Heights

REA, T. D., EISENBERG, M. S., SINIBALDI, G. & WHITE, R. D. 2004. Incidence of EMS-treated out-of-hospital cardiac arrest in the United States. Resuscitation, 63, 17-24

SARAVANAN, H. S. I. & VIGNESH, R. 2012. Symposium on HIV andInfectious Diseases. .BMC Infectious Diseases, 12, O1

SCHACHTER, J., CHERNESKY, M. A., WILLIS, D. E., FINE, P. M., MARTIN, D. H., FULLER, D., JORDAN, J. A., JANDA, W. & HOOK III, E. W. 2005. Vaginal swabs are the specimens of choice when screening for Chlamydia trachomatis and Neisseria gonorrhoeae: results from a multicenter evaluation of the APTIMA assays for both .infections. Sexually transmitted diseases, 32, 725-728

SHEPARD, C. W., FINELLI, L. & ALTER, M. J. 2005. Global epidemiology of hepatitis .Cvirus infection. The Lancet infectious diseases, 5, 558-567

SUZUMORI, N. & SUGIURA-OGASAWARA, M. 2010. Genetic factors as a cause of .miscarriage. Current medicinal chemistry, 17, 3431-3437

TILLÉ, Y. & WILHELM, M. 2017. Probability sampling designs: principles for choice of .design and balancing. Statistical Science, 32, 176-189

TOULLEC, D., PIANETTI, P., COSTE, H., BELLEVERGUE, P., GRAND-PERRET, T., AJAKANE, M., BAUDET, V., BOISSIN, P., BOURSIER, E. & LORIOLLE, F. 1991. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of .protein kinase C. Journal of Biological Chemistry, 266, 15771-15781

Appendices

Appendix (1): University of Gezira

Faculty of Medical Laboratory Sciences

Microbiology Department

Clinical Evaluation Form (Questionnaire)

General information:

Date: …/…/……… Time: _____ (am/pm) NO

Name: ______

Gender: ………………………………………….. Age: ……………………………years.

Tribe: ……..…………………………………….. Job: ………………………...……….

Occupation: …………………………………….. Education level: …………………….

Tall ……………………………………………….. Weight …………………………………….

Biomass index …………………………………………………………………………………….

Clinical information:

 Have you ever been pregnant before?  Yes……………….……No………………………….  Did you have miscarriage?  Yes……………….……No………………………….  Did you have miscarriage before that?  Yes…………………….No …………………………  If present how many time?  One ⃝ Two ⃝ Three ⃝ More ⃝  At what stage occur:  1st⃝ 2nd ⃝ 3rd⃝

 Did you family history of miscarriage?  Yes…………………….No…………………………  How long is it science your last miscarriage?  …………………………………………………………………….  Prior to pregnancy was your menstrual cycle regular?  Yes……………………….No………………………  Did you have Vaginal bleeding during pregnancy?  Yes……………………….No………………………  Did you have vaginal discharge before or during pregnancy?  Yes……………………….No………………………  Cigarette smoking?  Yes……………………….No………………………  Other chronic disease?  Thyroid………….Hypertension…………..Diabetes…………. . Anemia ………………..Other autoimmune-diseases ………………………… Preeclampsia …………………………………………………………..  Did you have ever been vaccinated against ……………………? Yes …………………………. No………………………………… What types of vaccine you have………………………………….. Lab results:

For both Wife and Husband

ABO system: ......

Rhuses factor: ……………………………………………………………………………………… Complete blood count

RBCs count: …... × 109/L. Hb: ……………g/dl. PCV: ….……%.

MCV: ……………....…fl. MCH: …..……….pg. MCHC: ……. %.

Platelet count: ……... × 106/L. MPV: …………...fl. PCT: ………. %.

TWBCs: ……..... × 106/L. RDW (CV): …… RDW (%): …..%.

Differential count:

Neutrophil: Absolute: ……………….…× 106/L. Relative: …………..%.

Lymphocyte: Absolute: ……………….…× 106/L. Relative: …………. %.

Monocyte: Absolute: ……………….…× 106/L. Relative: …………. %.

Eosinophil: Absolute: ……………….…× 106/L. Relative: …………. %.

Basophil: Absolute: ……………….…× 106/L. Relative: …………. %

PBP:

RBCs: ...…………………………………………………………………………………………

WBCs: ...………………………………………………………………………………..………

Platelets: ...………………………………………………………………………………………

Appendix (2) Informed consent

سم باهلل الرمحن الرحيم

جامعة الجزيرة

كلية علوم المختبرات الطبية

برنامج ماجستير علوم المختبرات الطبية

تخصص احياء دقيقه

……………………………………………………………………………………………………اإلسم

سوف يتم اخذ عينة دم من الوريد بحجم,5 مل وذلك بعد مسح منطقة الوريد بواسطه

المطهر وكل االدوات المستخدمه معقمه وليس هناك اثار جانبيه للعمليه ربما يحصل تورم بسيط في منطقة اخذ العينة وسوف يزول بعد فتره قصيره

أوافق أنا المذكور أعاله على التبرع بعينة الدم إلجراء هذه الدراسه

االمضاء ......

التاريخ......

IgG ` 2 3 4 5 6 7 8 9 10 11 12 A NC 3 11 19 27 35 43 51 59 67 75 83 B NC 4 12 20 28 36 44 52 60 68 76 84 C CUT.OFF 5 13 21 29 37 45 53 61 69 77 85 D CUT.OFF 6 14 22 30 38 46 54 62 70 78 86 E PC 7 15 23 31 39 47 55 63 71 79 87 F PC 8 16 24 32 40 48 56 64 72 80 88 G 1 9 17 25 33 41 49 57 65 73 81 89 H 2 10 18 26 34 42 50 58 66 74 82 90

CONTROL POSITIVE

CASE POSITIVE

Appendix ( 3): Layout shows the seropositivity and seronegativity for IgG antibody

Appendix(4) Micro teter plate after add stop solution.

IgM 1 2 3 4 5 6 7 8 9 10 11 12 A NC 3 11 19 27 35 43 51 59 67 75 83 B NC 4 12 20 28 36 44 52 60 68 76 84 C CUT.OFF 5 13 21 29 37 45 53 61 69 77 85 D CUT.OFF 6 14 22 30 38 46 54 62 70 78 86 E PC 7 15 23 31 39 47 55 63 71 79 87 F PC 8 16 24 32 40 48 56 64 72 80 88 G 1 9 17 25 33 41 49 57 65 73 81 89 H 2 10 18 26 34 42 50 58 66 74 82 90

CONTROL POSITIVE CASE POSITIVE

Appendix(5): Layout shows the seropositivity and seronegativity for IgM

Appendix(6) Microtiter plate before add stop solution.

Appendix (7): Easy wash for automated wash use for ELISA

Appendix (8): Photometric measurement for detection of ELISA cooler density

Appendix(9): Sysmex KX-21N Automated Hematology Analyzer