Epidemiology of Trichomonas vaginalis infection among infertile women in Gaza city, Palestine. Amal Y. Al Maqadma. Prof. Dr. Adnan I.

The Islamic University-Gaza Research & Postgraduate Affairs Faculty of Science Master of Biological Sciences Epidemiology of Trichomonas vaginalis infection among infertile women in Gaza city, Palestine. Submitted by: Amal Y. Al Maqadma Supervisor: Prof. Dr. Adnan I. Al Hindi Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Biological Sciences/Microbiology Faculty of Science 2014 AD 1436 AH

Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree of the university of other institute, except where due acknowledgment has been made in the text. Amal Y.H. Al Maqadma November, 2014. Copyright. All Rights Reserved: No part of this work can be copied, translated or stored in a retrieval system, without prior permission of the authors. I

Epidemiology of Trichomonas vaginalis infection among infertile women in Gaza city, Palestine. Abstract Background: Trichomoniasis is one of the most common curable sexually transmitted disease worldwide. Trichomoniasis is caused by the motile parasitic protozoan Trichomonas vaginalis (T. vaginalis). Methods: A descriptive analytical design was used to conduct this study and samples were selected from Al Basma medical center in Gaza city. A total of 120 endocervical swabs were collected from females attending the center for management of delayed conception. The samples were processed using PCR technique with Tv1-Tv2 primers. A structured questionnaire was conducted with all participants regarding sociodemographic data, risk factors and symptomatology. Results: The prevalence of T. vaginalis was 5.8%. Statistical significant relation was found between infection and age of patient, age at marriage, no history of previous vaginal infection and inguinal erythema. Higher infection rate was found among women who were unemployed, of preparatory educational level, from Mid zone, living in crowded house and married to smoker husband. Conclusions: We concluded that the prevalence of Trichomoniasis is attributed to iatrogenic causes mainly not to personal hygiene. Recommendations: We recommend healthcare provider to observe aseptic techniques during gynecological procedures. Keywords: T. vaginalis, PCR, Gaza, infertility. II

وبائية طفيل المشعرة المهبلية لدى النساء المصابات بالعقم في مدينة غزة فلسطين الملخص مقدمة: داء المشعرات واحد من أكثر األمراض المنتقلة جنسيا ويعتبر من أكثرها شيوعا عالميا وهو من األمراض القابلة للشفاء يسبب داء المشعرات طفيليات أولية متحركة تسمى المشعرات المهبلية. أساليب البحث: تم استخدام المنهج الوصفي التحليلي إلجراء هذه الدراسة وقد تم أخذ العينات من مركز البسمة الطبي في مدينة غزة حيث تم جمع 021 مسحة عنق رحم من السيدات الالتي يرتدن المركز بغرض معالجة تأخر اإلنجاب لديهن وقد تمت معالجة العينات باستخدام تقنية سلسلة الحمض النووي المتضخم أو تفاعل البوليميريز المتسلسل وقد تم إجراء استبيان منظم مع جميع المشاركات فيما يتعلق بالبيانات االجتماعية والصحية وعوامل الخطر واألعراض المحتملة. النتائج: أظهرت نتائج هذه الدراسة أن النسبة العامة للحاالت المصابة كانت %8.5 وأن ثمة عالقة ذات داللة إحصائية بين اإلصابة وعمر المريض وسن الزواج وعدم حدوث التهابات مهبلية سابقة ووجود احمرار أعلى الورك. كما وأظهرت النتائج أن أعلى معدل إصابة سجلت بين النساء العاطالت عن العمل والسيدات ذوات التعليم االبتدائي واإلعدادي من المحافظات الوسطى ويعشن في منزل مشترك مع أهل الزوج وكذلك المتزوجات من مدخنين. الخالصة: وخلصنا إلى أن انتشار داء المشعرات ربما يعزى إلى أسباب عالجية المنشأ وليس عادات النظافة بسبب الشخصية. التوصيات: نوصي األطباء باتخاذ تقنيات التعقيم أثناء إجراء الفحوصات والعمليات الجراحية النسائية. الكلمات المفتاحية: المشعرة المهبلية التقنيات الجزيئية قطاع غزة العقم. III

Dedication To my home Palestine To all martyrs To my beloved father and mother To my husband Mohammed To my sons, Mosaab and Ibrahim To my sister and brother To all my friends and teachers To Dr. Mofeed Al Mokhallalaty soul IV

Acknowledgments First of all, the praises and thanks be to Allah, the Lord of the Worlds. I have many people to thank for their help to complete this thesis: I would like to thank my beloved parents for their support and prayers till the dream became true. I deeply thank my supervisor, Prof. Adnan Al Hindi for the time, support and interest that have made the writing of this thesis possible and, whose support was vital for me to overcome all the difficulties I faced. Special gratitude is offered to all patients helped to conduct this thesis. I would like to express my sincere thanks to Dr. Bahaa Al Ghalayeni (Director of Al Basma Medical Center) for cooperation in producing this thesis. Also, I would like to thank Mrs. Aysha Qannan and Miss. Islam Al Kharty from Al Basma laboratory for their great help in the collection of samples. My special thanks go to my colleagues at the Genetic Diagnosis and Biology lab of the Islamic University of Gaza, Mrs. Heba Al Shorafa, Mr. Mohammmed Ashour, Mr. Mahmoud Al Hindi, Miss. Maysa Shubair, Miss. Dalal Abu Aazab, Mrs. Nisreen Badr and Mrs. Sanaa Al Taweel. I would like to thank the Master program coordinators and Faculty of Medicine at the Islamic University of Gaza. Finally, my sincere love and respect to my husband Mr. Mohammad Al Maqadma for his support and encouragement, also, my sons, Mosaab and Ibrahim, thank you for waiting. V

Table of Contents Declaration... I Abstract (English).... II Abstract (Arabic)....... III Dedication... IV Acknowledgments..... V Table of contents...vi List of tables... IX List of figures.. X List of abbreviations... XI Chapter One: Introduction... 1 1.1 Overview... 1 1.2 Objectives.. 2 1.2.1 General objective... 2 1.2.2 Specific objectives.. 2 1.3 Significance...2 Chapter Two: Literature Review. 3 2.1 Historical background...3 2.2 Classification 3 2.3 Morphology..4 2.4 Cell cycle and transmission..5 2.5 Growth factors and cultivation.6 2.6 Genome structure.7 2.7 Virulence factors..8 2.8 Pathogenesis and symptomatology..8 2.9 Epidemiology.....10 VI

2.10 Diagnostic methods.... 13 2.11 Risk factors... 15 2.12 Association with other STDs....16 2.13 Association with HIV virus......16 2.14 Complications in pregnancy........ 16 2.15 Association with infertility... 17 2.16 Association with family planning methods..... 18 2.17 Treatment.... 18 2.18 Control and prevention... 19 Chapter Three: Subjects and Methods...... 20 3.A Study population.. 20 3.B Study type... 20 3.C Ethical consideration.....20 3.1 Materials... 20 3.2 Methodology... 21 3.2.1 Sample collection... 21 3.2.2 Genomic DNA extraction... 21 3.2.3 Primers recostitution...22 3.2.4 Detection of extracted DNA... 23 3.2.5 Agarose gel electrophoresis... 23 3.2.6 Statistical analysis... 24 Chapter Four: Results... 25 4.1 Sociodemographic characters distribution of the study population.... 25 4.2 Residency status of the study population......25 4.3 Age at marriage, marriage duration and consanguinity of the study population...... 25 VII

4.4 Infertility status of the study population..... 25 4.5 Risk factors of T. vaginalis among the studied population..... 26 4.6 Symptomatology distribution of the study population.... 26 4.7 PCR results of endocervical swabs DNA.27 4.8 Frequency of T. vaginalis infection according to the sociodemographic characters... 27 4.9 Frequency of T. vaginalis infection according to residency status..... 28 4.10 Frequency of T. vaginalis infection according to age at marriage, duration of marriage and consanguinity...... 29 4.11 Frequency of T. vaginalis infection according to infertility status... 30 4.12 Frequency of T. vaginalis infection according to risk factors...... 30 4.13 Frequency of T. vaginalis infection according to infection symptoms.... 31 Chapter Five: Discussion..... 32 Chapter Six: Conclusion and Recommendations.....37 6.1 Conclusion... 37 6.2 Recommendations..,,,, 38 References... 39 Annexes... 51 Annex 1 Helsinki approval.. 51 Annex 2 A copy of the consent form...52 Annex 3 A copy of the questionnaire..53 Annex 4 Major equipments used in the study.55 Annex 5 Chemicals, kits and disposables used in the study......56 Annex 6 The group reviewed the questionnaire.57 VIII

List of Tables Table 2.1: Trichomonas species, host and habitat... 3 Table 2.2: Prevalence of T. vaginalis worldwide.... 11 Table 2.3: Prevalence of T. vaginalis in some Arabic countries..... 13 Table 2.4: Different T. vaginalis PCR assays, sensitivity and specificity...... 17 Table 2.5 Percentage of Infertility among married women 15-49 years by region, 2010..... 19 Table 3.1: Primers sequences and their characteristics... 25 Table 3.2: PCR reaction (20 µl)... 26 Table 4.1: Frequency of positive and negative PCR results for endocervical swab DNA.....33 Table 4.2: Frequency of positive DNA T. vaginalis infection according to Sociodemographic characters..... 34 Table 4.3: Frequency of positive DNA T. vaginalis infection according to residency status....35 Table 4.4: Frequency of positive DNA T. vaginalis infection according to age at marriage, marriage duration and consanguinity.. 36 Table 4.5: Frequency of positive DNA T. vaginalis infection according to infertility status... 36 Table 4.6: Frequency of positive DNA T. vaginalis infection according to risk factors.. 37 Table 4.7: Frequency of positive DNA T. vaginalis infection according to infection symptoms.... 38 IX

List of Figures Fig 2.1: Morphology of T. vaginalis (schematic). 4 Fig 2. 2: T. vaginalis life cycle... 6 Fig 2.3: Two T. vaginalis parasites, magnified (seen under a microscope 40x).. 16 Fig 3.1: PCR conditions....... 26 Fig 4.1: PCR product on agarose gel (312bp) obtained using T. vaginalis primers. 33 X

List of Abbreviations Abbreviation Term description Bp Base pair CDF Cell-detaching factors C o CDC HIV IVF IUI LBW Celsius Center of disease control and prevention Human Immunodeficiency Virus In vitro fertilization Intra uterine insemination Low birth weight µl Microliter Μm Micrometer Ml Milliliter MOH Ministry of health PCBS Palestinian Central Bureau of statistics pmol Picomole PCR Polymerase chain reaction PROM Premature rupture of membranes rpm Round per minute STD Sexually transmitted disease STI Sexually transmitted infection SPSS Statistical package for social sciences T. vaginalis Trichomonas vaginalis UTI Urinary tract infection WHO World Health Organization XI

CHAPTER ONE Introduction 1.1 Overview: Trichomoniasis is a sexually transmitted infection (STI) caused by the motile parasitic protozoan Trichomonas vaginalis (T. vaginalis). Trichomoniasis is one of the most common sexually transmitted disease (STD) in young, sexually active women (Garber, 2005). The World Health Organization (WHO) estimates that its prevalence ranges from 170 million to 190 million cases worldwide each year (Gross and Tyring, 2011). Humans are the only known host of T. vaginalis. Unlike many protozoan parasites, it possesses trophozoite form and lacks the cyst stage. Transmission occurs predominantly via sexual intercourse. The organism is most commonly isolated from vaginal secretions in women and urethral secretions in men (Workowski and Berman, 2006). Trichomoniasis can also be spread vertically from mother to baby during childbirth 5% of the time (Trichomoniasis. org., 2009). Women with Trichomoniasis may experience various symptoms, including a frothy yellow-green vaginal discharge and vulvar irritation. Men with Trichomoniasis may experience nongonococcal urethritis (Francis et al., 2008). A number of studies have highlighted the fact that at least 80% of T. vaginalis infections are asymptomatic. However, even asymptomatic infections are a public health concern. In addition to the risk of transmission to sex partners, T. vaginalis infection has been associated with as much as a 2.7-fold increase in the risk of Human Immunodeficiency Virus (HIV) acquisition, a 1.3-fold increase in the risk of preterm labour, infertility and a 4.7-fold increase in the risk of pelvic inflammatory disease (Poole and McClelland, 2013), prostate cancer (Sutcliffe et al., 2006) and cervical cancer (Gram et al., 1992). Trichomoniasis is thought to be widely underdiagnosed due to a variety of factors, including a lack of routine testing, (Van der Pol, 2007) (the low sensitivity of a commonly used diagnostic technique (wet mount microscopy), and nonspecific symptomatology. 1

Self-diagnosis and self-treatment or diagnosis by practitioners without adequate laboratory testing may also contribute to misdiagnosis (Krieger et al., 1988). 1.2 Objectives: 1.2.1 General objectives: To determine occurrence of T. vaginalis infection among infertile women in Gaza city, Palestine. 1.2.2 Specific objectives: 1. To identify T. vaginalis infection among infertile women. 2. To investigate risk factors that may contribute to Trichomoniasis infection in infertile women. 3. To compare clinical symptoms with molecular diagnosis of Trichomoniasis. 1.3 Significance As sexually transmitted diseases are considered a socially sensitive topic and rarely searched in our area and as Trichomoniasis is considered the most common parasitic sexually transmitted disease worldwide, it is important to estimate true prevalence of the disease using accurate methods. In addition, as Trichomoniasis was not studied among infertile population, this is the first study to estimate prevalence of T. vaginalis infection among infertile women in Gaza city based on molecular techniques. Furthermore, this study investigates the risk factors that may predispose to T. vaginalis infection especially in our community. 2

CHAPTER TWO Literature Review 2.1 Historical background: Donne, in 1836, was the first investigator to describe the protozoan T. vaginalis. Hohne, 1936, where he demonstrated a relationship between the presence of T. vaginalis and the development of symptoms localized to the vagina. Hohne also described the association of trichomoniasis with an increase in vaginal discharge. Trussell and Plass, in 1940, were able to fulfill Koch's postulates by inoculating vaginas of healthy volunteers with pure cultures of T. vaginalis and establishing acute symptomatic vaginitis (Faro, 2003). Different Trichomonas species are listed in Table 2.1. Table 2.1: Trichomonas species, host and habitat (Smyth, 1994). Species Host Habitat T. tenax Man Mouth T. gallinae Pigeon Oesophagus, crop T. muris Rat Caecum T. foetus Cattle Genital tract T. augusta Frog Rectum T. vaginalis Man Genital tract 2.2 Classification: T. vaginalis is a parasitic protozoan, and the taxonomic position is based on the classification scheme by Dyer, in which protozoa with the 9 + 2 flagellum fall into the phylum Zoomastigina. Phylum: Zoomastigina possess flagella. Class: Parabasalia presence of a parabasal body: Golgi associated with kinetosomes; axostyle (bundled microtubules); undulating membrane, an extension of the plasma membrane, enveloping the recurrent flagellum; occur in association with animals. Order: Trichomonadida (Kirby, 1947 emend. Honigberg, 1974) four to six flagella, free or attached to an undulating membrane; no true cysts. 3

Family: Trichomonadidae (Wenyon, 1926) presence of a cytostome, three to five free flagella (one flagellum on the margin of the undulating membrane); axostyle protruding through the posterior of the cell. Genus: Trichomonas four free flagella; one recurrent, along the outer margin of the undulating membrane; a costa at the base of the undulating membrane, and an axostyle extending through the cell. Species: Trichomonas vaginalis (Donné, 1836) (Dyer, 1990). 2.3 Morphology: T. vaginalis is a pear-shaped protozoan about 10-20 μm long and 2-14 μm wide. Five flagella emerge from a basal body at the anterior pole, four freely extend forwards and one extends backwards, forming the outer edge of the undulating membrane, which reaches back only just beyond the middle of the cell. An axial rod made up of microtubules (the axostyle) protrudes with its free tip from the posterior end of the cell. The oval cell nucleus lies near the upper pole of the protozoon. Trichomonads are anaerobic protozoa that posses hydrogenosome, which are specialized organelles producing H2 as a metabolite (Kayser et al., 2005). See fig 2.1. Fig 2.1 Morphology of T. vaginalis (schematic) (Kayser et al., 2005). 4

2.4 Cell cycle and transmission: Berger and Marr, (2006) stated that T. vaginalis resides in the female lower genital tract and the male urethra and prostate where it replicates by binary fission. The parasite does not appear to have a cyst form, and does not survive well in the external environment. T. vaginalis is transmitted among humans, its only known host, primarily by sexual intercourse, as shown in fig 2.2. Besides being transmitted sexually, there have been cases of nonsexual transmission of Trichomoniasis such as contaminated douche nozzles, the medical practitioners' specula, toilet seats, or swimming pool water. T. vaginalis would survive for 45 min in urine on wood, 6 hours if it was in vaginal exudate on wood (Whittington, 1957). Surprisingly, T. vaginalis would survive up to 25h on cloth used by woman infected with Trichomoniasis (Burch et al., 1959). Furthermore, Sherman et al., (1991) stated that T. vaginalis could be transmitted by artificial insemination with infected cryobanked human semen. The role of public swimming pools in spreading the disease is controversial yet there is little research done. The viability of T. vaginalis in samples of water was reexamined by Pereira-Neves and Benchimol. They concluded that T. vaginalis remains viable and infective in swimming pool water samples for several hours. The survival time is dependent on the cytotoxicity of the strain. The possible transmission of Trichomoniasis in public swimming pools may be low (Pereira-Neves and Benchimol, 2008). Although fetus is sterile, neonates was found to acquire Trichomoniasis through vulvovaginal contamination from infected mothers during birth by 0.6% (Al-Salihi et al., 1974). 5

Fig 2.2: T. vaginalis life cycle (CDC, 2009). 2.5 Growth factors and cultivation: Kostara et al., (1998) studied the different factors that affected the growth and survival of T. vaginalis in 2 growth media (Feinberg and Whittington media). He reported that the duration of survival and growth rate of T. vaginalis were inversely proportional to the inoculum density. The quicker exhaustion of nutrients and of other growth factors beside the production and accumulation of greater amounts of inhibitory by-products of metabolism by a denser growing population can explain this relation. Growth rate was also affected by the ph. The ph value of 6.4 was important in the beginning of growth while the value of 6.9 retards it. 6

The effect of temperature as a possible factor on the survival of T. vaginalis for shipment or routine laboratory maintenance was studied. Ten strains of T. vaginalis, and eight clinical isolates, were examined for viability when kept incubated at 37 degrees C o or removed from this temperature and held at different temperature degrees for increasing lengths of time without subculture or reincubation at 37 degrees C o. The order in which the strains remained viable without subculture was: 5 degrees C o, 8 to 10 days; 22 degrees C o, 4 to 8 days; 37 degrees C o, 4 to 6 days; 42 degrees C o, less than 2 days. Vials of medium with cells were also held at 22 and 5 degrees C o and then reincubated at 37 degrees C o. Cultures held at 22 degrees C o remained viable 6 to 8 days, whereas those stored at 5 degrees C o remained viable 10 to 14 days. These data show that T. vaginalis withstands a wide range of temperatures, particularly below normal growth temperatures without subcultures, beyond what would be expected in mailing cultures (Smith, 1983). T. vaginalis is an anaerobic obligate parasite in that it lacks the ability to synthesize many macromolecules de novo, particularly purines, pyrimidines, and many lipids. These nutrients are acquired from the vaginal secretions or through phagocytosis of host and bacterial cells (Heine and McGregor, 1993). The contributions of (Diamond et al., 1986) and (Linstead, 1981) to the development of media for trichomonads had enabled studies of the nutritional requirements of T. vaginalis. Culture media for T. vaginalis are thus required to include all the essential macromolecules, vitamins, and minerals. In particular, serum is essential for the growth of trichomonads, since it provides lipids, fatty acids, amino acids, and trace metals as Iron. 2.6 Genome structure: T. vaginalis genome is the first parabasalid genome to be described. Its genome is about 160 megabases in size with at least 65% of repeats and transposable elements. A core set of ~60,000 protein-coding genes were identified, which means T. vaginalis has one of the highest coding capacities among eukaryotes. Introns were found in 65 genes, transfer RNAs were found for all twenty amino acids, and about 250 ribosomal DNA were identified in this genome. There are six chromosomes in T. vaginalis. An interesting discovery was that the transcription machinery of this eukaryote appeared more metazoan than protozoan. The genome also showed there are 152 cases of possible prokaryote-to- 7

eukaryote lateral gene transfer. The genome helped with the discovery of unknown metabolic pathways, clarification of pathogenic mechanisms, and identification of unknown functions of organelles in T. vaginalis (Carlton et al., 2007). 2.7 Virulence factors: Pathogenesis of this parasite results from many factors playing a role in parasite-host cells interaction. A group of hydrolases, hemolysis factors, Adhesion proteins and proteinases are released from the cell (Lockwood et al., 1987). There are reports of other parasite products, described as cell-detaching factors (CDF) clearly implies that these parasite products could degrade proteins such as laminin, vitronectin, and other components of the extracellular matrix, thus effecting the release of host cells from tissue. In addition, the levels of secretory leukocyte proteases inhibitor in patients with T. vaginalis infections are significantly lower than those in uninfected patients (Draper et al., 1998). 2.8 Pathogenesis and symptomatology: The rise in the ph of the vagina, reduction (or complete loss) of Lactobacillus acidophilus and increase in the proportion of anaerobic bacteria are observed in cases of Trichomoniasis (McGrory et al., 1994). Several mechanisms as products, such as CDF or proteinases secreted by T. vaginalis, may destroy the lactobacilli (McGrory and Garber, 1992). It is still not clear why only a proportion of individuals infected with T. vaginalis become symptomatic while the rest remain asymptomatic. Strain variation and host factors may play a role in leading to symptomatic and asymptomatic infection (Kaul et al., 2004). Trichomoniasis in women usually occurs during the reproductive years. Infection before menarche or after menopause is generally rare, and symptoms are mild and transient (Rein, 1990). T. vaginalis is site-specific, usually surviving in only the urogenital tract of humans. Approximately 10% of vulvovaginitis is due to infection with T. vaginalis. The infected female patient may have no obvious clinical signs yet may still suffer vaginitis of varying severity. Clinical features that may develop in the infected woman include: purulent vaginal discharge, pruritis and dyspareunia. Leukorrhea and dysuria are occasionally seen. 8

The vaginal discharge may be malodorous, yellow, green or frothy. Punctuate haemorrhages of the cervix may result in strawberry-like appearance. Histological findings includes: increased vascularity and oedema of the squamous epithelium and inflammation in addition to the presence of the trichomonads. Up to one-third of infected females no histological findings were found (Collier et al., 1998). Krieger, (1998) found that although T. vaginalis infection is regarded primarily as a disease of women, it also occurs in men. Trichomoniasis in men is largely asymptomatic, and these men are considered asymptomatic carriers of T. vaginalis. Urogenital Trichomoniasis in men may be categorized into three groups: an asymptomatic carrier state, identified by investigation of sexual contacts of infected women; acute Trichomoniasis, characterized by profuse purulent urethritis; and mild symptomatic disease, which is clinically indistinguishable from other causes of nongonococcal urethritis. Krieger has shown that T. vaginalis is the cause of 11% of all cases of nongonococcal urethritis in males. The duration of infection is 10 days or less in most male patients. In symptomatic men, common complaints include scanty, clear to mucopurulent discharge, dysuria, and mild pruritus or burning sensation immediately after sexual intercourse (Petrin et al., 1998). Complications associated with Trichomoniasis include nongonococcal urethritis, prostatitis, balanitis, epididymitis, urethral disease, and infertility (Krieger, 1984). Symptoms may be related to protozoal load and it is possible, although not proven, that women with a high burden of infection are at a higher risk of complications. Alternatively, the duration of untreated infection may play a significant role in development of complications in females. Longitudinal cohort studies suggest that T. vaginalis can persist in asymptomatic, untreated women for at least 3 months (Van der Pol et al., 2005). In men, inoculation experiments in three volunteers in the 1950s demonstrated that T. vaginalis could be isolated at 4, 44 and 94 days following inoculation (Lanceley, 1953). A Zimbabwean study of symptomatic males with T. vaginalis found that 78% described symptoms for more than 4 weeks before diagnosis (Latif et al., 1987). It is likely, however, that in asymptomatic individuals the infection persists for longer, resulting in a potentially 9

large reservoir of asymptomatic infection, high rates of re-infection and a higher community prevalence (Sorvillo et al., 2001). 2.9 Epidemiology: Prevalence of T. vaginalis has been found to vary according to geographical location; study setting, for example sexual health clinic or community setting; presence or absence of symptoms; population studied, for example ethnic group, age and sex; and diagnostic techniques used. Direct comparisons between studies can therefore be difficult and can often not be generalized as shown in Table 2.2. Unlike Chlamydia trachomatis and Neisseria gonorrhoeae, the prevalence of T. vaginalis has been shown to increase with age in females and males. This may be due to the asymptomatic nature of infection and the persistence of untreated infection. Not surprisingly when sexual health clinics are studied, the prevalence of infection is higher (Wendel et al., 2003). In industrialized settings prevalence is high in prison inmates, intravenous drug users and sex workers. In both industrialized and resource-limited settings there are relatively few studies in the general population. In resource-limited settings antenatal or family planning attendees are therefore often used as an indicator of the prevalence in the general population (Johnston and Mabey, 2008). In comparison to developed countries, the prevalence of Trichomoniasis is reported as 15% or higher in developing countries where access to health care is limited. Consequently, it is likely that up to 25 million pregnant women globally are infected with Trichomoniasis (WHO, 2004). The prevalence of Trichomoniasis in neighboring islamic countries were also studied; where the prevalence ranges from 0.7% in Jordan to 36% in Egypt. Table 2.3 shows some prevalence studies in some islamic countries. 10

Table 2.2: Prevalence of T. vaginalis worldwide. Country Prevalence (%) Study (reference) ASIA India 8.5 Madhivanan et al., 2009 Sri Lanka 6.9 Fernando et al., 2011 AFRICA Zambia 27.1 Crucitti et al., 2011 EUROPE Greek 4.6 Piperaki et al., 2010 AMERICA Poland 26.6-78 Kurnatowska and Mamos, 2001 Portugal 31.2 Garcia et al., 2004 Ukraine 12.1 Landes et al., United Kingdom United States of America 2007 1.1 Oakeshott et al., 2011 Sample Size Reproductive health clinic (n=898) STDS clinic (n=346) School adolescent girls (n=460) Female attending gynecology hospital (n=502) Female (n=12000) Prison inmates female (n=211) HIV infected pregnant women (n=1050) Student female (n=183) 3.1 CDC, 2014 Women at reproductive age (n=4646) Argentina 4 Perazzi et al., 2010 Pregnant women in hospital (n=597) Diagnostic Method In Pouch TV Culture Not mentioned Wet mount, Culture, PCR Wet mount, culture Not mentioned Patients records PCR PCR Wet mount, culture 11

Table 2.3: Prevalence of T. vaginalis in some islamic countries. Country Prevalence (%) Egypt 7.7 Mahmoud et al., 1999 Study (reference) Sample Size Diagnostic Method 12.9 El-Shazly et al., 2001 36 Aboulghar et al., 2009 Symptomatic and asymptomatic women(n=450) Infertile and pregnant lady (n=280) Symptomayic and asymptomatic Childbearing age attending obstetrics and gynecology clinic (n=300) Wet mount, Pap smear, Culture, PCR Wet mount, Giemsa stain, culture Wet mount, Giemsa stain, In Pouch TV Iran 3.3 Nourian et al., 2013 Pregnant women (n=1000) Wet mount, culture Iraq 5.4 Al Saeed et al., 2011 Female with vaginal discharge (n=425) 20 Rahi et al., 2014 Symptomatic cases of Gynecology clinic (n=60) Jordan 0.7 Mahafzah et al., 2008 Kingdom of Saudi Arabia Women attending family planning and gynecology clinic (n=1275) 4 Al-Quaiz, 2000 Female with vaginal discharge attending primary care center (n=175) 0.7 Al-Zanbagi et al., 2005 Women with vaginal discharge (n=1767) Wet mount, H E stained, Pap smear, culture Wet mount and Whiff test In-Pouch TV kit Culture Not mentioned 1.43 Habib et al., 2009 Suspected cases (n=1469) Wet mount, culture and PCR Libya 1.2 Kassem and Majoud, 2006 Women attending gynecology department(n=2450) Nigeria 18.66 Jatau et al., 2006 Antenatal clinic attendee (n=300) Sudan 0.5 Abdelaziz et al., 2014 Pregnant from maternity hospital (n=200) Tanzania 10.7 Klinger et al., 2006 (n=1442) PCR Tunisia 5.6 Ben-Aissa et al., 1999 Turkey 4.5 KeleŞtemur et al., 2012 Pregnant women Infertile female (n=160) Wet mount and Giemsa stain Wet mount, culture Wet mount Not mentioned PCR Uganda 17.3 Tann et al., 2006 Antenatal clinic (n=250) Culture 12

In West Bank, (Houso et al., 2011) showed a prevalence rate of 13.6% of T. vaginalis among Palestinian women in the West Bank (n=1207), Palestine using culture method. In Gaza strip the available data about Trichomoniasis prevalence was investigated by one study. In 2006, cross sectional study used the method of Pap smear showed prevalence of 18.2% among pregnant Palestinian Women (n=430) (Al Hindi and Lubbad, 2006). 2.10 Diagnostic methods: T. vaginalis can be detected in both vaginal and cervical samples. No statistical difference in sensitivity and specificity estimates for vaginal and cervical specimens (Hardick et al., 2006). Viability of T. vaginalis is up to 20 min in vaginal secretion on cotton swab (Schwebke et al., 1999). Diagnostic methods of T. vaginalis include: a. Wet mount microscopy: The diagnosis of Trichomoniasis has traditionally depended on the microscopic observation of motile protozoa from vaginal or cervical samples and from urethral or prostatic secretions. T. vaginalis can be differentiated on the basis of its characteristic jerky movements. On occasion, flagellar movement can also be noted. The sensitivity of this test varies from 38% to 82% and is dependent on the inoculum size because fewer than 10 4 organisms/ml will not be seen. As well, the need for the specimen to remain moist and the experience of the observer are important variables. The size of the trichomonad is approximately the same as that of a lymphocyte (10 µm to 20 µm) or a small neutrophil; when not motile, a trichomonad can be difficult to differentiate from the nucleus of a vaginal epithelial cell as shown in fig 2.3. Motility is very dependent on the temperature of the specimen. At room temperature in phosphatebuffered saline, the organism will remain alive for more than 6 h; however, the motility of the organisms becomes significantly attenuated. Although wet mount examination is clearly the most cost-effective diagnostic test, the lack of sensitivity contributes to the underdiagnosis of the disease. Because viable organisms are required, delay in transport and evaporation of moisture from the specimen reduces motility and, consequently, diagnostic sensitivity (Garber, 2005). 13

Fig 2.3: Two T. vaginalis parasites, magnified (seen under a microscope 40x) (CDC, 2012). b. Culture: According to Mabey et al., (2006), at present, culture techniques are still regarded as the most sensitive and specific. Culture provides the gold standard against which other methods are judged. Culture media vary in efficiency but Diamond's TYM medium (sometimes with minor modifications) is among the best. Most samples will be positive within 48 hours but should be kept for 7 10 days before being finally discarded. A very convenient but expensive way of culturing specimens is the InPouch TV system which appears to be at least as sensitive as conventional tubed media (Sood et al., 2007). c. Immunological methods: There are an estimated eight serotypes observed in T. vaginalis (Ackers, 1990). Various techniques including agglutination, complement fixation, indirect hemagglutination, gel diffusion, fluorescent antibody, and enzyme-linked immunosorbent assay have been used to demonstrate the presence of antitrichomonal antibodies (Mathews and Healy, 1983). d. Molecular methods: DNA amplification techniques (especially PCR) have been increasingly used in clinical laboratories for T. vaginalis diagnosis. The use of PCR methods helps detect nonviable organisms and also has the ability to detect cells and target sequences in clinical samples that have undergone fixation or partial degradation (Riley et al., 1992). The technique was found to be superior in sensitivity and specificity to other conventional methods used for Trichomoniasis diagnosis as endocervical or high vaginal swab wet mount microscopy and 14

Pap smear, as stated by (Paterson et al., 1998). Several PCR assays have been developed by many investigators targeting many different genetic locations in T. vaginalis as shown in Table 2.4. Crucitti et al., (2003) conducted a study comparing the sensitivity of the different T. vaginalis PCR assays detection. The study showed that the sensitivity and specificity of different assays were less than reported in previous studies and he explained that strain variability and sample size may be the cause. Tv1 and Tv2 primers set targets conserved regions of the 18S ribosomal gene which showed no cross reaction with other trichomonads and bacteria present in vaginal flora. These primers detected T. vaginalis in urine as well as vaginal swabs. These primers are considered highly sensitive because it can detects small numbers of trichomonads that is not detected in culture (Mayta et al., 2000). Table 2.4: Different T. vaginalis PCR assays, sensitivity and specificity. Study (reference) PCR primers PCR Sensitivity/ Specificity (%)* Prevalence by gold standard (%) Madico et al., 1998 BTUB 9/2 97/98 6.6 Paterson et al., 1998 TvA5/TvA6 92.7/92.1 8.6 Ryu et al., 1999 Tv-E650 100/96.8 2.4 Lawing et al., 2000 Tv3/Tv7 89/97 Not mentioned Mayta et al., 2000 Tv1/Tv2 100/98 6.5 Caliendo et al., 2005 Tv1/Tv2 100/96.3 Not mentioned *positive culture or positive wet mount was used as the gold standard. 2.11 Trichomoniasis risk factors: Cotch et al., (1991) found that pregnant women colonized with T. vaginalis were significantly correlated to be black, cigarette smokers, unmarried, and less educated. Several behavioral factors associated with T. vaginalis included greater numbers of sexual partners both lifetime and in the last year, 5 years or more of sexual activity, and a history of gonorrhea. Other factors such as age, gravidity, income level, age at first coitus, and use 15

of antibiotics, alcohol, or douche during pregnancy were not independently associated with T. vaginalis colonization. In Nigeria, the prevalence of T. vaginalis infection among preganant women and the relationship between the sociodemographic and behavioral risk factors that helps to perpetuate the infection was studied. Trichomoniasis was more prevalent in the 20-24 years age group. Prevalence was recorded in pregnant women in the first trimester of pregnancy followed by those in the second and third trimesters respectively. Occurrence of infection by marital status shows that pregnant single women were more infected followed by married pregnant women. No T. vaginalis infection was recorded in the divorced and widowed group. Infection was more in pregnant women with primary school education, followed by those with secondary education qualification and in pregnant women with higher education. However, no T. vaginalis infection was recorded among pregnant women with no education and women with Quranic education in this study (Usanga et al., 2009). 2.12 Association with other STDs: A strong negative association between the presence of T. vaginalis and coinfection with Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) in female patients present with symptoms consistent with a sexually transmitted disease (STDs) ( White et al., 2005). However, Fule et al., (2012) stated that a women suffering from Trichomoniasis are at extreme risk of acquiring other STDs. 2.13 Association with HIV virus: T. vaginalis is one of the most common STDs among HIV-infected and un-infected women. Proportion of HIV-infected women diagnosed with T. vaginalis vaginitis ranges from 6 to 27% (Cohn and Clark, 2003) while Laga et al., (1994) recorded that T. vaginalis infection was associated with a 1.7 fold increased risk of HIV acquisition. 2.14 Complications in pregnancy: T. vaginalis infection during pregnancy can cross into the amniotic fluid and result in preterm labor. Preterm birth is associated with poor infant health and early deaths, admission of the newborn to neonatal intensive care in the first few weeks of life, prolonged hospital stay and long-term neurologic disability including cerebral palsy. Pregnant women with T. vaginalis are at increased risk for premature rupture of 16

membranes (PROM), preterm delivery and low birth weight infants (LBW). Trichomoniasis has been shown to be associated with increased risk of preterm birth; however, treatment has not consistently improved pregnancy outcome (Rasti et al., 2011). 2.15 Association with infertility: According to WHO, infertility is a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse (WHO, 2009). In Palestine, infertility prevalence is shown in Table 2.5. Table 2.5: Percentage of infertility among married women 15-49 years by region, 2010 (PCBS, 2010). Region Infertility % Secondary infertility Primary infertility Palestinian Territory 8.6 3.6 5.1 West bank 8.8 3.8 4.9 Gaza strip 8.3 3.0 5.3 Primary infertility: when a woman is unable to ever bear a child, either due to the inability to become pregnant or the inability to carry a pregnancy to a live birth she would be classified as having primary infertility. Thus women whose pregnancy spontaneously miscarries, or whose pregnancy results in a still born child, without ever having had a live birth would present with primarily infertility. Secondary infertility: when a woman is unable to bear a child, either due to the inability to become pregnant or the inability to carry a pregnancy to a live birth following either a previous pregnancy or a previous ability to carry a pregnancy to a live birth, she would be classified as having secondary infertility. Thus those who repeatedly spontaneously miscarry or whose pregnancy results in a stillbirth, or following a previous pregnancy or a previous ability to do so, are then not unable to carry a pregnancy to a live birth would present with secondarily infertile (Mascarenhas, 2012). 17

There is a few literature relating trichomonads to infertility. Benchimol et al., (2008) conducted a study aimed to show the effects provoked by T. foetus and T. vaginalis when in interaction with bovine and human sperm cells. Light microscopy, video microscopy, scanning, and transmission electron microscopy first revealed a tropism, then a close proximity followed by a tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed as well intense semen agglutination. The adhesion between trichomonads to the sperm cell occurred either by the flagella or sperm head. Motile parasites were observed during the next 12 h, whereas sperm cells in contact with the parasites rapidly became immotile. The parasites were able to maintain the sperm cells attached to their cell surface, followed by phagocytosis. This process began with a tight membrane-membrane adhesion and the incorporation of the sperm cell within an intracellular vacuole. Afterwards, the sperm cell was gradually digested in lysosomes. Many trichomonads were injured and/or died on making contact with the spermatozoa possibly due to necrosis. Results from this study demonstrated that both T. foetus and T. vaginalis interact with sperm cells provoking damage and death of these reproductive cells. T. vaginalis plays an important role in female infertility (El-Shazly et al., 2001). The risk of tubal infertility was almost twice as high in women who recalled previous Trichomoniasis compared with women with no such infection (Grodstein et al., 1992). 2.16 Association with family planning methods: The latest study about T. vaginalis was conducted in Jimma University Specialized Hospital, Southwest Ethiopia, showed that majority of the pregnant women who used pills (oral contraceptives) before pregnancy were more infected by T. vaginalis infection (Eshete et al., 2013). 2.17 Trichomoniasis treatment: Today, the standard treatment for Trichomoniasis is 250 mg of Metronidazole, given orally, three times a day for 7 days, or in a single 2-g dose. Both regimens are equally effective. Both the infected patient and sexual partner, whether symptomatic or asymptomatic, should be treated to prevent reinfection (Hager et al., 1980). Although current treatment protocols involving Nitroimidazoles are curative, Metronidazole resistance is on the rise. In 1989, the Centers for Disease Control estimated that 5% of all 18

T. vaginalis patient isolates had some level of resistance to metronidazole (Narcisi and Secor, 1996). This outline the need for research into alternative drugs. 2.18 Trichomoniasis Control and prevention: Control of T. vaginalis in industrialized settings recommends testing women attending clinics complaining of vaginal discharge. In addition, sexual partners of infected women should be treated on epidemiological grounds. This strategy will pick up women with symptomatic infection but will do little to reduce the community burden of infection due to high rates of asymptomatic disease. Where community prevalence of T. vaginalis and HIV is low this approach may be reasonable, but where prevalence is high, either within highrisk groups or within the community at large, screening should be considered. Due to high concordance rates among infected partners, high rates of re-infection and high rates of asymptomatic infection in both sexes, focusing screening on females alone is unlikely to be effective. Improving access to sexual healthcare, screening and/or periodic presumptive treatment of high-frequency transmitter groups, for example sex workers, is one approach to management of asymptomatic individuals (Johnston and Mabey, 2008). Vaccine development has been limited by a lack of understanding of the role of the host immune response to T. vaginalis infection. The lack of a good animal model has made it difficult to conduct standardized studies in drug and vaccine development and pathogenesis (Petrin et al., 1998). 19

CHAPTER THREE Subjects and Methods 3.A Study population The study population was married Palestinian women who attended Al Basma medical center in Gaza during the period from December 2013 to April 2014. A total of 120 females agreed to participate in the study, aged 18-45 yrs, non-menstruating (at the time of sample collection) and attended the center for attempting conception (mainly through assisted reproductive techniques). The patients who received antibiotics were excluded from the study. 3.B Study type The current study is a cross sectional descriptive analytical study and was conducted according to good clinical practice guidelines recommended by the CDC (CDC, 2007). A questionnaire included information like age, residence area, level of education, infertility status and symptoms complained by each patient was used (Annex 3). 3.C Ethical considerations 1. Ethical approval from health authorities: An ethical approval was obtained from Helsinki committee in Gaza strip (Annex 1). 2. Informed consent: A written consent form was obtained from each participant and the objectives of the study were fully explained (Annex 2). 3.1 Materials: The processing of samples was carried out in Al Basma medical center and the Biotechnology lab (Biology department) at the Islamic University- Gaza. The major equipments used in the study are listed in (Annex 4). Chemicals, kits and disposables used are listed in (Annex 5). 20

3.2 Methodology: 3.2.1 Sample collection: After speculum insertion, endocervical swab (Dacron tipped) was obtained by a specialized gynecologist. The swab was inserted 1 2 cm into the endocervical canal followed by two or three rotations. Swab was collected according to CDC guidelines and as indicated by the manufacturer (CDC, 2007). Swab was placed in a tube and labeled with patient name and date of collection and then sent to the lab for DNA processing. One ml of normal saline added to the swab and vortexed for 30 seconds. Then solution was transferred into 2 ml Eppendorf's tube then stored at 4 C for 7 days till DNA extraction stage. 3.2.2 Genomic DNA extraction: Before DNA extraction, tubes were taken from the freezer and left at room temperature till thawing. The tubes were centrifuged at 10,000 rpm for 15 mins. The supernatant was discarded, leaving approximately 25 µl of liquid and the deposit was vortexed and transferred to a 1.5 ml microtube. Genomic DNA was isolated from specimens (using Epicentre QuickExtract DNA extraction solution (Madison, USA). The extracted DNA by this kit is suitable for PCR analysis, as for genetic or environmental research and screening in humans and other organisms. Extraction was done according to the manufacturer protocol. One ml of 50mg/ml Proteinase K was diluted with 300 ml of Tissue and Cell Lysis solution for each sample. Then 300 ml of Tissue and Cell Lysis solution containing Proteinase K was added to the sample tube and was mixed thoroughly. The solution was incubated at 65 C for 15 min, Vortexed and mixed every 5 min. The sample was placed on ice for 3-5 min. Then, 150 µl of MPC Protein Precipitation Reagent was added to 300 ml of lysed sample, vortexed and mixed vigorously for 10 sec. The debris was pelleted by centrifugation for 10 min at 10,000 rpm in microcentrifuge. The supernatant was transferred to a clean microcentrifuge tube and the pellet was discarded. A total of 500 µl of Isopropanol was added to the recovered supernatant. Each tube was inverted 3040 times. The DNA was pelleted by centrifugation at 4 C for 10 min at 10,000 rpm in a microcentifuge. Isopropanol was 21

poured off carefully without dislodging the DNA pellet, which was rinsed twice with 75% Ethanol, being careful not to dislodge the pellet. The residual Ethanol was removed with a pipette and left for 20 min until drying. The total nucleic acids was suspended in 35 µl of TE buffer. Finally, the DNA was stored at -20 Cº until PCR analysis is carried out. 3.2.3 Primers reconstitution: The primer set Tv1 and Tv2 used is designed to amplify a DNA product of 312 bp according to Dwivedi et al, (2012). Positive controls were selected by pilot study which elaborated positive sample and matched the size of 312 bp that reported in literatures. Primers were received in a lyophilized (i.e. dried) state. Primer containers were first centrifuged at 13,000 rpm for 3 minutes, and then reconstituted with ultra-pure water (nuclease free water) to create a stock solution of each primer with final concentration 100 pmol/µl. A 10 µl aliquot was taken from stock primer and diluted with 90 µl nuclease free water to make 10 pmol/µl working solution. All primers sequences and their characteristics shown in Table 3.1. Table 3.1: Primers sequences and their characteristics. Primer Primer Sequence (5' to 3') Tm ( º C) For 100 pmol/µl dissolve in (µl) Tv1 TAA TGG CAG AAT CTT TGG AG 53.2 193 Tv2 GAA CTT TAA CCG AAG GAC TTC 55.9 260 After preparation of the PCR reaction (20 µl), amplification was carried out in a thermocycler (BioRad, USA Biometra). PCR reaction components and conditions are shown in Table 3.2 and Figure 3.1 respectively. 22

Table 3.2: PCR reaction (20 µl). Reagent Volume (μl) Final concentration Primer TV1 2 20 pmol Primer TV2 2 20 pmol Nuclease free water 4 - PCR Go Taq Green Master Mix 10 1X DNA sample 2 100ng Fig 3.1: PCR conditions. 3.2.4 Detection of extracted DNA: The quality of the isolated DNA was determined by running 8 μl of each sample with a 50 bp ladder on 2% agarose gel contained Ethidium bromide then the DNA sample was visualized on a Gel documentation system. 3.2.5 Agarose gel electrophoresis (2.0%): Dried agarose gel (1.2 gm) was dissolved in 60 ml 1x Tris-Acetate-EDTA buffer (2M Tris base 1M Glacial Acetic Acid, 0.05 M EDTA) by heating. Then, 2.5μl Ethidium Bromide was added and mixed, the gel was casted into a mold which was fitted with a well forming comb. The agarose gel was submerged in electrophoresis buffer within a horizontal electrophoresis apparatus. After amplification, each PCR product and a DNA ladder 50 bp size marker (Promega, Madison, WI, USA) were loaded into the sample corresponding wells to aid in fragment size determination. Electrophoresis was performed using 23

Electrophoresis power supply (Cleaver scientific Ltd, UK) at 70 volts for 50 mins at room temperature, and the DNA bands were visualized and documented using a UV transilluminator documentation system. 3.2.6 Statistical analysis: Statistical analysis was carried out using the statistical package for social sciences (SPSS) version 21 for windows. Frequency, means and standard deviation were calculated. Confidence intervals (95%) were reported when appropriate. Chi square test (χ²) and fisher exact tests were applied to compare sociodemograhic, infertility status, risk factors and signs and symptoms with T. vaginalis infection. 24

CHAPTER FOUR Results 4.1 Sociodemographic characters distribution of the study population: In the present study the subjects were married, non-pregnant women aged between 16 and 45 yrs (mean age, 27.6 ± 6.2 and median age, 27.0). Among the participants, the majority (59.2%) were 21-30 years old. About (28.3%) were 31-45 yrs old and (12.5%) were 16-20 yrs old. Residence areas were divided into 5 main areas, where the patients came from different locations to attend Al Basma medical center in Gaza city. Most cases (47.5%), were from Gaza city, followed by Gaza north (21.7%), Mid zone (15%), Khanyounis (10%), meanwhile the lowest number of cases (5.8%) were from Rafah. Most of the women (85.8%) were not working. The subjects reported various degrees of educational level. The majority were of University educational level (51.7%). About (35.8%) were of secondary education and (12.5) were pf primary and preparatory education. 4.2 Residency status of the study population: According to residency status, (64.2%) had independent home while the rest were living in house shared with husband family. Regarding number of subjects at the house, about (60%) had more than two subjects living at the same house. 4.3 Age at marriage, marriage duration and consanguinity of the study population: Most of subjects (53.3%) were married at the age of 15-20 years (Mean age, 21.6± 5.7, median 20). About (39.2%) were married at 21-30 yrs old and (7.5%) were married at age above 30 yrs. The duration of marriage mostly (66.6%) ranged from 1 to 10 years (Mean duration, 5.9 ± 4.9, median 4), followed by (19.2%) were married for >10 yrs and (14.2%) were of marriage duration for less than 1 yr. Most of women (76.7%) were married to a partner who was not relative to them. 4.4 Infertility status of the study population: It was found that (60%) of the subjects were cases of primary infertility with different causes. Causes including: male factor as low sperm count, unknown cause, cystic ovaries, fallopian tubes irregularity and absent male vas deference. The present study showed that 25

14 patient (11.7%) attended the center for infertility consultation before completing the first year of marriage. Thus, these patient are not cases of primary infertility and were labeled in the study as: delayed conception for less than 1 year. Out of 34 (28.3%) patient of secondary infertility, 15 patients reported history of previous abortion. Infertility status of patients is illustrated in Table 4.4. 4.5 Risk factors of T. vaginalis among the studied population: Most cases (95%) had no chronic disease and minority reported conditions like viral hepatitis, hypertension, thalassemia, anemia and skin allergy. About (97.5%) were not on regular medications. About (58.3%) of cases were non smoker but (41.7%) have a smoker husband. Majority of subjects (65.8%) reported no history of previous vaginal infection while (82.5%) denied history of urinary tract infection (UTI) in the last month of sample collection. Only (6.7%) of women were using vaginal wash in weekly regular bases. 4.6 Symptomatology distribution of the study population: Regarding infection symptoms, most cases had no symptoms. Minority of cases (29.2%) complained of abnormal vaginal discharge mostly of white color and with no odor. Also minority of cases complained of pruritis (12.5%), lower abdominal pain (15.8%), dysuria (10%), dyspareunia (17.5%), contact bleeding (2.5%) and vulvar erythema (96.7%). 26

4.7 PCR results of endocervical swabs DNA: In the present study the results showed that 7 (5.8%) of women were positive for T. vaginalis using PCR technique as shown in Table 4.1. and Figure 4.1 with (95% CI, 0.0265 to 0.1176). Table 4.1: Frequency of positive and negative PCR results for endocervical swab DNA. PCR results frequency % Positive 7 5.8 Negative 113 94.2 Total 120 100 Fig 4.1: PCR product on agarose gel (312bp) obtained using T. vaginalis primers. Lane L: 50 bp Ladder, lane P: Positive control (312 bp), lane N: Negative control. Lanes 1, 2, 3, 5, 7 and 8 are positive samples while lanes 4, 6 and 9 are negative. 4.8 Frequency of T. vaginalis infection according to the sociodemographic characters: The infection rate of T. vaginalis infection were higher among women aged 31-45 yrs and also in cases from Mid zone governorate while women from Gaza North and Rafah showed no infection. The infected subjects had no history of working. Regarding education, infection rate was highest in primary and preparatory educational level. Sociodemographic characters of the infected population are shown in Table 4.2 27

Table 4.2: Frequency of positive DNA T. vaginalis infection according to sociodemographic characters. Variable Positive PCR results No./n (%) χ², P-value Age group (yrs): 16-20 21-30 31-45 0/15 3/71 4/34 0 4.2 11.8 3.441, 0.179 Governorates: Gaza North Gaza Mid zone Khanyounis Rafah 0/26 3/57 3/18 1/12 0/7 0 5.3 16.7 8.3 0 6.060, 0.195 Occupation: Working Not working 0/17 7/103 0 6.8 1.227, 0.333 Education: Primary and Preparatory Secondary University P-value <0.05 considered a significant. n=total number of items. 2/15 3/43 2/62 18.2 7 3.2 2.406, 0.300 4.9 Frequency of T. vaginalis infection according to residency status: In cases were living in house shared with husband s family, the infection rate was (7%) compared to (5.2%) in women living in a separate house. Infection rate were highest among families with more than 2 subjects living in the same house. Residency status details are shown in Table 4.3. 28

Table 4.3: Frequency of positive DNA T. vaginalis infection according to residency status. Residency status Independent (separate) Shared with husband family Number of subjects in the same house 2 >2 Positive PCR results No./n 4/77 3/43 2/48 5/72 % χ², P-value 5.2 7 0.159, 0.488 4.2 6.9 0.405, 0.416 4.10 Frequency of T. vaginalis infection according to age at marriage, duration of marriage and consanguinity: Most cases were married at age of 15-20 years, highest infection rate was observed in women who were married at the age >30 years old (significant relation, P-value=0.001) and of marriage duration for more than 10 years. All the infected cases were married to a non-relative husband. Table 4.4. Table 4.4: Frequency of positive DNA T. vaginalis infection according to age at marriage, marriage duration and consanguinity. Variable Positive PCR results % χ², P-value Age at marriage (yrs) 15-20 21-30 > 30 Duration of marriage (yrs) < 1 1-10 > 10 Consanguinity Not relative Relative No./n 4/64 0/47 3/9 2/17 2/80 3/23 7/92 0/28 6.3 0 33.3 11.8 2.5 13 7.6 0 15.322, 0.001 4.884, 0.087 2.262, 0.147 29

4.11 Frequency of T. vaginalis infection according to infertility status: It was found that cases with delayed conception before completing first year of marriage was associated with higher rate of T. vaginalis detection (14.2%) as shown in Table 4.5. Table 4.5: Frequency of positive DNA T. vaginalis infection according to infertility status. Type of infertility complain Positive PCR results % χ², P-value No./n Delayed conception for < 1 yr 2/14 14.3 Primary infertility 3/72 4.2 Secondary infertility 2/34 5.9 2.185, 0.335 4.12 Frequency of T. vaginalis infection according to risk factors: The highest rate of T. vaginalis Infection were among passive smoker patients and among patients who denied history of previous vaginal infection with statistically significant relationship (P=0.04) or UTI in the last month. All infected cases were not using vaginal wash. Possible Risk factors in the infected cases are listed in Table 4.6. Table 4.6: Frequency of positive DNA T. vaginalis infection according to risk factors. Risk factors Positive PCR results % χ², P-value Smoking Active Passive Husband non smoker No./n 0/0 4/50 3/70 0 8 4.3 0.733, 0.318 History of vaginal infections Yes No History of UTI in the last month Yes No Weekly vaginal wash use Yes No 0/41 7/79 1/21 6/99 0/8 7/112 0 8.9 4.8 6.1 0 6.3 3.858, 0.049 0.053, 0.646 0.531, 0.609 30

4.13 Frequency of T. vaginalis infection according to infection symptoms: Out of seven patients who had no abnormal vaginal discharge, one patient complained of yellow discharge with bad smell. Highest infection rate were among patients who complained of pruritus and dyspareunia. The presence of vulvar erythema was statistically significant, (P=0.01). All infected cases denied any complain of lower abdominal pain, dysuria and bleeding after intercourse. Symptoms associated with T. vaginalis infection are shown in Table 4.7. Table 4.7: Frequency of positive DNA T. vaginalis infection according to self-reported symptoms. Symptoms Positive PCR % χ², P-value results No./n Abnormal vaginal discharge Yes (n=35) Color: White Green Yellow Black Smell: No odor Bad odor No 1/35 0/21 0/4 1/9 0/1 0/23 1/12 6/85 2.9 0 0 11.1 0 0 8.3 7.1 0.797, 0.340 2.974, 0.396 1.973, 0.343 Pruritus ( vulvar itching) Yes No 1/15 6/105 6.7 5.7 0.022, 0.617 Lower abdominal pain Yes No 0/19 7/101 0 6.9 1.398, 0.289 Dysuria ( burning urination) Yes No Dyspareunia (painful intercourse) 0/12 7/108 0 6.5 0.826, 0.469 Yes No Contact bleeding (bleeding after intercourse) Yes No Vulvar erythema Yes No 2/21 5/99 0/3 7/117 2/4 5/116 9.5 5.1 0 6 50 4.3 0.631, 0.354 0.191, 0.834 14.695, 0.017 31

CHAPTER FIVE Discussion Trichomoniasis is one of the most common curable STDs worldwide. Trichomoniasis contributes to well-documented female reproductive tract complications in addition to HIV transmission. For these reasons, accurate estimation of the prevalence of this parasite is needed. According to WHO women of reproductive age refers to all women aged 15 49 years. (WHO, 2006). According to Palestinian Central Bureau of statistics (PCBS) latest survey in 2010, infertile women of reproductive age estimated as 8.3% of women in Gaza strip (PCBS, 2010) as shown in table 2.5. The present study population included 120 infertile women. The present study showed a prevalence of 5.8% among women attending infertility management center in Gaza strip. The previous two studies (Houso et al., 2011), (Al Hindi and Lubbad, 2006) conducted in Palestine to estimate T. vaginalis prevalence used a different study population (pregnant women). Furthermore, these studies had larger sample size and used different examination techniques like culturing and Pap smear. A conventional method Pap smear estimated a prevalence of 18.5% among Palestinian pregnant women, however, Pap smear is considered less sensitive and specific than culture and PCR (Hobbs and Seña, 2013). When comparing this study with other studies conducted in neighboring countries (as shown in table 2.3), the prevalence is higher than Jordan which had prevalence rate of 0.7% (Mahafzah et al., 2008) but lower than that reported in Egypt which showed prevalence of 36% (Aboulghar et al., 2009). These differences may be due to socioeconomic status of patients studied. Worldwide, (as shown in table 2.2) the prevalence in this study was similar to Tunisia, Iraq, Turkey, Sirilanka and Greek. In Gaza, the prevalence is higher than Kingdom of Saudi Arabia, Libya, Jordan, Iran, Sudan, Argentina, United States of America and United Kingdom. However, our prevalence is lower than Nigeria, India, Zambia, Ukraine, 32

Poland, Portugal, Indonesia, Tanzania and Uganda. This difference can be attributed to many factors including sample size, study setting, techniques used, population socioeconomic status and religion. All these factors make the comparison between different studies very difficult. To make the comparison easier, looking for a study that matches our study methodology will be a suitable way. KeleŞtemur et al., (2012) from Turkey conducted a study among infertile women (n=160) and he used the same primers set we used (Tv1 and Tv2). He found that the prevalence rate was 4.5%. Therefore, our prevalence rate is higher than Turkey. The prevalence rate found by the present study is expected, as Palestine is considered islamic country where sexual relationships can be achieved by marriage according to religion and law. This also decrease the possibility of sexual transmission as a route of infection but does not prevent indirect transmission as toilet seats and medical specula. Indirect transmission is usually linked to poor hygiene level and poverty observed in Gaza strip. These issues highlight the importance of considering social and cultural background difference before interpreting results across different communities. This study is considered the first study in Palestine assessing T. vaginalis prevalence using molecular method (PCR) which considered highly sensitive and specific when compared to the culture (the gold standard). We could not assess the performance of PCR because we did not use a comparative reference test as culture. Although PCR is considered a rapid sensitive technique as their results can be available within four hrs of sample collection, it is still an expensive when compared to the cost of the conventional methods in relation to the limited resources of the MOH in Gaza strip. In this study, the rate of T. vaginalis infection were higher in women aged 31-45yr, which were similar to the result of (Stemmer et al., 2012) that showed that infection rate is increased as age is increased. The association with older age is in contrast with the association between age and other STDs, especially Chlamydia (which is found to be more common in young females). This prevalence may be due to reproductive hormone level in older women especially postmenopausal (Spinillo et al., 1997). The increased prevalence of infection in older women may be indicative of long standing subclinical infection according to Sutton et al., (2007). 33

The highest rate of infection were among women living in Mid zone, one of Gaza governorates. According to our knowledge there are no infertility management centers in the Mid zone, so most infertile women from Mid zone will attend the centers in Gaza city which is more close to them than other governorates. All infected women were unemployed. Unemployment was associated with infection according to Schneider et al., (1998). This can t be explained in our study. Higher detection rate of T. vaginalis infection was found in women with preparatory educational level. Similar results obtained by Mbizvo et al., (2001) who found a positive association between reproductive tract infections (as T. vaginalis) and lower educational level. Thus, it may be possible that better education was associated with early seeking of confirmation of diagnosis and treatment for STDs as explained by Fernando et al., (2012). When discussing residency, although the relationship between infection and crowded residency did not reach the significant level, infection rate was higher in women living in houses shared with husband's family. This may favor indirect transmission via use of common towel's and toilet seats in such houses. This also may explain increasing risk of infection when number of subjects living together in the same house is increased. As reported by Scheidell, (2014), crowding was associated with STDs. Poverty and crowding is a major problem in Gaza strip, where one of the cases reported that she is living in house shared by other 30 subjects from the husband s family. According to Safi and Elnamrouty, (2012), poverty in Palestine was associated with large household size (large number of children less than 18 yrs old or large number of unemployed adults more than 18 yrs) and with high dependency ratio (No. of family member/no. of bedroom). Many factors play a role in this problem. As Gaza strip is considered a war zone land in addition to the Israeli siege on Gaza, many materials as building materials are prohibited from importing. The low general economic status and unemployment of the husband is common. All these factors prevent the couples from having independent house and instead, they will live in a room (in father of law house) sharing kitchen and bathroom with minimal privacy and minimal hygienic conditions. 34

Age at marriage was found to be of considerable importance. Statistically significant results showed higher infection in cases of marriage at early age <20 yrs and old age >30. Kaestle et al., (2005) stated that younger ages at first intercourse were associated with higher odds of STI in comparison with older ages (and the relation between first sexual intercourse and STIs does not vary by race, ethnicity, or parental education). This is also supported by another study (Mbivzo et al., 2001) showed that infection is high among female with sexual debut < 20 yrs and most vaginitis is in cases at age of marriage < 20 years. As in many arabic conservative countries, which is the case in Palestine, early age marriage below 20 years old is common. According to PCBS, (2010), median age of marriage for Palestinian females is 19.5 years. The high infection rate among this group can be explained by that there is a biologic predisposition of the immature cervix to infection if exposed (Kahn et al., 2002). In the other hand, usually in old age marriage, women usually try to get pregnant soon after marriage which my lead here to attend conception consultation clinics earlier, and subsequently will undergo gynecological investigations (as hysteroscopy, IUI, IVF,..) which favor transmission of the parasite by inoculation through infected instruments. This also explain why in women with marriage duration >10 yrs, infection rate was high. In the present study all infected women were married to a relative husband. This observation is common in Palestine as most women are married to consanguineous partner. Higher detection rate of infection was among women who attended the center before completing the first year of marriage. This may be due to change in vaginal environment (mainly after intercourse) which decreases acidity of the vaginal ph. favoring the growth of such parasite (Goode et al., 1994). Although the cross sectional design is not the perfect design for defining risk factors, it can draw attention to some of them. Smoking is considered a risk factor for Trichomoniasis (Swartzendruber et al., 2013) (Zhang, 1996). This is observed in our results as infection detection were high among women married to smoker partner. 35

Statistically significant results showed denial of any history of previous vaginal infection in women infected with T. vaginalis. When further questioning the patients about the details of such infection, no clear answers were obtained about diagnosis and treatment. This may be due to inaccurate diagnosis of these episodes or insufficient informations delivered from physician to the patients and knowledge status of those patients. All patients denied any history of STDs, however, knowledge of STDs is limited in the community. Add to that point, Palestine is an islamic country, in which social embarrassment is a concern that may prevent patients from admitting STDs or vaginal infections if such diagnosis is present. In Gaza strip, there are neither electronic health recording system nor STDs clinics in MOH which make it impossible to get such information from patient files. Six patient out of seven reported no history of UTI in the last month of sample collection, although UTI is common in T. vaginalis (Swygard et al., 2004). All infected patients denied usage of vaginal wash weekly. This was different from the results obtained by Sutton et al., (2007) who found that infection rate is higher among female using vaginal douching. As explained by Rosenberg and Phillips, (1992), douching reduces the density of normal vaginal flora, so there may be a predisposition to colonization by such sexually transmitted pathogens. Similar to Wilkinson et al., (1997) findings, most of infected cases were asymptomatic. The signs and symptoms associated with the infection have a relatively low-positive predictive value for Trichomoniasis because of the frequent occurrence of these signs and symptoms among women with other infections (Sood and Kapil, 2008). Our finding is different from Sivaranjini et al., (2013) who found that among the clinical signs in women with vaginal discharge, having a profuse, malodorous, frothy and mucopurulent or purulent discharge were significantly associated with Trichomoniasis. Our results were different from that reported by Petrin et al., (1998) who recorded that the classic presentation of T. vaginalis is, that of a purulent, foul-smelling vaginal discharge which is associated with pruritis, dysuria and dyspareunia. However, these classic symptoms are only seen 20% of the time. Statistically significant results showed vulvar erythema is a significant complain among infected patients as supported by the findings of Wølner-Hanssen et al., (1989). 36

CHAPTER SIX Conclusion and Recommendations 6.1 Conclusion: The present study focused on detecting T. vaginalis among infertile women attending infertility center in Gaza strip using a sensitive and a specific molecular technique (PCR). The results of this study can be summarized as follows: The overall infection rate of T. vaginalis was 5.8% among infertile women in Gaza strip. Our results showed that there is a significant correlation between T. vaginalis infection and age at marriage, absence of history of previous vaginal infection and vulvar erythema (P<0.05). No significant correlation was observed between T. vaginalis infection and female personal hygiene practice and residency conditions which may favor iatrogenic cause as a source of infection. 37

6.2 Recommendations 1. We encourage health care workers to practice infection control measures during gynecological examination and procedures to avoid any source of infection. 2. We advise to establish electronic database for STDs and make them reportable diseases that ensure treatment of both husband and wife. 3. We recommend further studies to investigate parasite presence in medical equipments e.g; speculum. 4. We encourage further studies about STDs in men as this population has not been studied before. 5. It is advisable to initiate health education programs among physicians and patients about STDs especially Trichomoniasis. 38

References Abdelaziz Z.A., Ibrahim M.E., Bilal N.E. and Hamid M.E., 2014. Vaginal infections among pregnant women at Omdurman Maternity Hospital in Khartoum, Sudan. Journal of Infection in Developing Countries, 8 (4): 490-7. Aboulghar M., Aboushady O., Ahmed J. and Hanafy N., 2009. Diagnosis of Trichomonas Vaginalis Infection in Women of Childbearing Age at a University Setting Using OSOM A NewDiagnostic Technique. Egyptian Journal of Medical Microbiology, 18, No. 351. Ackers J. P., 1990. Immunologic aspects of human trichomoniasis. In: Honigberg B M, editor. Trichomonads parasitic in humans. New York, N.Y: Springer-Verlag; pp.36 52. Al Hindi A.I. and Lubbad A.M.H., 2006. Trichonamas vaginalis infection among Palestinian women: Prevalence and trends during 2000-2006. Turkish Journal of Medical Sciences, 36 (6): 371-375. Al-Quaiz, J. M., 2000. Patients with vaginal discharge: a survey in a University primary care clinic in Riyadh City. Journal Annals of Saudi Medicine, 20 No. 3/4:302-306. Al-Saeed W. M., 2011. Detection of Trichomonas vaginalis by different methods in women from Dohok province. Eastern Mediterranean Health Journal, 17 (9): 706 709. Al-Salihi F.L., Curran J.P. and Wang J., 1974. Neonatal Trichomonas vaginalis: report of three cases and review of the literature. Pediatrics, 53 (2): 196-200. Al-Zanbagi N., Salem H. and Braiken F., 2005. Trichomonasis among women with vaginal discharge in Jeddah City, Saudi Arabia. Journal of the Egyptian Society of Parasitology, 35 (3): 1071-1080. Ben Aissa-Hamzaoui R., Kouki S., Ben Hamida A., Kechrid A. and Gueddana N., 1999. Prevalence of sexually transmitted diseases among pregnant Tunisian women and validation of a clinical algorithm proposed by WHO for the management of sexually transmitted diseases. Contraception Fertilite Sexualite, 27 (11): 785-90. Benchimol M., de Andrade Rosa I., da Silva Fontes R. and Burla Dias AJ., 2008. Trichomonas adhere and phagocytose sperm cells: adhesion seems to be a prominent stage during interaction. Parasitology Research, 102 (4): 597-604. Berger S. and Marr J., 2006. Human Parasitic Diseases Sourcebook. Jones and Bartlett's, pp. 399. Burch T.A., Rees C.W. and Reardon L.V., 1959. Epidemiological studies on human trichomoniasis. American Journal of Tropical and Medical Hygiene, 8 (3): 312 318. 39

Caliendo A.M., J. A. Jordan, A. M. Green, J. Ingersoll, R. J. Diclemente, and G. M. Wingood, 2005. Real-Time PCR Improves Detection of Trichomonas vaginalis Infection Compared with Culture Using Self-Collected Vaginal swabs. Infectious Diseases in Obstetrics and Gynecology, 13, no. 3: 145-150. Carlton J.M., Hirt R.P., Silva J.C., Delcher A.L., Schatz M., Zhao Q., Wortman J.R., Bidwell S.L., Alsmark C.M., and Besteiro S., 2007. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science, 315 (5809): 207-211. Center of Disease Control and Prevention Cdc.gov. Trichomoniasis, life cycle, parasites and health, 2009. http://www.dpd.cdc.gov/dpdx/html/frames/sz/trichomoniasis/body_tricho moniasis_page1.htm. Last accessed 24/9/2013. Center of disease control and prevention, 2014. Trichomoniasis. Trichomoniasis statistics. http://www.cdc.gov/std/trichomonas/stats.htm. Center of Disease Control and Prevention. Cdc.gov. STD fact sheet, 2012. http://www.cdc.gov/std/trichomonas/stdfact-trichomoniasis.htm, last accessed in 2/8/2013. Center of Disease Control and Prevention. Healthcare Infection Control Practices Advisory Committee (HICPAC), 2007. Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Setting. Cohn S. E. and Clark R. A., 2003. Sexually transmitted diseases, HIV, and AIDS in women. Medical Clinics of North America, 87, no. 5: 971 995. Collier L.,Belows A.,Sussmam M., Cox F., Kriere J. and Wakelin D., 1998. Topley and Wilson's.Microbiology and Microbial Infections, ed.9 th. Volume 5. Parasitology. Arnold, 203-2011. Cotch M.F., Pastorek J.G.2nd, Nugent R.P., Yerg D.E., Martin D.H. and Eschenbach D.A., 1991. Demographic and behavioral predictors of Trichomonas vaginalis infection among pregnant women. The Vaginal Infections and Prematurity Study Group. Obstetrics and Gynecology, 78 (6): 1087-92. Crucitti T., Jespers V., Mulenga C., Khondowe S., Vandepitte J. and Buvé A., 2011. Non-sexual transmission of Trichomonas vaginalis in adolescent girls attending school in Ndola, Zambia. Plose One, 6 (no. 1),: e16310. Crucitti T., Van Dyck E., Tehe, Abdellati S., Vuylsteke B., Buve A. and Laga M., 2003. Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens. Sexually Transmitted Infections, 79: 393-398. Diamond L.S., 1986. In vitro cultivation of the Trichomonadidae: a state of the art review. Acta Univ. Carol.-Biol. 30: 221 228. 40

Draper D., Donohoe W., Mortimer L., and Heine R. P., 1998. Cysteine proteases of Trichomonas vaginalis degrade secretory leukocyte protease inhibitor. Journal of Infectious Diseases, 178 (3): 815-819. Dwivedi S., Nuzhat Husain N., Singh R. and Malla N., 2012. 18S ribosomal DNA based assay for Trichomonas vaginalis infection in symptomatic and asymptomatic women in India. Asian Pacific Journal of Tropical Disease, 133138. Dyer, B. 1990. Phylum Zoomastigina Class Parabasalia, p. 252-258. InL. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman (ed.), Handbook of Protoctista. Jones and Bartlett, Boston, Mass. El-Shazly A.M., El-Naggar H.M., Soliman M., El-Negeri M., El-Nemr H.E., Handousa A.E., Morsy T.A., 2001. A study on Trichomoniasis vaginalis and female infertility. Journal of Egyptian Society of Parasitology, 31(2): 545-53. Eshete A., Mekonnen Z., and Zeynudin A., 2013. Trichomonas vaginalis Infection among Pregnant Women in Jimma University Specialized Hospital, Southwest Ethiopia. ISRN Infectious Diseases, vol. 2013, Article ID 485439, 5 pages. Faro S., 2003. Sexually Transmitted Diseases in Women, 1 ST ed. Lippincott Williams and Wilkins, 112. Fernando S., Herath S., Chaturaka Rodrigo C. and Lalani Rajapakse S., 2012. Clinical features and sociodemographic factors affecting Trichomonas vaginalis infection in women attending a central sexually transmitted diseases clinic in Sri Lanka. Indian Journal of Sexually Transmitted Disease and AIDS, 33 (1): 25 31. Fernando S., Herath S., Chaturaka Rodrigo C. and Senaka Rajapakse S., 2011. Improving diagnosis of Trichomonas Vaginalis infection in resource limited health care settings in Sri Lanka. Journal of Global Infectious Disease, 3 (4): 324-328. Francis S.C., Kent C.K., Klausner J.D., Rauch L., Kohn R., Hardick A., 2008. Prevalence of rectal Trichomonas vaginalis and Mycoplasma genitalium in male patients at the San Francisco STD clinic. Sexually Transmitted Diseases, 35 (9): 797-800. Fule S.R., Fule R.P. and Tankhiwale N.S., 2012. Clinical and laboratory evidence of Trichomonas vaginalis infection among women of reproductive age in rural area. Indian Journal of Medical Microbiology, 30 (3): 314-316. 41

Garber G.E., 2005. The laboratory diagnosis of Trichomonas vaginalis. The Canadian Journal of Infectious Diseases and Medical Microbiology, 16 (1): 3538. Garcia A., Exposto F., Prieto E., Lopes M., Duarte A. and Correia da Silva D., (2004). Association of Trichomonas vaginalis with sociodemographic factors and other STDs among female inmates in Lisbon. Journal of STD and AIDS, 15 (9): 615-618. Goode, M. A., Grauer K., and Gums J. G., 1994. Infectious vaginitis. Selecting therapy and preventing recurrence. Postgraduate Medical Journal, 96: 85-98. Gram I.T., Macaluso M., Churchill J. and Stalsberg H., 1992. Trichomonas vaginalis (TV) and human papillomavirus (HPV) infection and the incidence of cervical intraepithelial neoplasia (CIN) grade III. Cancer Causes Control, 3 (3): 231-236. Grodstein F., Goldman M.B. and Cramer D.W., 1992. Relation of Tubal Infertility to History of Sexually Transmitted Diseases. Oxford Journals, Medicine, American Journal of Epidemiology, 137 (5): 577-584. Gross G. and Tyring S., 2011. Sexually Transmitted Infections and Sexually Transmitted Diseases, 1 st ed. Springer, 2011. Habib F., El-Aal A., Yamany N., Mohamadein A., Hamed O., Bahashwan A., Shehata N. and El-Hosseiny L., 2009. Feasibility of a nested PCR for the diagnosis of vaginal trichomoniasis: Study in Al-Madinah Al-Munwarrha, Saudi Arabia. International Journal of Medicine and Medical Sciences, 1 (5): 206-210. Hager W. D., Brown S. T., Kraus S. J., Kleris G. S., Perkins G. J., Henderson M., 1980. Metronidazole for vaginal trichomoniasis: seven-day vs. single-dose regimen. The Journal of the American Medical Association, 244: 1219 1220. Hardick A., Hardick J., Wood B.J. and Gaydos C., 2006. Comparison between the Gen-Probe transcription-mediated amplification Trichomonas vaginalis research assay and real-time PCR for Trichomonas vaginalis detection using a Roche LightCycler instrument with female self-obtained vaginal swab samples and male urine samples. Journal of Clinical Microbiology, 44: 4197 4199. Heine P. and McGregor J., 1993. Trichomonas vaginalis: a re-emerging pathogen. Clinical Obstetrics and Gynecology, 36: 137 144. Hobbs M. and Seña A., 2013. Modern diagnosis of Trichomonas vaginalis infection. Sexually Transmitted Infections, 89 (6): 434 438. 42

Houso et al., 2011: Houso Y., Farraj M. A., Ramlawi A., and Essawi T., 2011. Detection of Trichomonas vaginalis in Vaginal Swab Clinical Samples from Palestinian Women by Culture. ISRN Microbiology, vol. 2011, Article ID 872358, 4 pages. Jatau E. D., Olonitola, O. S. & Olayinka, A., 2006. Prevalence of Trichomonas Infection among Women Attending Antenatal Clinics in Zaria, Nigeria. Annals of African Medicine, 5, No 4: 178-181. Johnston V. J. and Mabey D. C., 2008. Global epidemiology and control of Trichomonas vaginalis. Hospital for Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK. Current Opinion in Infectious Diseases, 21: 56 64. Kaestle C.E., Halpern C.T., Miller W.C. and Ford C.A., 2005. Young age at first sexual intercourse and sexually transmitted infections in adolescents and young adults. American Journal of Epidemiology, 161 (8): 774-80. Kahn J.A., Rosenthal S.L., Succop P.A., et al., 2002. Mediators of the association between age of first sexual intercourse and subsequent human papillomavirus infection. Pediatrics, 109:E5. Kassem H.H. and Majoud O.A., 2006. Trichomoniasis among women with vaginal discharge in Benghazi city, Libya. Journal of the Egyptian Society of Parasitology, 36 (3): 1007 16. Kaul P., Gupta I., Sehgal R. and Malla N., 2004. Trichomonas vaginalis: random amplified polymorphic DNA analysis of isolates from symptomatic and asymptomatic women in India. Parasitology International, 53 (3): 255 62. Kayser F., Bienz K., Echert J. and Zinkernagel R., 2005. Medical Microbiology 10 th ed. Georg Thieme Verlag, 481. Klelestemur N., Kaplan M., Ozdemir E. and Erensoy A., 2012. The Frequency of Trichomonas vaginalis, Gardnarella vaginalis and Candida ssp. Among Infertile Men and Women with Vaginitis. Kafkas Univ Vet Fak Derg, 18 (Suppl-A): A47-A52, Sayfa: A47-A52. Klinger E.V., Kapiga S.H. and Sam N.E., Aboud S., Chen C.Y., Ballard R.C. and Larsen U., 2006. A community-based study of risk factors for Trichomonas vaginalis infection among women and their male partners in Moshi urban district, Northern Tanzania. Sexually Transmitted Disease, 33: 712 718. Kostara I., Carageorgiou H., Varonos D. and Tzannetis S., 1998. Growth and survival of Trichomonas vaginalis. Journal of Medical Microbiology, 47 (6): 555-60. 43

Krieger J.K., 1984. Prostatitis syndromes: pathophysiology, differential diagnosis, and treatment. Sexually Transmitted Diseases, 11: 100 112. Krieger J.N., Tam M.R., Stevens C.E., Nielsen I.O., Hale J., Kiviat N.B., Holmes K.K., 1988. Diagnosis of trichomoniasis. Comparison of conventional wet-mount examination with cytologic studies, cultures, and monoclonal antibody staining of direct specimens. Journal of American Medical Association, 259 (8): 1223-7. Krieger JN., 1998. Trichomoniasis in men: old issues and new data. Sexually Transmitted Diseases, 22 (2): 83-96. Kurnatowska A. and Mamos A.R., 2001. Prevalence of Trichomonas vaginalis Donné in women of Lódź population in 1955-1999 years. Wiad Parazytol, 47 Suppl 1:9-12. Laga, M., Manoka A., Kivuvu M., Malele B., Tuliza M., Nzila N., Goeman J., Behets F., Batter V., Alary M.et al., 1993. Non-ulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: results from a cohort study. AIDS, 7: 95-102. Lan P.T., Lundborg C.S., Mogren I., Phuc H.D. and Chuc N.T. Lack of knowledge about sexually transmitted infections among women in North rural Vietnam. BMC Infectious Diseases, 9:85. Lanceley F., 1953. Trichomonas vaginalis in the male. The experi- mental infection of a few volunteers. British Journal of Venereal Diseases, 29 (4): 213 217. Landes M., Thorne C., Barlow P., Fiore S., Malyuta R. and Martinelli P., 2007. Prevalence of sexually transmitted infections in HIV-1 infected pregnant women in Europe. Europian Journal of Epidemiology, 22 (12): 925-36. Latif AS, Mason PR, Marowa E., 1987. Urethral trichomoniasis in men. Sexually Transmitted Diseases, 14 (1): 9-11. Lawing L. F., Hedges S. R., and Schwebke J. R., 2000. Detection of Trichomonosis in Vaginal and Urine Specimens from Women by Culture and PCR. Journal of Clinical Microbiology, 38 (10): 3585 3588. Linstead D., 1981. New defined and semi-defined media for cultivation of the flagellate Trichomonas vaginalis. Parasitology, 83 (Pt 1): 125 137. Lockwood B., North M. J., Scott K. I., Bremner A. F., Coombs G. H., 1987. The use of a highly sensitive electophoretic method to compare the proteinases of trichomonads. Molecular and Biochemical Parasitology, 24 (1): 89-95. Mabey D., Ackers J. and Adu Sarkodie Y., 2006.Trichomonas vaginalis infection. Sexually transmitted infections, 82 (4): iv26 iv27. 44

Madhivanan P., Bartman M., Pasutti L., 2009. Prevalence of Trichomonas vaginalis infection among young reproductive age women in India: implications for treatment and prevention, Sexual Health, 6, no. 4, pp. 339 344. Madico G., Quinn T.C., Rompalo A., McKee K.T. and Gaydos C.A., 1998. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples. Journal of Clinical Microbiology, 36 (11): 3205-10. Mahafzah A.M., Al-Ramahi M.Q., Asa d A.M. and El-Khateeb M.S., 2008. Prevalence of sexually transmitted infections among sexually active Jordanian females. Sexually Transmitted Diseases, 35 (6): 607 10. Mahmoud M.S., Abdel-Aziz S.S., El-Sherif E.A. and Swidan KH., 1999. Diagnosis of symptomatic and asymptomatic Trichomonas vaginalis infection by applying one tube nested PCR to vaginal discharge. Journal of the Egyptian Society of Parasitology, 29 (3): 1031-46. Mascarenhas M., Flaxman S., Boerma T., Vanderpoel S. and Stevens G., 2012. Trends in prevalence. National, regional, and global trends in infertility: a systematic analysis of 277 health surveys. Plos Medicine, 9 (12): e1001356. Mathews H M and Healy G R., 1983. Evaluation of two serological tests for Trichomonas vaginalis infection. Journal of Clinical Microbiology, 17: 840 843. Mayta H., Robert H. Gilman and Vivar A., 2000. 18S Ribosomal DNA-Based PCR for Diagnosis of Trichomonas vaginalis. Journal of Clinical Microbiology, 38 (7): 2683-2687. Mbizvo E.M., Msuya S.E., Stray-Pedersen B., Sundby J., Chirenje Z.M. and Hussain A., 2001. Determinants of reproductive tract infections among asymptomatic women in Harare, Zimbabwe. Central Africa Journal of Medicine, 47 (3): 57 64. McGrory T. and Garber G. E., 1992. Mouse intravaginal infection with Trichomonas vaginalis and role oflactobacillus acidophilus in sustaining infection. Infection and Immunity, 60: 2375 2379. McGrory T.K., Meysick C., Lemchuk-Favel L. and Garber G., 1994. The interaction of Lactobacillus acidophilus and Trichomonas vaginalis in vitro. Journal of Parasitology 80: 50 54. Narcisi E.M. and Secor W.E., 1996. In vitro effect of tinidazole and furazolidone on metronidazole-resistant Trichomonas vaginalis. Antimicrobial Agents and Chemotherapy, 40 (5): 1121-5. Nourian A., Shabani N., Fazaeli A. and Mousavinasab N., 2013. Prevalence of Trichomonas vaginalis in Pregnant Women in Zanjan, Northwest of Iran. Jundishapur Journal of Microbiology, 6 (8): e7258.a Q. 45

Oakeshott P., Ahmed J., Hay P.E., Reid F., Kerry S.R., Aghaizu A. and Tong CY, 2011. Trichomonas vaginalis among multiethnic female UK students. Sexually Transmitted Infection, 87 (5): 369. Central Bureau of statistics (PCBS), 2010. Paterson B.A., Tabrizi S.N., Garland S.M., Fairley C.K. and Bowden F.J., 1998. The tampon test for trichomoniasis: a comparison between conventional methods and a polymerase chain reaction for Trichomonas vaginalis in women. Sexually Transmitted Infections, 74 (2): 136 139. Perazzi B., Menghi C. and Coppolillo E., 2010. Prevalence and comparison of diagnostic methods for Trichomonas vaginalis infection in pregnant women in Argentina. Korean Journal of Parasitology, 48 (1): 61 65. Pereira-Neves, A. and Benchimol M., 2008. Trichomonas vaginalis: In vitro survival in swimming pool water samples. Experimental Parasitology, 118:438441. Petrin D., Delgaty K., Bhatt R.. and Garber G., 1998. Clinical and Microbiological Aspects of Trichomonas vaginalis. Clinical Microbiology Review, American Society of Microbiology, 11 (2): 300-317. Piperaki E., Theodora M, Mendris M, Barbitsa L, Pitiriga V, Antsaklis A. and Tsakris A., 2010. Prevalence of Trichomonas vaginalis infection in women attending a major gynaecological hospital in Greece: a cross-sectional study. Journal of Clinical Pathology, 63 (3): 249-53. Poole D.N. and McClelland R.S., 2013. Global epidemiology of Trichomonas vaginalis. Sexually Transmitted Infections, doi:10.1136/sextrans-2013-051075. Rahi A., Ali A. and Khamesipour A., 2014. Comparison of Real time PCR with traditional tools for diagnosis of Trichomonas vaginalis in patient with vaginal discharge at Kut city, Iraq. Journal of Advances in Biology, 5 no.3. Rasti S., Behrashi M., Mousavi GH. and Monieri R., 2011. Complications of trichomoniasis on the pregnant women. Jundishabur Journal of Microbiology, 4 (1). Rein, M. F., 1990. Clinical manifestations of urogenital trichomoniasis in women, p. 225-234. In B. M. Honigberg (ed.), Trichomonads parasitic in humans. Springer- Verlag, New York, N.Y. 46

Riley D.E., Roberts M.C., Takayama T., Krieger J.N., 1992. Development of a polymerase chain reaction based on diagnosis of Trichomonas vaginalis. Journal of Clinical Microbiology, 30 (2): 465 472. Rosenberg M.J. and Phillips R.S., 1992. Does douching promote ascending infection?. Journal of Reproductive Medicine, 37 (11): 930-8. Ryu J.S., Chung H.L., Min D.Y., Cho Y.H., Ro Y.S. and Kim SR., 1999. Diagnosis of trichomoniasis by polymerase chain reaction. Yonsei Medical Journal. 40 (1): 56-60. Safi S. and Elnamrouty KH., 2012. Building Logistic Regression Model to Identify Key determinants of poverty in Palestine. IUG Journal of Natural and Engineering Studies, Vol.20, No.1; 85-102 2012. Scheidell J., 2014. Independent and synergistic effects of low education and poverty on sexually transmitted infection in Haiti. American Public Health Association 142 nd annual meeting and expo November 18, 2014: 2:30 PM - 2:42 PM. Schneider H., Coetzee D.J., Fehler H.G., Bellingan A., Dangor Y., Radebe F., Ballard R.C., 1998. Screening for sexually transmitted diseases in rural South African women. Sexually Transmitted Infecionst, 74 Suppl 1:S147-52. Schwebke J., Venglarik M., and Morgan S., 1999. Delayed versus immediate bedside inoculation of culture media for diagnosis of vaginal trichomonosis. Journal of Clinical Microbiology, 37:2369-2370. Sherman J.K., Hostetler T.L., Mchenry K. and Daly J.J., 1991. Cryosurvival of Trichomonas vaginalis during cryopreservation of human semen. Cryobiology, 28 (3): 246 250. Sivaranjini R., Jaisankar T. J., Thappa D. M., Kumari R., Chandrasekhar L., Malathi M., Parija S. and Habeebullah S., 2013. Trichomoniasis: How do we diagnose in a resource poor setting?. Indian Journal of Sexually Transmitted Diseases, 34 (1): 25 31. Smith R.F., 1983. Viability of Trichomonas vaginalis in vitro at four temperatures. Journal of Clinical Microbiology, 18 (4): 834 836. Smyth J.D., 1994. Introduction to Animal Prasitology, 3rd ed. Cambridge university press, 1994. 47

Sood S. and Kapil A., 2008. An update on Trichomonas vaginalis. Indian Journal of Sexually Transmitted Diseases, 29 (1): 7 14. Sood S., Mohanty S., Kapil A, Tolosa J. & Mittal S., 2007. InPouch TV culture for detection of Trichomonas vaginalis. Indian Journal of Medical Research, 125 (4): 567-71. Sorvillo F, Smith L, Kerndt P, Ash L., 2001. Trichomonas vaginalis, HIV, and African- Americans. Emerging Infectious Diseases, 7: 927 932. Spinillo A., Bernuzzi A.M., Cevini C., Gulminetti R., Luzi S. and De Santolo A., 1997. The relationship of bacterial vaginosis, Candida and Trichomonas infection to symptomatic vaginitis in postmenopausal women attending a vaginitis clinic. Maturitas, 27 (3): 253-60. Stemmer S.M., Adelson M.E., Trama J.P., Dorak M.T. and Mordechai E., 2012. Detection rates of trichomonas vaginalis, in different age groups, using real-time polymerase chain reaction. Journal of Lower Genital Tract Diseases, 16 (4): 352-7. Sutcliffe S., Giovannucci E., Alderete J.F., Chang T.H., Gaydos C.A., Zenilman J.M., De Marzo A.M., Willett W.C. and Platz E.A., 2006. Plasma antibodies against Trichomonas vaginalis and subsequent risk of prostate cancer. Cancer Epidemiology, Biomarkers and Prevention, 15 (5): 939-45. Sutton M., Sternberg M, Koumans E.H., McQuillan G., Berman S. and Markowitz L., 2007. The prevalence of Trichomonas vaginalis infection among reproductive-age women in the United States, 2001-2004. Clinical Infectious Diseases, 45 (10): 1319-26. Swartzendruber A., DiClemente R. J., Sales J. M., Brown J. L. and Rose E. S., 2013. Correlates of Incident Trichomonas Vaginalis Infections Among African American Adolescent Females. Sexually Transmitted Infection, 89: A33. Swygard H., Seña A.C., Hobbs M.M. and Cohen M.S., 2004. Trichomoniasis: clinical manifestations, diagnosis and management. Sexually Transmitted Infections, 80: 91-95. Tann C., Mpairwe H., Morison L., Nassimu K., Hughes P., Omara M., Mabey D., Muwanga M., Grosskurth H. and Elliott A., 2006. Lack of effectiveness of syndromic management in targeting vaginal infections in pregnancy in Entebbe, Uganda. Sexually Transmitted Infection, 82 (4): 285-289. 48

Tanudyaya F., Rahardjo E., Bollen L., Madjid N., Daili F., Priohutomo S., Morineau G., Nurjannah, RoselindaA.; Anartati, S.; Purnamawati, E. and Mamahit, R. S., 2010. Prevalence of sexually transmitted infections and sexual risk behavior among female sex workers in nine provinces in Indonesia. Southeast Asian Journal of Tropical Medicine and Public Health, 41 (2): 463473. Trichomoniasis.org. Trichomoniasis, transmission of trichomoniasis, 2009. http://trichomoniasis.org/causes/index.aspx. Last accessed 5/8/2013. Usanga V., Abia-Bassey L., Inyang-etoh P., Udoh S., Ani F., Archibong E., 2009. Trichomonas vaginalis Infection Among Pregnant Women In Calabar, Cross River State, Nigeria. The Internet Journal of Gynecology and Obstetrics, 14 (2). Van Der Pol B., 2007. Trichomonas vaginalis infection: the most prevalent nonviral sexually transmitted infection receives the least public health attention. Clinical Infectious Diseases, 44 (1): 23-25. Van Der Pol B., Williams J.A., Orr D.P., Batteiger B.E., Fortenberry J.D., 2005. Prevalence, incidence, natural history, and response to treatment of Trichomonas vaginalis infection among adolescent women. Journal of Infectious Diseases, 192 (12): 2039-44. Wendel K.A., Erbelding E. J, Gaydos C.A., Rompalo A.M., 2003. Use of polymerase chain reaction to define the prevalence and clinical presentation of Trichomonas vaginalis in men attending an STD clinic. Sexually Transmitted Infections, 79: 151 153. White M.J., Sadalla J.K., Springer S.R. and Counselman F.L., 2005. Is the presence of Trichomonas vaginalis a reliable predictor of coinfection with Chlamydia trachomatis and/or Neisseria gonorrhoeae in female ED patients?. American Journal of Emergency Medicine, 23 (2): 127-130. Whittington M.J., 1957. Epidemiology of infections with Trichomonas vaginalis in the light of improved diagnostic methods. British Journal of Venereal Diseases, 33 (2): 80 91. Wilkinson D., Ndovela N., Harrison A., Lurie M., Connolly C., and Sturm A.W., 1997. Family planning services in developing countries: an opportunity to treat asymptomatic and unrecognised genital tract infections?. Genitourinary Medicine, 73 (6): 558 560. Wølner-Hanssen P., Krieger J.N., Stevens C.E., Kiviat N.B., Koutsky L., Critchlow C., DeRouen T., Hillier S. and Holmes K.K., 1989. Clinical manifestations of vaginal trichomoniasis. Journal of American Medical Association, 261 (4): 571-6. 49

Workowski K.A. and Berman S.M., 2006. Sexually transmitted diseases treatment guidelines. MMWR Recomm Rep. Aug 4 2006; 55(RR-11): 1-94. World Health Organization, 2004. Interventions for trichomoniasis in pregnancy. http://apps.who.int/rhl/rti_sti/gwacom/en/index. html. Last accessed in 3/8/2013. World Health Organization, 2006. Reproductive Health Indicators. Guidelines for their generation, interpretation and analysis for global monitoring. WHO: 9. World Health Organization, sexual and reproductive health. Revised glossary on Assisted Reproductive Terminology (ART).The International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) Revised Glossary on ART Terminology, 2009. Zhang Z. F., 1996. Epidemiology of Trichomonas vaginalis. A prospective study in China. Sexually Transmitted Diseases, 23 (5): 415-24. 50

Annex 1 Helsinki approval 51

Annex 2 The consent form دراسة علمية حول االلتهابات المهبلية الطفيلية السالم عليكم: أنااا الباحثااة الطبيبااة: أماال يوساام المقادمااة ملتحقااة الدراسات العليا / قسم األحياء الدقيقة. بالجامعااة اإلسااالمية بغاازة فااي كليااة أقااوم بااءجراء بحااث )بموافقااة مركااز البساامة الطبااي يهاادإ إلااى تحديااد نساابة انتشااار االلتهابات المهبلية الطفيلية في مدينة غزة ومعرفة مساببات للسايدات اللاواتي لاديهن مشااكل فاي اإلنجاب. سيشمل البحث تعبئة استبيان من خالل المقابلة المباشرة مع السيدة المشاركة في الدراسة وأخذ مسحة مهبلية منها حيث ستقوم الباحثة بتحليلها بتقنيات جينية جزئية لمعرفة إصابتها من عدمها. أختي الكريمة: إن نتائج الفحوصات المخبرية والمعلومات التي سوف يتضمنها فقط االستبيان سوف تستخدم للغرض البحثي وسريتها محفوظة ولن يتم نشرها بأي صورة من الصور وهي في النهاية إفادة لصحتك وسوف يتم تزويدك بنتائج التحاليل التي تخصك شاكرة لك تعاونك معنا في إنجاح هذا البحث مع كل االحترام والتقدير. أنااا الساايدة:... الموقعااة أدناااه أوافااق علااى المشاااركة بهااذا البحث. توقيع السيدة:... التاريخ:.../.../ 2102 م. 52