Occurrence of Rectal Gonorrhoea and Chlamydia and Antibiotic Susceptibility Profile Among Men Who Have Sex with Men at the Kenyan Coast

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OCCURRENCE OF RECTAL GONORRHOEA AND CHLAMYDIA AND ANTIBIOTIC SUSCEPTIBILITY PROFILE AMONG MEN WHO HAVE SEX WITH MEN AT THE KENYAN COAST

CAROLINE JUMWA NGETSA (BSC)

P150/CE/25905/2014

A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (INFECTIOUS DISEASES) IN THE SCHOOL OF MEDICINE OF KENYATTA UNIVERSITY

OCTOBER, 2018

DECLARATION

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DEDICATION

I dedicate this work to my family; loving husband Oscar, wonderful sons Lloyd and

Hans and my lovely daughter Stacey. For your patience and love during this worthy course, God bless you!

ACKNOWLEDGEMENT

Firstly, I would like to thank God for his goodness and mercy in granting me the strength to finish this worthy course in my life. I am deeply indebted to my supervisors Dr. Margaret Muturi and Dr. Nelson Menza both of Department of

Medical Laboratory Sciences, Kenyatta University, without their guidance, support, good nature and patience I would not have made it this far. The doors to their offices remained open all the times when I had trouble with this write up.

My gratitude also goes to my mentor and project director, Prof. E. Sanders for his guidance, encouragement and recommendation to applying for the International Aids

Vaccine Initiative (IAVI) Advance Degree Support scholarship. My special gratitude also goes to the IAVI for funding the project that culminated in to this thesis. To my colleagues; Peter Mugo, Amin Hassan and Simon Chengo, Eunice Nduati, Ian Oyaro and all IAVI project Kilifi staff big thank you for helping me in this write up and putting things into perspective in this world of science writing.

My special gratitude goes to my laboratory colleagues John Mwambi, Clara Karani,

Benedict Chengo and all Bacto staff in Microbiology department in helping with the hard and laborious laboratory work and tolerating my many times away from the laboratory. Finally, to my husband Oscar and kids your awesomeness in putting up with me during this very important time of my life. May Almighty God bless you all abundantly!

TABLE OF CONTENTS

Title Page

DECLARATION ...... ii DEDICATION ...... iii ACKNOWLEDGEMENT ...... iv TABLE OF CONTENTS ...... v LIST OF TABLES ...... viii LIST OF FIGURES ...... ix LIST OF APPENDICES ...... x ABBREVIATIONS AND ACRONYMS ...... xi ABSTRACT ...... xii 1.0 INTRODUCTION...... 1 1.1 Background information ...... 1 1.2 Problem Statement ...... 2 1.3 Justification ...... 3 1.4 Research Questions ...... 3 1.5 Objectives of Study ...... 4 1.5.1 General Objective ...... 4 1.5.2 Specific Objectives ...... 4 1.6 Significance of the Study ...... 5 2.0 LITERATURE REVIEW ...... 6 2.1 Sexually Transmitted Infections ...... 6 2.2 Sexual Transmitted Infections among men who have sex with men ...... 6 2.3 Asymptomatic gonorrhoea and chlamydia infections in men who have sex with men ...... 7 2.3.1 Gonorrhoea Infection ...... 7 2.3.2 Chlamydia Infection...... 8 2.4 Prevalence and occurrence of N. gonorrhoea and C. trachomatis ...... 9 2.5 Rectal Cellular Changes during infection ...... 9 2.6 Diagnosis of N. gonorrhoea and C. trachomatis ...... 10 2.6.1 Neisseria gonorrhoea...... 10

2.6.2 Chlamydia trachomatis ...... 11 2.7 Treatment of gonorrhoea and chlamydia ...... 12 2.7.1 History...... 12 2.7.2 Presumptive treatment of gonorrhoea and chlamydia infections ...... 14 2.8 Antibiotic resistance profiles of N. gonorrhoea ...... 14 3.0 MATERIALS AND METHODS ...... 16 3.1 Study Site ...... 16 3.2 Study Design ...... 16 3.3 Ethical Consideration ...... 16 3.4 Sampling Technique ...... 17 3.5 Study Population ...... 17 3.5.1 Inclusion criteria ...... 18 3.5.2 Exclusion criteria ...... 18 3.6 Enrolment of Participants ...... 18 3.7 Sample Size Determination...... 18 3.8 Sample Collection ...... 20 3.8.1 Rectal Swab ...... 20 3.8.2 Blood Collection ...... 21 3.9 Laboratory Methods ...... 21 3.9.1 Direct Smear for the detection of N. gonorrhoea ...... 21 3.9.2 Gene Xpert Chlamydia trachomatis / Neisseria gonorrhoea assay...... 22 3.9.2.1 Assay Quality Control...... 22 3.9.2.2 Assay Procedure ...... 23 3.9.3 Culture ...... 23 3.9.3.1 Gram stain ...... 24 3.9.3.2 Oxidase test ...... 25 3.9.3.3 Catalase Test ...... 25 3.9.3.4 Analytical Profile Index for Neisseria and Haemophilus ...... 26 3.9.3.5 Antibiotic susceptibility Test ...... 26 3.9.4 HIV Test...... 27 3.10 Data Analysis ...... 28

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4.0 RESULTS ...... 30 4.1 Demographic Characteristics of the Study Population ...... 30 4.1.1 Sexual Transmitted Infection risk characteristics of participants ...... 32 4.1.2 Risk for sexual exposure ...... 34 4.2 Occurrence of C. trachomatis and or N. gonorrhoea at Baseline ...... 36 4.2.1 Symptomatic and Asymptomatic rectal chlamydia and gonorrhoea ...... 36 4.3 Recurrence and reinfection of rectal gonorrhoea and chlamydia ...... 37 4.4 Comparison of Gram stain and Gene Xpert in the detection of rectal gonorrhoea and chlamydia ...... 39 4.5 Antibiotic sensitivity of N. gonorrhoea ...... 42 5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ...... 45 5.1 Discussion ...... 45 5.1.1 Demographic characteristics of participants at baseline ...... 45 5.1.2 Occurrence of asymptomatic rectal gonorrhoea and chlamydia among MSM at the Kenyan Coast ...... 49 5.1.3 Recurrence and reinfection of Neisseria gonorrhoea and Chlamydia trachomatis ...... 51 5.1.4 Comparison of Gram stain and Gen Xpert in the detection of rectal gonorrhoea and chlamydia trachomatis infections ...... 51 5.1.5 Performance of Gram stain against Gene Xpert RNA testing for detection of N. gonorrhoea ...... 52 5.1.5 Antibiotic sensitivity profile of N. gonorrhoea among MSM at the Kenyan Coast ...... 52 5.2 Conclusions ...... 55 5.3 Recommendations ...... 55 REFERENCES ...... 57 APPENDICES ...... 64 Appendix I: Consent Form...... 64 Appendix II: The map of Kenya showing the location of Kilifi County ...... 80 Appendix III: Ethical Approval ...... 81 Appendix IV: Risk Assessment Questionnaire ...... 82

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LIST OF TABLES

Table 4.1.a: Baseline Socio-demographic characteristics and their association with occurrence of rectal Neisseria gonorrhoea and Chlamydia trachomatis in MSM……………………………………………………….……...31 Table 4.1.b: Baseline STI risk characteristics of MSM and occurrence of rectal Neisseria gonorrhoea and Chlamydia trachomatis in MSM…………33 Table 4.1.c: Baseline sexual exposure risk characteristics and occurrence of rectal Neisseria gonorrhoea and Chlamydia trachomatis in MSM…………35 Table 4.2: Baseline prevalence of rectal C. trachomatis and or N. gonorrhoea among MSM at the Kenyan Coast…………………………………...36 Table 4.3: Prevalence of rectal C. trachomatis and or N. gonorrhoea among MSM at the Kenyan Coast at 6-months follow up, recurrence and or reinfection…………………………………………………………….36 Table 4.4a: Comparison of Gram stain and Gene Xpert in detection of NG at Baseline ……………………………………………………………...41 Table 4.4b: Comparison of Gram stain and Gene Xpert in detection of NG at Follow up……………………………………………………………..42

LIST OF FIGURES

Figure 4.1: Candle extinction jar with inoculated modified Thayer-Martin agar plates………………………………………………………………….37

Figure 4.2a: Proportion of Rectal Polymorphonuclear neutrophil cells populations

(PMN) Cells with CT and or NG at Baseline………………………...39

Figure 4.2b: Proportion of Rectal Polymorphonuclear neutrophil cells populations

(PMN) Cells with CT and or NG at Follow up………………………40

Figure 4.3: Sensitivity by disc diffusion test……………………………………..43

Figure 4.4: Sensitivity by minimum inhibition concentration……………………44

LIST OF APPENDICES

Appendix i: Consent……………………………………………………………….64

Appendix ii: Map of Kenya………………………………….……………………..80

Appendix iii: Ethical Approval…………………………………………...………...81

Appendix iv: Risk Assessment Questionnaire……………………………………...82

ABBREVIATIONS AND ACRONYMS

AST Antimicrobial Sensitivity Test

BSAC British Society for Antimicrobial Chemotherapy

CDC Centre for Disease Control

CT/NG Chlamydia trachomatis/Neisseria gonorrhoea

DNA Deoxyribonucleic Acid

HIV Human Immunodeficiency Virus

MSM Men who have Sex with Men

NAAT Nucleic acid amplification test

PMN Polymorphonuclear Neutrophils

TMM Thayer Martin Modified agar

RAI Receptive Anal Intercourse

STI Sexually Transmitted Infection

WHO World Health Organization

UK United Kingdom

USA United States of America

EDTA Ethylenediaminetetraacetic acid

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ABSTRACT

Men who have sex with men experience a high burden of Chlamydia trachomatis and Neisseria gonorrhoea infections which remain largely undiagnosed in the context of syndromic and presumptive treatment. In Kenya, limited data is available to inform on occurrence and recurrence of rectal chlamydia and gonorrhoea and antibiotic sensitivity on Neisseria gonorrhoea among Men who have sex with men who report receptive anal intercourse. The aim of this study was to determine the occurrence, recurrence and reinfection of rectal Neisseria gonorrhoea and Chlamydia trachomatis infections among Men who have sex at the Kenyan Coast. This study also compared the performance of Gram stain and Gene Xpert in the detection of chlamydia and gonorrhoea and determined the antibiotic sensitivity profile of N. gonorrhoea. The study was carried out in Kemri-Wellcome Trust Program Mtwapa Research clinic employing a longitudinal cohort study design and purposeful sampling technique. Men who had sex with men enrolled in an ongoing larger cohort studies and who reported receptive anal intercourse in the past six months were involved in this study. Three rectal swabs were collected and analysed by Gram stain, culture on Thayer Martin medium and molecular point of care technique by use of the Gene Xpert analyser. All gonorrhoea isolates were subjected to antibiotic sensitivity test. Results in this study were analysed using Stata version 13.0 (Stata Corp LP, college station, United States of America) and significance level set at P<0.05. Out of 104 MSM assessed at baseline, 21% had chlamydia or gonorrhoea, 12% had chlamydia only, 8% had gonorrhoea only and 2% had both chlamydia and gonorrhoea. Out of all the infections detected at baseline, 20% were asymptomatic and had <10 PMN cells (p=0.0039, 95% CI 0.02, 0.015). At 6 months follow-up, 82 (79%) those assessed contributed 39.1 person-years with a median follow-up time of 5.7 (interquartile range: 5.4-6.2). Similar findings of occurrence and recurrence were obtained as of the baseline with a high rate of chlamydia or gonorrhoea infection at 53.7 (95% CI, 35.0– 82.4) per 100 person-years. All isolated N. gonorrhoea were sensitive to ceftriaxone and cefixime, but resistant to ciprofloxacin. chlamydia or gonorrhoea infection was associated with being paid for sex [aOR, 4.2, p=0.01]. Therefore, this study showed a high burden of asymptomatic rectal chlamydia and or gonorrhoea co-infections among MSM at the Coastal Kenya with high recurrence of these infections over time. Gram stain method had very low specificity and sensitivity for rectal gonorrhoea and polymornonuclear neutrophils cells should not be used as indicator for infections in men who have sex with men. Macrolides and third generation cephalosporins antibiotics were highly sensitive to gonorrhoea isolates. The high prevalence of asymptomatic chlamydia and gonorrhoea suggest the need for regular screening or presumptive treatment. Screening by Gene Xpert should be considered as a valuable addition to routine screening of chlamydia and gonorrhoea in healthcare settings and treatment of gonorrhoea. Intervention programs should follow new guidelines that recommend macrolides and third generation cephalosporins and avoid quinolones, tetracyclines and penicillins.

1.0 INTRODUCTION

1.1 Background information

Chlamydia trachomatis and Neisseria gonorrhoea infections are problematic sexually transmitted infections (STIs) affecting huge numbers of the general population and high-risk subgroups (Goldenberg et al. 2012). It is estimated that nearly 20 million new STI occur every year in the USA (CDC 2015). A total of 1,441,789 cases of

Chlamydia and 350,062 cases of gonorrhoea which is an increase of 2.8% and 5.1% since 2013 were reported in the United States in 2014 (CDC 2015).

Men who have sex with men (MSM) are at high risk of contracting and transmitting human immunodeficiency virus (HIV) and other sexually transmitted infections such as syphilis, hepatitis B, chlamydia and gonorrhoea (Baral et al., 2007) and as such they have been recognized as a crucial population requiring targetted HIV prevention interventions (Rebe et al. 2015). Bodily sites of sexual infection in MSM may include but are not limited to oro-pharyngeal and anorectal sites commonly referred to as extragenital sites. Sexually transmitted infections (STIs) at these sites may be symptomatic or asymptomatic (Rieg et al., 2008).

There is a disproportionate high STI infection burden among MSM who practice receptive anal intercourse (RAI) with the increased complexity for the asymptomatic infections. Therefore, the routine practice of urogenital only testing can miss these asymptomatic infections hence will more likely remain undetected and untreated where syndromic management is advocated (Rebe et al. 2015).

In Kenya, studies have shown that men who have sex with men (MSM) are at disproportionately high risk of STI infection (Sanders et al., 2007). A study involving

43 MSM reported that 11 (26%) were detected as either Neisseria gonorrhoea or

Chlamydia trachomatis infection. Out of 43 participants, only 2 reported STI symptoms while the rest of the infections were asymptomatic (Sanders et al., 2010). A study carried out in 2013 in a cohort of 'high-risk' MSM in Coastal Kenya showed a high burden of N. gonorrhoea and C. trachomatis with incidence of 39.7% per 100 people (Sanders et al., 2014). This showed that the cases of N. gonorrhoea and C. trachomatis are still high among the MSM in Coastal Kenya and studies have shown that increase in multidrug resistance threatens their treatment (Lagace-Wiens et al.

2012). Therefore, this study determined the occurrence and recurrence of asymptomatic Neisseria gonorrhoea and Chlamydia trachomatis after treatment among MSM cohort at the Kenyan Coast. In addition, this study also determined the antibiotic sensitivity profile of Neisseria gonorrhoea.

1.2 Problem Statement

The threat of sexually transmitted infections (STIs) has increased between men who have sex with men and continue to cause increasing concerns in their management and treatment. Neisseria gonorrhoea and C. trachomatis have been associated with increased morbidity in MSM and asymptomatic infections which have resulted in the increase in spread and chances of HIV acquisition. World Health Organization recommends presumptive treatment to MSM reporting receptive anal intercourse, multiple sex partners or partner with an STI. However, there is no documented information in Kenya on how the presumptive treatment to MSM is practised. This problem may be aggravated by lack of information on antibiotic sensitivity profile and

reinfections among MSM. The study therefore investigated the occurrence and recurrence of gonorrhoea and chlamydia infections after treatment, compared the performance of Gram stain and Gene Xpert in detecting rectal gonorrhoea and

Chlamydia and the antibiotic sensitivity patterns of N. gonorrhoea among MSM.

1.3 Justification

Extragenital STIs are common in MSM and remain largely undetected and untreated where syndromic management is advocated especially in resource-limited settings.

Untreated STIs may increase the risk for HIV acquisition and reproductive health complications (WHO, 2011) hence negatively impacting on one‘s health. Moreover,

MSM living with HIV may be more likely to become infected with Chlamydia trachomatis and or Neisseria gonorrhoea and experience worse health outcomes.

Current efforts to diagnose and treat STIs are inadequate among MSM in Coastal

Kenya due to the low rate of extragenital screening for STIs. Even if a person with an

STI does get tested, their infection may not be diagnosed because they are not tested in the correct body site such as the rectum. Information obtained from this study informed on occurrence and recurrence of asymptomatic rectal STI and frequency of presumptive treatment. Data obtained from this study is helpful to the Ministry of

Health in informing on the diagnosis, treatment and management of rectal chlamydia and gonorrhoea infections among MSM in Kenya.

1.4 Research Questions

1. What is the occurrence of rectal gonorrhoea and chlamydia infections among

MSM at the Kenyan Coast?

2. What is the recurrence and reinfection of rectal gonorrhoea and chlamydia

infections after treatment?

3. How does the Gram stain performance compare with the Gene Xpert in the

detection of rectal gonorrhoea and chlamydia among MSM?

4. What is the antibiotic susceptibility profile of N. gonorrhoea among MSM?

1.5 Objectives of Study

1.5.1 General Objective

To determine the occurrence of rectal gonorrhoea and chlamydia infections and antibiotic susceptibility profile among men who have sex with men at the Kenyan

Coast.

1.5.2 Specific Objectives

1. To determine the occurrence of N. gonorrhoea and Chlamydia trachomatis

among MSM at the Kenyan Coast.

2. To determine the recurrence and reinfection of Neisseria gonorrhoea and

Chlamydia trachomatis after treatment among MSM at the Kenyan Coast.

3. To compare the performance of Gram stain and the Gene Xpert in the detection

of rectal gonorrhoea and chlamydia among MSM at the Kenyan Coast.

4. To determine the antibiotic sensitivity profile of N. gonorrhoea among MSM at

the Kenyan Coast.

1.6 Significance of the Study

Data obtained from this study informed on the potential to improve the detection of rectal gonorrhoea and chlamydia infections between men who reported receptive anal intercourse which were mainly asymptomatic and would have been missed where syndromic management of infections is advocated.

The study allowed for the selection of the right antibiotic for treatment of infected individuals to reduce the severity of complications of untreated disease and transmission. Further, the study compared the performance of the Gram stain and the

Gene Xpert in the detection of rectal gonorrhoea and chlamydia among MSM and informed on best method for the diagnosis of rectal chlamydia and gonorrhoea.

2.0 LITERATURE REVIEW

2.1 Sexually Transmitted Infections

Sexually transmitted infections (STIs) are important public health problem, especially in developing countries (Maina, Kimani, and Anzala 2016) with Chlamydia trachomatis and Neisseria gonorrhoea infections being the most common bacterial sexually transmitted infections (STIs) worldwide (Marangoni et al. 2015).

Anal and pharyngeal intercourse have been increasingly recognized as a component of the sexual repertoire of many couples, leading to the creation of selective high-risk networks for STIs transmission (Workowski et al. 2010). It is reported that 53% of chlamydial infections and 64% of gonococcal infections in a selected population of men who practice sex with other men (MSM) were localized at non urethral sites

(Kent et al. 2005).

2.2 Sexual Transmitted Infections among men who have sex with men

In developed countries, the occurrence of sexually transmitted diseases (STDs) among men who have sex with men (MSM) has been on the rise since late 1990s (Truong et al., 2006), a trend characteristically due to an increase in the practice of anal receptive intercourse (Wexner 1990). Subgroups of men who have sex with men (MSM) participate in unsafe sexual behaviours for example, having sex with many partners of unknown HIV status, performing unsafe oral and rectal sex; often leading to infections at these extragenital sites (CDC, 2014). This can be a public health problem because of the potential for enhancing HIV transmission (CDC 1998). In low and middle-income countries STDs are a significant burden to the health care system,

further fueled by a lack of systematic surveillance and population-based studies of

STD prevalence (Cunha et al. 2015).

2.3 Asymptomatic gonorrhoea and chlamydia infections in men who have sex with men A disease is considered asymptomatic or subclinical if a patient is a carrier for a disease or infection but experiences no symptoms. A condition might be asymptomatic if it fails to show the noticeable symptoms with which it is usually associated (Tattersall 2001). The asymptomatic infections may develop symptoms later, resolve itself or become benign or the affected person may act as a reservoir in the community that perpetuates transmission unknowingly spreading the infection to others. Diagnosis and treating asymptomatic infections is therefore paramount to preventing development of medical problems later and spread of infections (Tattersall

2001).

2.3.1 Gonorrhoea Infection

Gonorrhoea is a purulent infection of the mucous membrane surfaces resulting from infection with the Gram-negative diplococcus Neisseria gonorrhea (Wong, 2016).

This very common sexually transmitted disease (STD) can infect both men and women especially among young people ages 15-24 years (CDC, 2014). Transmission is by direct inoculation of infected secretions from one mucous membrane to another.

The primary sites of infection among MSM are the mucous membranes of the urethra, rectum and pharynx for those who practice oral sex also known as extragenital sites

(CDC, 2014). Rectal infection with gonorrhoea may cause symptoms in both men and women that may include; rectal pain, mucopurulent rectal discharge, anal itching,

soreness, bleeding and painful bowel movements. However, infection remain asymptomatic in 90% of the cases in the rectum and oro‐pharynx in both men and women (Bignell et al., 2006).

2.3.2 Chlamydia Infection

Chlamydia infection is caused by the bacterium Chlamydia trachomatis, a gram- negative, obligate intracellular parasite that is linked to several diseases causing one of the most common sexually transmitted diseases in the world (Schilling, 2005). For

Chlamydia species to exist and proliferate, they require to be inside mammalian cells as they are obligate intracellular bacteria. Thus, Chlamydia cannot be cultured on conventional bacteriological medium (Scidmore 2005). People with chlamydia infections trasmit it to their partners through exchange of infected bodily fluids by having unsafe vaginal, oral or anal sexual intercourse (CDC 1998). It can cause cervicitis in women and urethritis in men while in people who practice anal intercourse it can cause proctitis in both men and women which can lead to serious complications such as proctocolitis (CDC 1998).

The World Health Organization estimates 131 million new cases of C. trachomatis genital infection occur annually. Globally, infection is most prevalent in young women and men (14-25 years), likely driven by asymptomatic infection, inadequate partner treatment and delayed development of protective immunity (O'Connell and

Ferone 2016).

2.4 Prevalence and occurrence of N. gonorrhoea and C. trachomatis

The global estimates for the total number of new cases in adults stood at 105.7 million cases of C. trachomatis and 106.1 million cases of N. gonorrhoea in 2008. In the same report, Africa‘s total number of new cases was estimated to be 8.3 million cases of C. trachomatis and 21.1 million cases of N. gonorrhea (WHO, 2016). However, this report has no data on global incidence and prevalence of rectal C. trachomatis and N. gonorrhoea infections among MSM. In 2009, in a small study involving 43

HIV-1 negative MSM in coastal Kenya, documented a high burden of STIs among

MSM, with a reported prevalence of 12% for gonorrhoea and 44% for chlamydia

(Sanders et al. 2010). Most these infections were asymptomatic. None of the men in the study reported urethral or rectal discharge, although one man had dysuria and another had rectal pain.

2.5 Rectal Cellular Changes during infection

Proctitis, which is inflammation of the rectum may occur during anorectal STIs.

These are commonly the result of anal receptive intercourse and a high proportion of cases of proctitis are caused by gonorrhoea and chlamydia (Klausner, Kohn, and Kent

2004). A report by Klausesner (2004), defined proctitis as the presence of ≥5 polymorphonuclear cells per high power field on a rectal Gram stain. While Ivens et al., (2007) reported a positive association with chlamydia infection in those who indicated that they had symptoms, HIV-positive patients and in smears which showed greater than ten polymorphonuclear (PMN) cells/ high power field (Ivens et al. 2007).

2.6 Diagnosis of N. gonorrhoea and C. trachomatis

2.6.1 Neisseria gonorrhoea

The diagnosis of gonorrhoea is through finding the causative organism in Gram stained smear of the genital and extragenital material through microscopy, culturing or detection of nucleic material by molecular techniques (Holt et al., 1994). The sensitivity and specificity for Gram stained urethral smears is 90% to 95% respectively, while rectal smears have a low sensitivity but a high specificity when performed by experienced personnel (Grover et al., 2006). This sensitivity is even lower for asymptomatic infections.

Diagnosis by culturing N. gonorrhoea requires special media for its isolation and hence enriched Theyer-Martins Modified (TMM) agar base with lysed or chocolatised horse blood was used. Theyer-Martins Modified (TMM) agar is a special medium that is used for culturing and isolation of Neisseria gonorrhoea selectively. It contains inhibiting antibiotics such as vancomycin, colistin, nystatin, and trimethoprim lactate

(VCNT) that suppress most Gram-negative diplococci, Gram-negative bacilli, Gram- positive microorganisms and yeast hence selective isolation of Neisseria gonorrhoea is achieved, while dextrose enhances the growth of N. gonorrhoea (Ng and Martin

2005).

Neisseria gonorrhoea can be differentiated from other Neisseria species,

Moraxella species, Kingella species and other commensals based on its ability to grow on appropriate selective and nonselective media, produce acid from glucose, maltose, lactose, sucrose and fructose, reduce nitrate, produce polysaccharide from sucrose and exhibit DNase production (Ng and Martin 2005). The diagnostic yield of

culture may be reduced if lubricants with antibacterial agents are used during anoscopy, making water the recommended lubricant in this setting (Whitlow 2004).

While culturing of the microorganism is the surest way of isolating and identifying the causative microorganism, and allowing further antimicrobial susceptibility profiling, not many local settings can afford this intense but important procedures.

2.6.2 Chlamydia trachomatis

Molecular techniques such as the Gene Xpert C. trachomatis and N. gonorrhoea

(CT/NG) assay (Cepheid, Sunnyvale, California) is a newly developed real-time point of care polymerase chain reaction (PCR) based assay for the simultaneous detection of C. trachomatis and N. gonorrhoea DNA (Goldenberg et al. 2012). The Gene Xpert assay utilizes a special CT/NG cartridge, which permits all PCR processes including sample preparation, extraction, amplification and detection of C.trachomatis and or

N.gonorrhoea DNA all in a closed automatic system. The assay takes approximately one and a half hours giving qualitative results. For N. gonorrhoea to be indicated as a positive result, the assay matches two different built in molecular targets that must be positive and a sample adequacy control, which detects the presence of a single-copy human gene (Goldenberg et al. 2012). A study done in the UK in 2012 validated Gene

Xpert C.trachomatis and N.gonorrhoea assay for use with rectal samples comparing performance characteristics against those of the commonly used Aptima Combo 2 assay (GenProbe, San Diego, California). Results showed that the specificity of the

GeneXpert assay for N. gonorrhoea is similar to that found for other nucleic acid amplification tests (NAATs) in previously published studies (>99.3%), suggesting that cross-reactivity is not a significant problem for this target in this high-prevalence population (Goldenberg et al. 2012). Due to its very rapid turnaround time and ease of

use, the GeneXpert chlamydia and gonorrhoea test is well suited for near-patient testing which will help in improving patient care by providing early treatment to the infected.

2.7 Treatment of gonorrhoea and chlamydia

2.7.1 History

During the pre-antimicrobial era (before 1930), treatment of gonorrhoea involved receiving different systemic types of balsams, urethral irrigations, chemical compunds and hyperthermia and rest while refraining from alcohol and sexual activites. Living a healthier lifestyle with fresh air and well balanced diet was advocated (Oriel et al.,

1943). The antimicrobial era started just before 1930 and sulfonamides were the first antimicrobials used for treatment of gonorrhoea; these initially cured 80 to 90% of gonorrhoea cases (Van Slyke et al.,1941). Unfortunately, Clinical resistance to sulphonamides started showing up in many gonococcal strains by 1944 and by late

1940s, 90% of gonococcal isolates were resistant to sulfonamides in vitro

(Kampmeier 1983).

Penicillin was discovered by Alexander Fleming accidentally in the late 1920‘s. This compound produced by a fungus could lyse staphylococci and other bacteria causing many infectious diseases (Unemo and Shafer 2014). However, correct documentation of the ―wonder drug‖ efficacy for gonococcal urethritis never begun until 1943

(Mahoney et al., 1943). Thereafter, penicillin marked a new era in the treatment of gonorrhoea and many other infectious diseases (Mahoney et al., 1943).

In 1976, two types of β-lactamase-encoding plasmids emerged which originated from

South-east Asia and sub-Saharan Africa. These caused high-level resistance to penicillin and the speedy international spread of these strains was of great concern

(Unemo and Nicholas 2012). This led to penicillin being abandoned as a first-line antimicrobial. Currently, gonococcal strains with plasmid and/or chromosomally mediated resistance to penicillin are common globally (Unemo 2012). Quinolones which are synthetic antimicrobials were discovered in the 1960s. These included but not limited to fluoroquinolones, ciprofloxacin and ofloxacin which were previously recommended for gonorrhoea treatment, and ciprofloxacin in particular was widely used to treat gonorrhoea from the mid to late 1980s onwards. Initially, low doses, for example 250 mg, of ciprofloxacin were used, but clinical failures were already reported by 1990 (Gransden et al. 1990).

Ciprofloxacin-resistant gonococcal strains were subsequently rapidly exported internationally or emerged independently (Su and Lind 2001) Due to high levels of fluoroquinolone resistance, many Asian and European countries removed ciprofloxacin as a first-line treatment in the early to mid-2000s (Unemo and Shafer

2011). In 2007, the fluoroquinolones were abandoned from the CDC-recommended treatment regimens for gonorrhoea, with no exceptions (CDC 2006). Currently, the prevalence of fluoroquinolone-resistant gonococcal strains is high worldwide (Unemo

2012).

In Kenya, N. gonorrhoea is treated with cefexime 400mg start in combination with azithromycin 2g start (NASCOP, 2016). This dual antimicrobial treatment regimen appears to be highly effective currently and user friendly as both drugs are taken as

start doze orally and may be administered while observing the patient (NASCOP,

2016).

2.7.2 Presumptive treatment of gonorrhoea and chlamydia infections

In 2011, the World health organization (WHO) recommended periodic screening or presumptive treatment (PT) for N. gonorrhoea and C. trachomatis for MSM who report receptive anal intercourse (RAI) in past 6 months and have either multiple partners or a partner with an STI (WHO, 2011). However, the frequency of screening or presumptive treatment is not stated in the guidelines.

2.8 Antibiotic resistance profiles of N. gonorrhoea

Antibiotic resistance is the ability of bacteria to resist the effects of the drugs used to treat them and therefore they are not killed hence continue to reproduce.

Antimicrobials have been used to treat gonorrhoea successfully for the last 70 to 80 years, however high prevalence of multidrug resistant N. gonorrhoea strains to sulphonamides, penicillin, earlier cephalosporins, tetracyclines, macrolides and fluoroquinolones have been reported worldwide (Unemo and Shafer 2014).

Neisseria gonorrhoea has an exceptional capability to modify its genetic material. It is naturally competent for transformation (transfer of partial or whole genes) throughout its whole life cycle and can efficiently change its genome through all types of mutations (Unemo and Shafer 2014). These machineries are often used by the N. gonorrhoea to speedily adjust and endure in the often-hostile environments at different anatomical sites in the human body. In this way, the gonococcus has transformed and acquired almost all known physiological mechanisms of

antimicrobial resistance to all classes of antibiotics recommended and or used for treatment (Unemo and Shafer 2014). The transformation by the gonococcus include antibiotic destruction or modification by enzymatic means, target modification or protection that reduces affinity for the antibiotics, reduced influx of antibiotics and increased efflux of antibiotics (Unemo and Shafer 2014).

Most genetic antimicrobial resistance (AMR) determinants in N. gonorrhoea are situated chromosomally. The AMR determinants that cause high-level resistance to penicillin and tetracycline, respectively, are known to be plasmid mediated (Ashford,

Golash, and Hemming 1976). Antimicrobial resistance has appeared and expanded with every release of new classes of antibiotics for the treatment of gonorrhoea. Due to the extensive resistance, older and inexpensive antibiotics have lost their efficacy in treatment of infections (WHO, 2016). Therefore, there is need for ongoing surveillance for antibiotic resistance which is essential despite lacking public health capacity to ensure timely response to arising cephalosporin and multidrug-resistant N. gonorrhoea strains.

3.0 MATERIALS AND METHODS

3.1 Study Site

The study was conducted in Mtwapa at the Kenya Medical Research Institute

(KEMRI) HIV/STI clinic facility. The study area is a town located in Kilifi County.

The town is situated approximately 16 kilometres north-east of Mombasa on the Mombasa-Malindi road. The locals are the Mijikenda community. However, the town hosts people from other Kenyan communities and from all other parts of the world. Mtwapa has grown into a 24-hour economy due to both local and foreign tourism crammed with extravagance lifestyle with alcohol, sex and drugs making it a more suitable site for the study.

3.2 Study Design

A longitudinal cohort study design was adopted. Volunteers who were already enrolled in a larger cohort observational study and were eligible were followed up once after 6 months.

3.3 Ethical Consideration

Ethical approval was sought from the Kenya Medical Research Institute Scientific

Steering Committee (KEMRI SSC Protocol number 894) (Appendix III). Informed consent was obtained from every participant. The participants were informed of the objectives, risks and benefits of the study. The risk included minor pain during blood sample collection as it involved venepuncture with a needle while benefits included regular counselling and testing for HIV-1, screening and treatment of certain STDs and hepatitis B virus (HBV) vaccination. Those that were found to be HIV infected

were provided with antiretroviral therapy (Ng and Martin) and opportunistic infections managed clinically.

The participants were informed that they could withdraw from the study whenever they wanted to and that their withdrawal was not going to interfere with participation in other on-going benefits. After giving consent, three rectal swabs were collected.

The procedure was carried out in a private examination room while anonymity of records and confidentiality of all gathered information from participants were maintained.

3.4 Sampling Technique

This study adopted a purposeful sampling technique where all MSM who reported receptive anal intercourse in the last 6 months were recruited to participate in the study. These were the MSMs who were already in an existing cohort for the last 10 years in both HIV positive and negative epidemiological studies. Since the MSMs were few, all those who were available and willing to participate in the study were enrolled.

3.5 Study Population

All MSM aged between 18-49 years, who had consented by filling in a consent form

(Appendix I) in the ongoing cohort studies and who reported RAI in the past 6 months were invited to participate in this study.

3.5.1 Inclusion criteria

Men who have sex with men aged 18 to 49 years who reported receptive anal intercourse in the past 6 months before STI assessment and consented were included in this study.

3.5.2 Exclusion criteria

Men who have sex with men below 18 and above 49 years and who did not report receptive anal intercourse in the past 6 months before STI assessment were excluded from this study.

3.6 Enrolment of Participants

The participants risk to acquiring HIV and other STI infections were assessed by a counsellor using risk assessment questionnaire (Appendix IV). Participants who consented by filling the consent form (Appendix I) were enrolled to participate in this study. Screening was done at scheduled cohort visits at two-time points: month 0

(enrolment) and month 6. At each screening visit, 3 rectal swabs were collected through a proctoscopy procedure.

3.7 Sample Size Determination

The study recruited participants from larger ongoing cohorts which had 256 MSM participants. Based on preliminary screening of 43 volunteers, the prevalence of asymptomatic NG and CT was 23% (Sanders et al. 2010).

The minimum sample size was determined using the Raosoft Sample Size Calculator.

Raosoft, Inc. 2004, Seattle http://www.raosoft.com/samplesize.html

Formula;

n = N x/((N-1)E2 + x)

c 2 x = Z( /100) P(1-p)

(N - n)x E = Sqrt[ /n(N-1)]

Where, n = sample size

E = margin of error (5%)

N = the population size (256) All available MSM in the larger cohort

P = the estimated prevalence of rectal N. gonorrhoea and C. trachomatis (23%)

Z (c/100) = critical value for the 95% confidence level c. (1.96)

Calculation

c 2 x = Z( /100) P(1-p)

x = 1.962 x 0.23 (1-0.23) x = 0.6803

Therefore, n = N x/((N-1)E2 + x) n = 256 x 0.6803/ (256-1) 0.052 + 0.6803 n = 174.1568/ 1.3178

n = 132.16 ~ 133 participants

Although the study required a minimum of 133 participants, all cohort participants who met the inclusion criteria were included in this study (n= 153). The decision to include all who met the criteria was based on the fact that the study had two-time points; baseline and 6- months follow up. Therefore, this was to improve on the turn up numbers 6 months later as it was foreseen that some of the participants would fail to come back for their appointment.

3.8 Sample Collection

3.8.1 Rectal Swab

Rectal swabs were collected through a proctoscopy examination by a clinical officer in a private clinical room. A sterile swab was inserted 2-3cm in the rectum, avoiding faecal material, was rotated slowly for 10 seconds to allow absorption of secretion into the swab (Ng and Martin 2005). This process was repeated 3 times to collect three rectal swabs. The first swab was inoculated on Thayer Martin modified (TMM) agar, a second swab was immersed in the CT/NG collection kit buffer, capped and labelled and the third swab, a direct smear for Gram staining was collected by gently rolling onto the slide to preserve cellular morphology and over an area less than 1 cm2

(Ng and Martin 2005).

The three collected samples were processed using the Gram‘s stain for the direct smear, the Gene Xpert analyser (Cepheid) for the CT/NG swab and culture and sensitivity for the swab that was inoculated on the TMM agar. Positive results were communicated back to the clinic within 2 hours for the Gene Xpert test, 30 minutes

for the Gram stain and 3 days for the culture to aid with clinical management. The participants with positive results were traced for treatment.

3.8.2 Blood Collection

Blood sample collection was performed according to WHO guidelines (WHO, 2010).

Visible large median cubital vein was selected and the area of puncture was thoroughly cleaned using 70% alcohol. The vein was punctured using a vacuteiner needle and pressurised tube system that allowed automatic flow of blood into the tube.

A 10 ml tube with ethlenediaminetetraacetic acid (EDTA) anticoagulant was used to collect blood for HIV test and other routine tests for that scheduled visit in the ongoing larger cohort.

3.9 Laboratory Methods

3.9.1 Direct Smear for the detection of N. gonorrhoea

Smears were prepared and stained by Gram stain per Woods & Walker (1996). The

Gram‘s stained smears were examined microscopically under oil immersion (1000x magnification), the presence of polymorphornuclear leukocytes specifically neutrophils containing intracellular Gram-negative kidney-shaped diplococci were required for the presumptive diagnosis of gonorrhoea (LCDC, 1998). The polymorphornuclear neutrophils were counted in each field and upto three field that were examined, an average was calcuated and reported as a whole number.

3.9.2 Gene Xpert Chlamydia trachomatis / Neisseria gonorrhoea assay

The Xpert CT/NG assay, performed on the Gene Xpert Instrument Systems, was a qualitative in vitro real-time PCR test for the automated detection and differentiation of genomic DNA from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoea

(NG) to aid in the diagnosis of chlamydial and gonorrhoeal diseases (Tabrizi et al.

2013). The real-time PCR used 5 primer pairs for the targeted sequences which included CT1, NG2, NG4, Specimen Processing Control (SPC) and Sample

Adequacy Control (SAC). (Cepheid AB, Rontgenvagen 5 SE-171 54 Solna, Sweden).

The Gene Xpert worked by capturing the sample and SPC nucleic acid after cell lysis during sample preparation. The eluted DNA was mixed with the dried down reagent bead causing simultaneous amplification and detection of the CT and or NG infections.

3.9.2.1 Assay Quality Control

The Gene Xpert had an internal control system in each cartridge that utilised

Specimen Processing Control (SPC) and Sample Adequacy Control (SAC). The SPC contained the genomic DNA of Bacillus globigii. This control verified adequate sample processing, lysis, presence of the organism and detected inhibition. For the assay to pass, a positive result was obtained in a negative sample and was either positive or negative in a positive sample. While the SAC contained

Hydroxymethylbilane synthase that ensured human cells were added in the sample chamber of the cartridge. This was positive in a negative sample and either positive or negative in a positive sample.

The other internal quality control was the Probe Check Controls (PCC). This was performed automatically by the analyser. Before the PCR step, fluorescent signal was measured on all probes and compared with the pre-established factory settings. The check was to monitor bead rehydration, reaction tube filling, probe integrity and dye stability (Cepheid AB, Rontgenvagen 5 SE-171 54 Solna, Sweden).

3.9.2.2 Assay Procedure

The CT/NG swab in CT/NG buffer was processed per the manufacturer‘s instructions

(Cepheid AB, Rontgenvagen 5 SE-171 54 Solna, Sweden). Briefly, the CT/NG swab in the buffer was gently inverted 3 to 4 times to ensure adequate mixing of sample material present on the swab and the buffer. 1 ml of the well mixed buffer was transferred to the CT/NG cartridge using a sterile pasture pipette. The cartridge lot number was scanned; volunteer details were logged and the assay started on the analyser. Assay run time was one and half hours after which results of C. trachomatis and N. gonorrhoea RNA by the Gene Xpert machine were displayed as either detected or undetected for both single and co-infections.

3.9.3 Culture

Culture of N. gonorrhoea was done on Thayer Martin Modified (TMM) agar containing nutrients and antibiotics such as vancomycin, colistin, nystatin, and trimethoprim that facilitated its growth while inhibiting the growth of contaminating bacteria and fungi using CLSI guidelines (Patel 2016). The inoculated TMM agar plates were placed in an improvised candle extinction jar, the candle was lit and the jar capped, this cut out supply of atmospheric oxygen creating an environment

conducive for the growth of Neisseria gonorrhoea. The jar was then incubated at

37oC to await transportation to the KEMRI Microbiology laboratory in Kilifi.

Plate 1: Candle extinction jar with inoculated modified Thayer-Martin agar plates (Source: Connie, R. M., 2011).

Upon arrival in Kilifi the same day of collection, the plates were further incubated at

o 36-38 C in 3-7% CO 2 incubator overnight. The following day, plates were examined for any sign of growth. Those plates that had not grown or showed tiny growth were re incubated for further 24 hrs. Those colonies that showed typical colonial characteristics of N. gonorrhoea as smooth, round, moist, uniform grey/brown were taken through further tests for their identification (Tabrizi et al. 2013).

3.9.3.1 Gram stain

The test was performed per the procedure of Woods & Walker (1996). Smears made from suspected colonies were stained using the Gram staining procedure. This differential stain was used to provide valuable preliminary information concerning

identification of the suspected colony. The smears were examined under the oil immersion objective and the presence of gram negative diplococci suggested that the test organism was suspected to be N.gonorrhoea which warranted more tests for its identification.

3.9.3.2 Oxidase test

The oxidase test uses the tetramethyl derivative of the oxidase reagent (1% aqueous solution of N, N, N, N-tetramethyl-1, 4-phenylenediamine) (Ng and Martin 2005) that was prepared in-house. The test was performed by putting a drop of reagent on a filter paper, culture colony was then applied to the filter paper using a wooden applicator stick. A dark-purple colour change within ten seconds indicated a positive sample.

Quality control to check the performance of the oxidase reagent was done using

Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 as a positive and negative control strains respectively.

3.9.3.3 Catalase Test

This was performed using 3% hydrogen peroxide where a drop of the reagent was placed in the centre of a clean glass slide and the suspected colony was picked with a loop and emulsified in the reagent (Daniel & Petrishko, 1973). A positive reaction was indicated by production of bubbles within one to two seconds. N. gonorrhoea gave a positive reaction. Quality control to check the performance of the catalase reagent was done using Staphylococcus aureus ATCC 25923 as positive control strain and Streptococcus pyogenes ATCC 19615 as a negative control strain.

3.9.3.4 Analytical Profile Index for Neisseria and Haemophilus

Neisseria gonorrhoea was differentiated from other Neisseria species, Moraxella species, Kingella species and other commensals by use of the API NH (Analytical

Profile Index Neisseria and Haemophylus) strips (BioMérieux). These consisted of 10 microtubes containing dehydrated substrates, which enabled the performance of 12 identification tests that were enzymatic reactions and sugar fermentations (Barbe et al.

1994).

The reactions produced after a 2-hour incubation at a temperature of 35-37oC resulted in spontaneous colour changes while others were revealed by the addition of reagents.

The reading of the reactions was performed visually by scoring all the reactions and apiwebTM identification software was used to identify the obtained numerical profile.

Quality control to check the performance of the API NH strips was done using

Neisseria gonorrhoea ATCC 19424 and Staphylococcus aureus ATCC 25923 as a positive and negative control strains respectively.

3.9.3.5 Antibiotic susceptibility Test

Antibiotic susceptibility test was performed on all Neisseria gonorrhoea isolates per

Clinical and Laboratory Standards Institute (CLSI) guidelines using the disk diffusion method and the minimum inhibitory concentration (MIC) by E-test method

(Patel 2016). Neisseria gonorrhoea ATCC 49226 was used as a quality control strain. Gonococcal (GC) agar base and 1% defined growth supplement were utilised for both the disk diffusion and E-test assays on 100mm or 150mm petri dishes respectively. Direct colony suspension, equivalent to a 0.5 McFarland standard prepared in Mueller-Hinton broth using colonies from an overnight growth on TMM

agar was inoculated on the GC agar and incubated at 36°C ± 1°C with 5% CO2 for

20–24 hours. The antibiotics which included tetracycline 30µg, ceftriaxone 30µg, penicillin 10µg (Oxoid, UK) were seeded on the 100mm plate for the disk diffusion.

E-test strips (AB Biodisk, Solna, Sweden), a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic. The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein were seeded on the GC agar in 150mm petri dishes. The tested drugs included cefixime 5µg, penicillin 10units and ciprofloxacin 5µg. Zones of inhibition were measured and compared to the CLSI guidelines breakpoints where a drug was determined to be sensitive, intermediate or resistant.

3.9.4 HIV Test

Human immunodeficiency virus (HIV) test was performed as per the manufacturer‘s instruction using two different rapid test kits in parallel; Determine HIV 1/2 (Alere,

Japan) and Unigold HIV 1/2 (Trinity Biotech, UK) in parallel. Briefly, blood sample was added to the sample pad and a drop of chase buffer added to enhance blood flow by capillary action through the sample pad. As the sample moved through, it reconstituted and mixed with the embedded antigen conjugate. The mixture continued to migrate through the solid phase to the immobilised recombinant antigens and synthetic peptides at the patient window site. Presence of antibodies to HIV-1 and or

HIV-2 in patient sample triggered binding to the antigen at the patient window forming a red line. The mixture continued to migrate to the control window where a red line formed to indicate that the kit was working well. Where the patient sample

did not have the antibodies to HIV 1 and or HIV 2, no binding of antigen took place in the patient window hence there was no formation of a red line.

Human immunodeficiency virus (HIV) test was interpreted as positive where there was presence of a red line in both the patient window and the control window for both

Determine and Unigold HIV test. An individual was given a positive result where both the two rapid tests gave a concordant positive result and likewise for a negative result. Quality control of the HIV test was performed utilising known HIV positive and negative samples for both positive and negative controls respectively.

3.10 Data Analysis

Data was presented in tables and figures. Prevalence of CT and NG was calculated

0 using the formula of Le and Boen., 1995 ( x 100% ), Where: O = The number of P individuals with the disease, P = The population at risk of having the infection during the study period. The 95% confidence intervals (CI) for prevalence were calculated using test for proportion.

Associations of prevalent CT or NG with baseline socio-demographics and behavioural characteristics for MSM reporting RAI was assessed using Pearson‘s χ2 test and Fisher‘s exact test for variables that were not normally distributed. Odds ratios were used to estimate the associations between participant characteristics and

CT or NG infections using logistic regression. Factors associated with CT or NG at P

< 0.2 in the univariate logistic regression analysis were included in a logistic multivariable model.

The 6-month CT or NG incident rates were calculated as the number of cases divided by person years of follow-up and expressed as incidence per 100 person-years.

Associations of baseline and CT and or NG with symptoms and asymptomatic infections, polymorphornuclear neutrophils (PMN) count in Gram stained smears and antibiotic susceptibility tests were assessed using Fisher‘s exact test with P values of

2-sided and significance was set at P ≤ 0.05. Stata 13.0 (StataCorp LP, College

Station, TX) was used for analysis. Performance of the gram stain was assessed by calculating the sensitivity and specificity using Gene Xpert as gold standard, combining baseline and follow-up samples and mcnemmar test for paired proportions to compare the proportion of positives according to Gene Xpert and the number of positives according to Gram stain.

4.0 RESULTS

4.1 Demographic Characteristics of the Study Population

This was a two-time point study conducted from April to July 2016 at the KEMRI

HIV/STI Research clinic in Mtwapa. During the study period, 256 MSM participants had been enrolled in a larger ongoing observational study. One hundred and fifty- three MSM (60%) reported receptive anal intercourse in the past 6 months hence met the inclusion criteria and were therefore invited to participate in this study. Although the minimum required participants in this study was 133, all the 153 were targeted as this was a unique cohort and having more participants would take care of the expected attrition during 6-months follow up. Those that met the inclusion criteria and came for the baseline sample collection were 68% (104/153), while 25% (39/153) declined the proctoscopy exam and 7% (10/153) were lost to follow up in the larger ongoing studies.

Out of 104 MSM assessed at baseline, 63% were within 18-24 years‘ age group p value (χ2) 0.622 [aOR] 95% CI 0.5 (0.0–6.5), 32% were within 25-35 years‘ age group p value (χ2) 0.803 [aOR] 95% CI 0.7 (0.6-9.2), while only 3.8% were above 35 years‘ age group. Majority 87% were never married p value (χ2) 0.978 [OR] 95% CI

1.0 (0.3-4.0) and 49% had secondary education while those participants who attended tertiary education were only 9.6% p value (χ2) 0.603 [OR] 95% CI 1.3 (0.5-3.4). The participants who had a Christian religious background were 51%, Muslims were 26% p value (χ2) 0.819 [OR] 95% CI 0.9 (0.3-2.6) while 23% belonged to other, p value

(χ2) 0.237 [OR] 95% CI 0.4 (0.1-1.7). Most participants 58% were self-employed p value (χ2) 0.275 [OR] 95% CI 2.1 (0.5-8.2), 17.3% had a formal employment p value

(χ2) 0.09 [OR] 95% CI 3.8 (0.8-18.1) and 25% were unemployed (Table 4.1.a).

Table 4.1.a: Baseline Socio-demographic characteristics and their association with occurrence of rectal Neisseria gonorrhoea and Chlamydia trachomatis in MSM.

Socio-demographic All CT NG CT/NG Adjusted characteristics (n = 104) (n = 14) (n = 10) (n = 22) Unadjusted analysis P analysis OR P Value AOR Value n (%) n (%) n (%) n (%) (95% CI) (95% CI) Age group (years)‡ 18-24 66 (63.5) 9 (13.6) 6 (9.1) 13 (19.7) 0.6 (0.0 – 0.7 (0.1-7.7) 0.798 6.5) 0.622 25-34 34 (32.7) 5 (14.7) 3 (8.8) 8 (23.5) 0.4 (0.6 – 0.9 (0.1-10.2) 0.948 9.2) 0.803 35+ 4 (3.8) 0 (0.0) 1 (25.0) 1 (25.0) Reference Reference Education Primary/none 43 (41.3) 6 (14.0) 4 (9.3) 9 (20.9) Reference Secondary 51 (49.0) 8 (15.7) 6 (11.8) 13 (25.5) 1.3 (0.5-3.4) 0.603 Higher/tertiary 10 (9.6) 0 (0.0) 0 (0.0) 0 (0.0) - Marital status Never married 90 (86.5) 12 (13.3) 9 (10.0) 19 (21.1) Reference Ever married 14 (13.5) 2 (14.3) 1 (7.1) 3 (21.4) 1.0 (0.3-4.0) 0.978 Religion Christian 53 (51.0) 7 (13.2) 7 (13.2) 13 (24.5) Reference Muslim 27 (26.0) 5 (18.5) 2 (7.4) 6 (22.2) 0.9 (0.3-2.6) 0.819 Other/None 24 (23.1) 2 (8.3) 1 (4.2) 3 (12.5) 0.4 (0.1-1.7) 0.237 Employment None 26 (25.0) 2 (7.7) 1 (3.8) 3 (11.5) Reference Self 60 (57.7) 10 (16.7) 5 (8.3) 13 (21.7) 2.1 (0.5-8.2) 0.275 Formal 18 (17.3) 2 (11.1) 4 (22.2) 6 (33.3) 3.8 (0.8-18.1) 0.090 CT: Chlamydia trachomatis; NG: Neisseria gonorrhoea, ‡ Included in the multivariable analysis adjusted for prevalent CTNG, age and transactional sex a priori.

Out of the 39 (25%) who declined testing at baseline, all their social demographic characteristics were not different from the 104 participants who were tested. These would not have changed the prevalence of chlamydia and gonorrhoea among MSM at baseline.

4.1.1 Sexual Transmitted Infection risk characteristics of participants

About one out of four (26.9%) participants had reported having sex with men exclusively; however, majority 73% had reported having sex with both men and women p value (χ2) 0.131; [aOR] 95% CI 2.1 (0.9-6.5). Half the study participants reported receiving money or goods for sex p value (χ2) 0.008; [aOR] 95% CI 4.4 (1.5-

13.3) while 93 % did not pay for any sex in the past 3 months p value (χ2) 0.648; [OR]

95% CI 0.6 (0.1-5.3). Participants who reported use of alcohol in the past one month were 40%, p value (χ2) 0.586; [OR] 95% CI 1.3 (0.5-3.4). While the entry criteria for this study was having reported receptive anal intercourse in the past 6 months among

MSM, 61.5% of the participants also reported insertive anal intercourse in the past three months p value (χ2) 0.82; [OR] 95% CI 1.1 (0.4-3.0) In the multivariable analysis, CT or NG infections were independently associated with being paid for sex

(adjusted odds ratio [aOR], 4.4; 95% CI, 1.5–13.2) adjusted apriori for age at baseline

(Table 4.1.b).

Table 4.1.b: Baseline STI risk characteristics of MSM and occurrence of rectal Neisseria gonorrhoea and Chlamydia trachomatis in MSM

All CT NG CT/NG Unadjusted Adjusted P Value P Value Risk characteristics (n = 104) (n = 14) (n = 10) (n = 22) analysis OR analysis AOR n (%) n (%) n (%) n (%) (95% CI) (95% CI) Sex with‡ MSME 28 26.9 7 25 3 10.7 9 32.1 2.3 (0.9-6.2) 0.101 2.1 (0.9-6.5) 0.131 MSMW 76 73.1 7 9.2 7 9.2 13 17.1 Reference Reference Received payment for sex in past 3 months‡* No 52 50 2 3.8 3 5.8 5 9.6 Reference Reference Yes 52 50 12 23.1 7 13.5 17 32.7 4.6 (1.5-13.6) 0.006 4.4 (1.5-13.3) 0.008

Paid for sex in past 3 months No 97 93.3 14 14.4 9 9.3 21 21.6 Reference Yes 7 6.7 0 0 1 14.3 1 14.3 0.6 (0.1-5.3) 0.648

Alcohol use in past month No 62 59.6 4 6.7 8 13.3 12 20 Reference Yes 42 40.4 9 20.5 2 4.5 10 22.7 1.3 (0.5-3.4) 0.586 IAI in past 3 months No 40 38.5 4 8.2 5 10.2 8 16.3 Reference Yes 64 61.5 10 18.2 5 9.1 14 25.5 1.1 (0.4-3.0) 0.82 IAI: Insertive Anal Intercourse, MSME: Men exclusive, MSMW: Both Men and Women, CT: Chlamydia trachomatis; NG: Neisseria gonorrhoea. * P-value≤ 0.05, ‡ Included in the multivariable analysis adjusted for prevalent CTNG, age and transactional sex a priori.

4.1.2 Risk for sexual exposure

One in four (25%) participants reported unprotected sex in the past one week, 42% reported all protected sex and 32% reported no sexual activity in the past one week p value (χ2) 0.951; [OR] 95% CI 1.0 (0.3-3.3). While in the past three months, no condom use was reported by 72% of the MSM participants p value (χ2) 0.363; [OR]

95% CI 0.6 (0.2-1.9). Group sex was rarely reported (1.9%) while 65% reported multiple sexual partners in the past one month p value (χ2) 0.683; [OR] 95% CI 1.3

(0.3-5.3). Those participants who were HIV infected were 34.6% p value (χ2) 0.772;

[OR] 95% CI 0.8 (0.2-3.2) and only one (1%) had not been circumcised. (Table

4.1.c).

Table 4.1.c: Baseline sexual exposure risk characteristics and occurrence of rectal Neisseria gonorrhoea and Chlamydia trachomatis in MSM Adjusted All CT NG CT/NG Unadjusted P Value analysis P Value (n = 104) (n = 14) (n = 10) (n = 22) analysis OR Exposure characteristics AOR n (%) n (%) n (%) n (%) (95% CI) (95% CI) Sexual exposure and condom use in past week‡ No activity 34 32.7 5 11 1 2.3 6 13.6 Reference All protected 44 42.3 3 7.9 5 13.2 8 21.1 1.0 (0.3-3.3) 0.951 Any unprotected 26 25 6 27 4 18.2 8 36.4 2.1 (0.6-7.0) 0.238

Condom use for anal in past 3 months‡+ No 75 72.1 11 15 8 10.5 18 23.7 Reference Yes 26 25 3 11 2 7.1 4 14.3 0.6 (0.2-1.9) 0.363 Total sex partners in past month None 15 14.4 3 6.8 0 0 3 6.8 Reference One 21 20.2 0 0 2 7.1 2 7.1 0.4 (0.1-2.9) 0.38 Two or more 68 65.4 11 34 8 25 17 53.1 1.3 (0.3-5.3) 0.683 Group sex in past 3 months No 102 98.1 14 14 10 9.7 22 21.4 - Yes 2 1.9 0 0 0 0 0 0 - HIV Status Negative 68 65.4 11 16 6 8.8 15 22.1 Reference Positive 36 34.6 3 8.3 4 11.1 7 19.4 0.8 (0.2-3.2) 0.772 Circumcision status No 1 1 0 0 1 100 1 100 - Yes 103 99 14 14 9 8.7 21 20.4 - CT: Chlamydia trachomatis; NG: Neisseria gonorrhoea, * P-value≤ 0.05, ‡ Included in the multivariable analysis adjusted for prevalent CTNG, age and transactional sex a priori, + 3 participants did not report anal sex in the last 3 months but reported within the past 6 months

4.2 Occurrence of C. trachomatis and or N. gonorrhoea at Baseline

One hundred and four (104) MSM who reported receptive anal intercourse were examined for rectal chlamydia and gonorrhoea infections. The number of infections in relation to the population at risk involved at baseline was calculated to give the percentage prevalence of rectal chlamydia and gonorrhoea among MSM who reported

RAI. Twenty-two out of 104 (21.15%) MSM either had chlamydia or gonorrhoea, 8

(7.7%) had rectal gonorrhoea only, 12 (11.53%) had chlamydia only while 2 (1.92%) had co-infections with both chlamydia and gonorrhoea (Table 4.2).

Table 4.2: Baseline prevalence of rectal C. trachomatis and or N. gonorrhoea among MSM at the Kenyan Coast.

Infections Positive at baseline n = 22 95% CI

NG 8 (7.7%) 3.9 – 15.3

CT 12(11.53%) 6.9 – 20.0

CT and NG 2 (1.92%) 0.7- 4.6

Total 22 (21.15%) 13.3 – 29.0

CT: Chlamydia trachomatis, NG: Neisseria gonorrhoea, n: number of positive cases, CI: confidence Interval

4.2.1 Symptomatic and Asymptomatic rectal chlamydia and gonorrhoea

Thirteen (12.5%) of those who tested positive for CT only and nine (8.7%) who tested positive for NG only were asymptomatic at baseline. However, all CT and NG infections were asymptomatic at follow up. Overall, asymptomatic infections were

20.2% at baseline and 26.8% at 6 months follow up p= 0.196, Fisher's exact test

(Figure 4.1).

Figure 4.1: Proportions of rectal CT and or NG in symptomatic and asymptomatic infections at baseline and follow up.

CT (Chlamydia trachomatis), NG (Neisseria gonorrhoea)

4.3 Recurrence and reinfection of rectal gonorrhoea and chlamydia

The second time point of sample collection was done after 6 months. Out of 104 participants examined at baseline, about 79% (82) returned for the second appointment; these contributed 39.1 person-years (PY) with a median follow-up time of 5.7 [interquartile range (IQR): 5.4-6.2].

Twenty-one (26.3%) had Chlamydia trachomatis or Neisseria gonorrhoea infection,

12 (15%) had Chlamydia trachomatis, 12 (15%) had Neisseria gonorrhoea infections, and 3 (4%) were co-infected with both Chlamydia trachomatis and Neisseria gonorrhoea. The rate of Chlamydia trachomatis or NG infection was 53.7 (95% CI,

35.0–82.4) per 100 person-years (PY). Out of 21 with Chlamydia trachomatis or

Neisseria gonorrhoea at follow-up, six (7.3%) had prevalent Chlamydia trachomatis

or Neisseria gonorrhea and 15 (18.3%) had no Chlamydia trachomatis or Neisseria gonorrhoea at baseline, P = 0.2 (Table 4.3).

Of the 104 participants included in the baseline analysis, 22 (21.2%) participants did not return for the 6-month follow up visit. A comparison of the baseline prevalence estimate of rectal chlamydia and gonorrhoea was done. Results suggested that participants who did not come back for the 6-month follow up visit had a higher prevalence of baseline chlamydia and gonorrhoea (n=7, [30.4%]) compared to those who came back for the 6-months follow up visit (n=15, [ 18.5%]), though the difference did not achieve statistical significance (p=0.217).

Table 4.3: Prevalence of rectal C. trachomatis and or N. gonorrhoea among MSM at the Kenyan Coast at 6-months follow up, recurrence and or reinfection.

Infections Positive 6 months n 95% CI Positive at baseline = 21 and 6 months follow up n =21 NG 9 (11%) 7.2 – 22.8 0 ()

CT 9(11%) 7.2 – 22.8 4(4.9%)

CT and NG 3 (4%) 0.4- 7.9 1(1.2%)

Total 21(26%) 13.3 – 35.9 5 (6.1%)

CT: Chlamydia trachomatis, NG: Neisseria gonorrhoea, n: number of positive cases, CI: confidence Interval

4.4 Comparison of Gram stain and Gene Xpert in the detection of rectal gonorrhoea and chlamydia A total of 104 Gram stained smears were examined for rectal polymorphonuclear neutrophils (PMN) cells populations at baseline. Of the examined smears, 87% had no

PMN cells, 11% had <10 PMN cells while only 2% had >10PMN cells. Only the 2% which had >10 PMN cells were considered as positive for infection by Gram stain.

The Gene Xpert detected 8 (7.7%) NG, 12 (11.53%) CT and 2 (1.92%) CT and NG co-infections. Out of the total 14(13.46%) CT and 10(9.7%) NG detected at baseline, only 1.9% P-Value (χ2) 0.024 and 1% P value (χ2) = 0.0039; [95% CI 0.02- 0.15] had

>10 PMN cells for CT and NG respectively (Figure 4.2a).

Figure 4.2a: Proportion of Rectal Polymorphonuclear neutrophil cells populations (PMN) Cells with CT and or NG at Baseline

CT: Chlamydia trachomatis, NG: Neisseria gonorrhea

At follow up, total of 82 Gram stained smears were examined for rectal polymorphonuclear neutrophils (PMN) cells populations. Of the examined smears,

68% had no PMN cells, 24% had <10 PMN cells while only 7% had >10PMN cells.

Only the 7% which had >10 PMN cells were considered as positive for infection by

Gram stain. The Gene Xpert detected 9(11%) Neisseria gonorrhea, 9(11%)

Chlamydia trachomatis and 3(4%) Chlamydia trachomatis and Neisseria gonorrhea co-infections. Out of the total 12(14.6%) Chlamydia trachomatis and 12(14.6%)

Neisseria gonorrhea detected by the Gene Xpert at follow up, only 2.4% and 3.7% had >10 PMN cells for Chlamydia trachomatis and Neisseria gonorrhea respectively

P-Value(χ2) 0.0016 95% CI (0.04, 0.20) (Figure 4.2b).

Figure 4.2b: Proportion of Rectal Polymorphonuclear neutrophil cells populations (PMN) Cells with CT and or NG at Follow up

CT: Chlamydia trachomatis, NG: Neisseria gonorrhea

Sensitivity and specificity of Gram stain when Gene Xpert was used as the gold standard were 10% and <1%, P value (χ2) 0.0039 [95% CI (0.016-0.306)] for baseline

(Table 4.4a). At follow up, sensitivity and specificity of Gram stain when Gene Xpert was used as a gold standard were 25% and <1%, P value (χ2) 0.0039; [95% CI (0.03-

0.19)] (Table4.4b).

Table 4.4a: Comparison of Gram stain and Gene Xpert in detection of NG at Baseline.

Tests Gene Xpert

Positive Negative Total

Gram Stain Positive 1 0 1

NG (a) (c)

Negative 9 94 103

(b) (d)

Total 10 94 104

P value (χ2) 0.0039; 95% CI (0.02-0.15)

Sensitivity = a / (a+b) * 100

= 1/ (1+9) *100

= 10%

Table 4.4b: Comparison of Gram stain and Gene Xpert in detection of NG at Follow up.

Tests Gene Xpert

Positive Negative Total

Gram Stain NG Positive 3 0 3 (a) (c)

Negative 9 70 79 (b) (d)

Total 12 70 82

P value (χ2) 0.0039; 95% CI (0.03-0.19)

Sensitivity = a / (a+b) * 100

= 3 / (3+9) *100

= 25 %

4.5 Antibiotic sensitivity of N. gonorrhoea

Out of all 186 cultures done at both baseline and 6 months follow up, six (3.2%) cultures were positive for N. gonorrhoea and had been detected by the Gene Xpert.

These N. gonorrhoea isolates were tested for antibiotic sensitivity using disc diffusion against penicillin, tetracycline and ceftriaxone. Penicillin showed 33% resistance,

67% were intermediate and none was sensitive. Tetracycline showed 100 % resistance while ceftriaxone showed 100% sensitivity (P value <0.001) Fisher‘s exact test

(figure 4.3).

Figure 4.3: Sensitivity by disc diffusion test (P value <0.001) Fisher‘s exact test

Minimum inhibition concentration (MIC) was also performed on all the six isolates against penicillin, ciprofloxacin and cefixime to complement and confirm sensitivity of the antimicrobial test by the disc diffusion method. The same results as those of disc diffusion were obtained for penicillin. Ciprofloxacin showed 100% resistance while cefixime showed 100% sensitivity.

Figure 4.4: Sensitivity by minimum inhibition concentration (P value <0.001)

5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

5.1 Discussion

5.1.1 Demographic characteristics of participants at baseline

This study focused on men who had sex with men (MSM) who reported receptive anal intercourse. The data obtained showed that the prevalence of rectal gonorrhoea and or chlamydia infections among the different age groups were 19.7%, 23.5% and

25.0% for 18-24 years, 25-34 years and >35 years respectively. Although there were differences in prevalence in the different age groups and >35 years group showed the highest prevalence; however, the differences seen were not significantly different P value = 0.622; 95% CI (0.0 – 6.5) and 0.803; 95% CI (0.6-9.2). This showed that all age groups of MSM reporting receptive anal intercourse continued to engage in risky behaviour. This means that all age groups were at risk of getting rectal chlamydia and gonorrhoea infections. This study agrees with other studies done on MSM showing no independent association between age and STI acquisition (Plankey et al., 2009). The findings of this study suggest that MSMs continue to engage in risky sexual behaviour and therefore, increased vigilance with CT and NG screening is paramount to detect and treat infections that remain asymptomatic yet act as reservoirs for transmission.

Majority 75% of the MSM in this study engaged in some economic activities through self-employment (57.7%) or formal employment (17.3%) while 25 % were not employed. The MSM who were formally employed carried the bigger burden (33.3%) of the rectal CT and or NG infections followed by the self-employed (21.7%) while the non-employed had the least (11.5%) of the infections. This could be because they had the economic power to engage in risky behaviour such as buying alcohol and sex

from other unreached MSM networks outside the cohort. Although the study revealed high burden of rectal CT and or NG among the employed compared to the non- employed, there was no significant difference between these groups (P value =

0.090). Therefore, this study showed that being employed was not associated with CT and or NG acquisition, hence the risk of acquiring rectal CT/NG does not depend on being employed or not.

The study showed that there were more chlamydia infections among MSM who reported having sex exclusively with men (MSME) (24.1%) than their counterparts who reported having sex with both men and women (MSMW) (9.3%, P=0.101).

However, results in this study showed that this was not significant and that both

MSME and MSMW were at risk of acquiring rectal CT and NG. Research has shown that having unprotected receptive penile anal sex exclusively with men increases the risk of infection acquisition (Rebe et al. 2015). In addition, behavioral and sexual network factors such as having multiple sexual partners, having unprotected sex with a partner of unknown HIV status and larger social sexual networks drives disease transmission among MSM. A study done in S. Africa on 200 MSM also showed similar findings (Rebe et al. 2015).

Receiving payment for sex was strongly associated with rectal chlamydia and gonorrhoea infections [OR 4.4 (1.5-13.2), p=0.014]. Other similar research has shown that MSM who receive payment for sex are more likely to participate in unprotected anal sex with those paying for sex thus increasing the risk of acquiring these infections (Beyrer et al. 2010). Being paid for sex reduces power to negotiate for condom use hence increasing vulnerability to STIs acquisition. Other reasons to

engage in transactional sex may include the increased burden of psychosocial morbidities, stigma and discrimination (Prado Cortez, Boer, and Baltieri 2011).

Previous studies have also shown that reasons for MSM engaging in transactional sex are varied and include economic pressures as well as pleasure and excitement, especially in regions where MSM‘s behaviour is highly stigmatized (Oldenburg et al.

2015). This agrees with results of a study done in S. Africa by Rebe et al., ((Rebe et al. 2015) which reported a high burden of STI among MSM who engaged in transactional sex.

In addition, transactional sex could also be as a result of addiction to substance use and alcohol intake. This may incline these men to engage in transactional sex in order to alleviate the economic pressure being faced due to lack of employment hence increased sexual risk for STI acquisition (Burnette et al. 2008). This suggests that

MSM who engage in transactional sex may require targeted STI prevention interventions incorporated with mental health programs for wholesome health care provision. Moreover, agendas to empower MSM in reducing social susceptibility and other barriers including demanding for safe sex are paramount to minimize acquisition of sexually transmitted infections (Bamgboye, Badru, and Bamgboye

2017).

Out of the 42% who reported use of alcohol in the past one month, 25% had chlamydia and or gonorrhoea. Results in this study did not show any significant difference between those who reported use of alcohol and those who did not

(p=0.648). This could have been due to recall bias since this was self-reported as the question asked if there was alcohol use in in the past one month. Although this was

the case in this study, other studies have found that the use of alcohol and/or party drugs by some MSM may play a role in enabling risky behaviours, rendering users vulnerable to STI by not being able to negotiate for safe sex (Briggs 2016). In India,

MSM experience multiple and complex challenges, including stigmatization and discrimination, which may put them at risk for alcohol use and unprotected sex

(Yadav et al. 2014). MSM who drink alcohol are more likely to engage in risky behaviour including having sex with partners of known HIV status without protection.

Therefore, policies on interventions to reduce incidence of STIs should include focusing on drug and alcohol use in MSM.

This study showed that uncircumcised participants in this cohort were at greater risk of acquiring gonorrhoea infection compared to their circumcised counterparts. Other studies have shown that circumcision was effective in decreasing HIV acquisition among adult heterosexual males in several sub-Saharan African countries, recent clinical trials in South Africa, Kenya and Uganda showed that adult circumcision reduced HIV acquisition by 50–60% (Begley, 2008) Results from other studies have shown that the tissues beneath the foreskin is rich with Langerhans‘ cells and CD4+ T lymphocytes which are target cells for HIV virus (Patterson et al, 2002). The removal of the foreskin therefore removes cells targeted by the HIV virus. Studies have suggested that sustained tears of the delicate foreskin during sexual acts may be used as the entry points of STI causing organisms such as HIV virus, Chlamydia trachomatis and Neisseria gonorrhoea (Walton et al., 2012).

Men who had sex with men (MSM) who were HIV infected in this study were 35% and the other 65 % were HIV uninfected. Rectal CT and or NG infections were more among the HIV uninfected MSM (22%) compared to those MSM who were HIV infected (19%). The low frequency of CT and NG observed in the HIV infected

MSM could probably have been because they heeded to the counsellor‘s advice in using protection to prevent STI acquisition which could worsen their health outcome.

Although this was the case, there was no significant difference between those who were HIV infected and those who were HIV uninfected (P value = 0.772). The results in this study concur with results in a study done by Rebe K. et al., 2015 that showed no significant difference in the CT and or NG infection among those MSM who were

HIV infected and those who were uninfected (P value = 0.91).

5.1.2 Occurrence of asymptomatic rectal gonorrhoea and chlamydia among MSM at the Kenyan Coast This study reported a high burden of rectal Neisseria gonorrhoea and or Chlamydia trachomatis co-infection where 21.2% (95% CI 13.3 - 29.0%) MSM participants tested positive at baseline and 26.3% (95% CI16.6 -35.9%) tested positive at six months follow up. Similar findings were reported in other parts of sub Saharan Africa where in 2015 in South Africa, Rebe et al reported 24% prevalence for NG/CT at any anatomical site among a cohort of 200 participants while another study in Coastal

Kenya by Sanders (2013) reported 26% (95% CI 14–41%) prevalence for NG and or

CT among a cohort of 43 participants. For unknown reasons, prevalence did not reduce at follow up as was expected. Given that these were cohort participants who received prevention interventions such as condoms, lubricants, counselling on risk reduction, STI screening and treatment, a decline in the prevalence at follow up was

expected but was not achieved. The participant‘s risks remained high probably because they interacted with networks outside the cohort who were not reached by the prevention interventions. This study has therefore shown that there is need to scale up prevention interventions to reach MSM networks outside the cohort population.

Majority of the rectal NG and or CT infections in this study were asymptomatic (P- value 0.001; 95% CI 0.04, 0.17). These results suggest that the asymptomatic case were significantly associated with rectal CT and or NG infections thus indicating that rectal infection with CT and or NG may not always present with symptoms. Rebe et al., 2015 in Cape Town in a study among 200 MSM showed that 19% of the infections were asymptomatic. Therefore, in resource limited settings where laboratory diagnosis is lacking and STIs are managed syndromically, these infections would remain untreated in high risk MSM reporting receptive anal intercourse.

Moreover, the inability to diagnose asymptomatic STIs in MSM leads to under- treatment, inadequate STI control and possibly propagate transmission of HIV in this high HIV-risk population. Transmission of HIV is presumed to be facilitated by these infections through disruption or inflammation of the genital epithelium as reported by

Rehle et al., (2004).

The study reported risky sexual behaviours among MSM at the Kenyan Coast.

Enrolled participants had two or more partners in the past one month, there was low condom use, transactional sex was common, use of alcohol and a third of the participants were HIV infected. Almost a quarter had been diagnosed and treated for an STI in the past 12 months. These data demonstrate the need for effective HIV and quality STI prevention interventions that target MSM.

5.1.3 Recurrence and reinfection of Neisseria gonorrhoea and Chlamydia trachomatis Enrolled participants in this study contributed to 39.1 person-years with a median follow up time of 5.7 months (IQR 5.4-6.2). The current study revealed that about a quarter of the participants were re-infected with either CT and or NG during this study period with an infection rate of 53.7 (95%CI, 35.0-82.4) per 100 person years. This rate is much higher than that reported by Sanders (2013) in Kenya which was 39.7

(95% CI 24.3 to 64.8) per 100 person-years.

Infected participants were given directly observed treatment with cefixime 400mg and

2g azithromycin as a single doze. Extra treatment was given to them to take to their partners, however the study could not determine whether the partners were given the treatment or not. This uncertainty could have contributed to the reinfection of the participants or participants may have acquired new partners.

5.1.4 Comparison of Gram stain and Gen Xpert in the detection of rectal gonorrhoea and chlamydia trachomatis infections In many cases, the pattern of inflammation provides important clues to diagnosis and helps to guide in the diagnosis of infectious diseases. This study observed that majority of the rectal smears had <10 polymorphonuclear neutrophils (PMN) cell even with CT and or NG infections detected by the Gene Xpert (P-Value 0.082). This means that PMN cells were not associated with rectal CT and NG infections. This could have been attributed to the fact that majority of the detected infections were asymptomatic as well. Therefore, this study has revealed that low numbers of PMN cells as seen in these asymptomatic infections does not always indicate absence of infection. In low resource setting where highly sensitive molecular diagnosis is

lacking together with syndromic management of infections, all these infections would have been missed hence remain undetectable and untreated. This therefore suggests that regular screening for MSM who practice receptive anal intercourse is paramount.

5.1.5 Performance of Gram stain against Gene Xpert RNA testing for detection of N. gonorrhoea Performance of Gram stain against Gene Xpert was assessed for the detection of rectal gonorrhoea infection at both baseline and 6 months follow up. This study revealed that Gram stain missed 9 out of 10 (90%) of all rectal gonorrhoea infections that were detected by Gene Xpert at baseline, P value (χ2) = 0.0039 (95% CI 0.02,

0.15). Similar results were obtained at 6 months follow up, with very low sensitivity and specificity of 9% (95% CI 0.016, 0.306) and 1% (95% CI 0.97, 1) respectively.

Therefore, Gram stain had very low sensitivity and specificity in the detection of rectal STI infections as compared to the Gene Xpert. These results were consistent with results in a study done in Burkina Faso among 127 prostitutes, Gram stain results for the detection of gonorrhoea were considered unrealistically low because of low sensitivity of the test (Damiba, Vermund, and Kelley 1990).

5.1.5 Antibiotic sensitivity profile of N. gonorrhoea among MSM at the Kenyan Coast Only about a quarter of the gonorrhoea infections among those that were detected by the Gene Xpert were culture positive. The very low detection rate of N. gonorrhoea by culture was also reported by Bromhead et al (2013) New Zealand. Culture being the cheaper and affordable method had very low detection rate hence missed three- quarters of the gonorrhoea infections. This could have been due to very low numbers of the bacteria present as most of the infections were asymptomatic.

Therefore, molecular point of care diagnostics, such as the Gene Xpert, allowed screening of at risk individuals with rapidly generated results within 90 minutes. The ability to detect chlamydia was an added advantage given the hard task of culturing the organism. While the crucial factor in decreasing STI include both enhancing detection and time taken from diagnosis to treatment (Geiger et al. 2016). In Kenya and other resource limited countries, the high cost of reagents and specialized equipment remains a substantial obstacle in aquiring appropriate molecular tests for the detection of STIs. However, efforts to culture the N. gonorrhoea should not be discarded as this will make monitoring of circulating genotypes and drug resistance possible.

Interestingly, results in this study showed that all N. gonorrhoea isolates were resistant to at least one of the following antibiotic drugs: penicillin, tetracycline or fluoroquinolones which are inexpensive and readily available. Emergency of flouroquinolone resistance in Kenya in 2012 as reported by Philippe et al., was at

53.2%. This study has shown that resistance has increased to 100%. This could have been as a result of over use of the over the counter available and affordable drugs, ability of N. gonorrhoea to develop resistance to antimicrobial therapies, genetic mutations and or acquisition of genetic material from closely related bacteria species which could have resulted in antibiotic-resistant N. gonorrhoea (Ashford, Golash, and Hemming 1976). Plasmid mediated resistance to penicillin could be conferred by extrachromosomal genes encoding for β-lactamase that destroys penicillin (Ashford,

Golash, and Hemming 1976) while resistance to tetracyclines could occur when the organism acquires an extrachromosomal gene from streptococcus, the tetM gene that

allows for ribosomal protein synthesis that is normally impaired by tetracycline

(Phillips 1976).

These isolates however, remained fully sensitive to the two classes of antibiotics recommended by the WHO for the treatment of gonorrhoea, namely macrolides and third generation cephalosporins (WHO, 2016). During the last decade, N. gonorrhoea strains with decreased susceptibility or resistance to extended spectrum cephalosporins have spread internationally and their presence have been documented globally (Unemo and Nicholas 2012; Unemo 2012). Targeted screening involving key populations, with subsequent bacteriological culture and antibiotic susceptibility testing for further monitoring of resistance is therefore a preferred strategy.

Participants diagnosed with gonorrhoea and chlamydia infections were dual treated with 400mg cefixime and 2g azithromycin. This was administered as a single doze observed therapy. In most settings worldwide, ceftriaxone is the last remaining option for empirical first-line antimicrobial monotherapy (Unemo and Nicholas 2012). Due to this fact, there is fear that gonorrhoea might become untreatable using antimicrobial monotherapy and in response to this concern, recommendations to use dual-antimicrobial therapy have been introduced (WHO, 2016).

The fewer number of time points evaluated in this study limited the study to making strong conclusions on frequency of screening MSM who report receptive anal intercourse or giving presumptive treatment as recommended by the World Health

Organization.

5.2 Conclusions

i. There is a high burden of asymptomatic rectal CT and or NG co-infections

among MSM at the Coastal Kenya with high recurrence of these infections

over time.

ii. Gram stain method should be phased out as a diagnostic test for rectal

gonorrhoea and PMN cells should not be used as an indicator of infections. iii. N. gonorrhoea isolated from the MSM at the coastal Kenya were highly

sensitive to macrolides and third generation cephalosporins antibiotics among

MSM at the Kenyan Coast. iv. Gram stain had very low sensitivity and specificity in the detection of

asymptomatic rectal gonorrhoea infection.

5.3 Recommendations

i. The high prevalence of asymptomatic CT and NG suggest the need for regular

screening or presumptive treatment.

ii. Screening by Gene Xpert should be considered as a valuable addition to

routine screening of CT and NG in healthcare settings

iii. For treatment of NG, programs should follow new guidelines that recommend

macrolides and third generation cephalosporins and avoid quinolones,

tetracyclines and penicillins.

iv. Further studies on:

a) Circulating genotypes of N. gonorrhoea among MSM will be of help to

determine their resistance mechanism to determine the best infection

control strategy.

b) The recurrence of rectal infections at shorter time points which would help

to determine required frequency for screening or offering treatment

presumptively to this high- risk population. c) Age <18 years who are sexually active to give more comprehensive data

on the burden of STI among this population.

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APPENDICES

Appendix I: Consent Form

SSC-894-amended_6th rev, E Sanders, 21 April 2015 Study number |5|__|__|__|__|

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Screening and enrolment Consent Form for

“HIV-UNINFECTED OPEN COHORT STUDY”

INTRODUCTION

Investigator‘s statement: I am (------Name of Researcher) from name institution (KEMRI or University of Washington (UW)—based at the Ganjoni clinic in Mombasa). I would like to tell you about a study being conducted by researchers from KEMRI, in Kilifi, and UW. I would like to find out if you would be willing to participate.

GENERAL INFORMATION

During the past two decades, a new infectious disease called AIDS which is caused by the human immunodeficiency virus type 1 (HIV-1) has appeared in countries throughout the world, including Kenya. This disease is primarily spread to adults through sexual contact. In the research study that I will tell you about, we are attempting to learn more about HIV-1 infection and other sexually transmitted diseases (STDs) among persons who may be at risk for infection with HIV-1. We also want to learn about what factors increase and decrease the risk of acquiring the virus.

The planned research study aims to find out the number of people (with high risk behaviour) who will become infected with HIV over a period of 1 year after they have received regular counselling and testing. This study will aim to screen over 3000

volunteers and have in follow up to 1000 persons at any point in time who are at high risk for HIV infection, but are not infected with HIV.

After one year of participation, and if you meet one of the criteria below, you may continue participation in the study. At any time, you are free to leave the study.

We are specifically interested to enrol persons aged 18-49 years, who meet one or more of the following criteria:

1) Persons who have a regular sexual partner who has tested positive for the HIV virus

2) Persons who exchange sex for payment in cash or in kind (either money or other support such as gifts, food, housing, etc.)

3) Persons who are diagnosed with a sexually transmitted infection

4) Persons who have multiple (more than 2) sexual partners in the past month.

5) Persons who practise anal sex

Learning about new HIV infections in these groups will be useful in designing larger,

HIV-prevention vaccine trials in the future (i.e. it will teach us how many people we need to involve in future work looking at how well HIV preventative vaccines work).

Many experts believe that HIV vaccines offer hope in controlling the spread of HIV disease. At this time there is no HIV vaccine that can protect you against HIV/AIDS.

If you participate in this study, you will receive counselling, periodic HIV-1 testing, and diagnosis and treatment of STDs. By learning more about what causes HIV-1 transmission, we hope that new infections can be prevented in the future. You will be

asked about your risk of acquiring HIV infection through a questionnaire. Risks for

HIV infection will also be assessed through an audio computer-assisted self-interview

(ACASI).

It is important that you know the following: your participation is voluntary, and you have every right to choose to take part in this study or not. Your future medical care will not be affected by your decision to choose or not choose to participate and you may decide to stop being part of the study at any time. SSC-894-amended_6th rev, E

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PROCEDURES

Screening for HIV: First, you will be given a study number and we will ask you to give written consent that we can test you for HIV. We will ask you to give contact details, and a counsellor will ask some questions about your risk behaviour and interest to make regular follow up visits. You will be given the opportunity to assess your risk behaviour through ACASI.

Then, we need to find out if you have antibodies to the HIV-1 virus, as the cohort study is following people who are HIV-1 negative. An antibody is the body‘s reaction to the virus causing AIDS. The antibody can be found in the blood. Therefore, if you agree to be screened, we will withdraw two tablespoons of blood to do a test for the antibody to the AIDS virus. We will also use this blood to look for antibodies against syphilis. You will learn your HIV test result immediately. You will be given an appointment within 2 weeks, to learn the results from the syphilis test. If you have an

STI you will be examined and treated.

If the rapid test is ―negative‖ we will do a specialized test that can detect if you may be infected with the HIV virus. We will only do the specialised point-of-care RNA or p24ag test when you meet specific risk criteria. You will also be tested for RNA or p24ag when you present with symptoms or signs compatible with an acute retroviral syndrome (e.g. reported fever, fatigue, body pains, vomiting, nausea, diarrhoea, sore throat, general ulcer disease, or discordant rapid test results). The test will provide results within 2 hours, and you will be invited to await the results. The period that one is HIV virus positive but antibody negative is called ―window period‖. This period usually lasts 2-3weeks and is the reason we are using a specialized test. If the RNA or p24ag test is positive and you are not able to wait for the results, we will contact you immediately and ask you to return for additional testing as soon as possible.

You will be contacted if the result of your syphilis test or the specialised HIV test is positive. We will provide treatment for syphilis and discuss with you how we can assist in contacting your recent sex partners for STD treatment. When you learn the test results, you will also be counselled to increase your understanding of HIV transmission, to reduce your risks of getting or transmitting HIV, and to notify your sexual partners if your test result is positive. We will provide you with a free supply of condoms, and KY jelly if you practise anal sex. For your participation in the screening and your return visit, you will receive 200 KSh per study visit to cover your time and bus fare.

(For women only): Pregnancy test: If you are a woman your urine will be tested to see if you are pregnant. If you are pregnant, you will be referred to ANC at a government health facility of your choice. If you are pregnant and infected with HIV, you will

receive HIV care and offered antiretroviral therapy to reduce the risk of giving the

HIV virus to your baby. Pregnancy testing will be offered at each scheduled visit.

If you are found to be HIV-infected during your screening visit you will be asked to register at the CCRC (Family Clinic) at Kilifi District Hospital, or any other CCC in

Mombasa, Malindi, or private provider of your choice. You will also be offered an opportunity to learn about a research study involving HIV-1 positive individuals at our clinic in Mtwapa or Kilifi.

Regardless of this study, we will offer to test your blood for CD4 cells, and a doctor will examine you. You will be given an appointment within 2 weeks to learn the results from these tests, including the syphilis test and the CD4 cell count, receive additional counselling. A doctor will advise you on where to access comprehensive care, including antiretroviral medication if the doctor decides that you should start such treatment.

Enrolment into Open cohort study of HIV-uninfected volunteers

If you are HIV-uninfected and willing to take part in the open cohort study, you will be given an enrolment appointment, and retain your study number. A counsellor will review your contact details, ask some questions about your risk behaviour and interest to make regular follow up visits. We will also ask you about your medical history, symptoms, and sexual behaviour. Once a year, you will be asked to perform the audio-computer-assisted self-interview (ACASI) about depression, substance use, stigma and other mental health challenges. SSC-894-amended_6th rev, E Sanders, 21

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You will be asked to return at monthly intervals at subsequent visits. At each follow- up visit, a 15 to 20 minute interview regarding recent sexual behaviour and symptoms will be conducted. Participants who can read and write may be asked to take an additional 15-20 minutes to perform the audio-computer-assisted self-interview. A physical examination and evaluation for STDs will be performed. For women, this includes a speculum pelvic examination. For individuals who report anal receptive sex, we will also offer an examination by proctoscopy to identify anal ulcers or proctitis, and collect a quarterly rectal swab for gonorrhoea screening, and drug sensitivity testing (i.e. testing to find out if the prescribed drugs for gonorrhoea are effective). If you have an STD at any time during follow up, you can come to the clinic to be examined, and receive treatment.

At each visit, two table spoons of blood will be obtained for HIV-1 testing. You will receive your rapid HIV test result immediately. At each visit, counselling will be provided and a free supply of condoms made available. For women only, at each visit your urine will collected for a pregnancy test.

For your participation in the long-term study, you will receive 400 KSh per scheduled study visit to cover for your time and bus fare. If you report to the clinic on your assigned appointment date you will receive an additional Ksh 50, If you report specific risk behaviour that in the opinion of the counsellor would increase your chance of acquiring HIV-1, or present with symptoms compatible with an acute retroviral syndrome, your blood sample will be subjected to the specialised HIV test testing for RNA or p24ag. You will receive 400 KSh per scheduled monthly study visit, or Ksh 450 if you report on your appointment date.

If you develop HIV-1 infection during the course of the study, you will receive counselling and information about treatment options for HIV infection. You will be invited to participate in another study that monitors your recent HIV infection, and offers antiretroviral treatment (Ng and Martin) and care. We will ask questions about your understanding of the infection and care uptake.

Benefits to you

If you participate in this study, you will receive regular counselling and testing for

HIV-1 and screening and treatment of certain STDs. We will also provide treatment for minor medical illnesses. If you are a woman you will have a pregnancy test at each scheduled visit.

If you report regular anal sex or use intravenous drugs we will offer hepatitis B virus

(HBV) vaccination.

All specimens obtained in this study will be stored for further research. Your specimens will be used in future studies of HIV-1, or other infectious diseases including the body‘s response to infection.

It is important to consider carefully whether you want to have the HIV antibody test.

The test is voluntary. You do not have to have the test, however, it is required if you wish to join the study. You should be tested only if you are well informed about the testing. Please listen to what I tell you about the test so that you can make a good decision.

If the first HIV test is NEGATIVE you probably do not have the HIV-1 virus. If the specialised HIV test is also negative you are most likely uninfected with HIV-1.

However, each time you have unprotected sex you are at risk of getting HIV.

If you test POSITIVE (either by the first HIV test (rapid antibodies) or by a specialised HIV test (RNA or p24 antigen), you most probably have the HIV-1 virus.

That means you can pass it to others. If you are rapid antibody negative and RNA or p24 antigen positive you have an acute HIV-1 infection and you are very likely to pass on the virus when you have unprotected sex. This does not mean that you have

AIDS, which is the most advanced stage of HIV-1 infection, but that you will be eligible to start ART and care according to NASCOP recommendations. SSC-894- amended_6th rev, E Sanders, 21 April 2015 Study number |5|__|__|__|__|

4 APPROVED 06 MAY 2015

False results are rare. Unclear results are also rare. When a test result does not seem to make sense, we do the test again. If the specialised test is positive we will test you for rapid antibodies to confirm infection approximately within 10 days that the specialised test was positive. If you test positive while you are in the study, we will collect 3 tablespoons of blood (30 ml) to do the following additional tests:

n your blood

-cell count‖)

- typing or tissue typing)

You will be informed about your CD4 count and the amount of virus in the blood as soon as these are available. We will also offer enrolment in the care cohort at KEMRI, or any other CCC of your choice.

The benefits of being tested are very personal. If you are worried about AIDS, you might feel better if you have a negative test. Sometimes knowing that the test is positive can relieve stress. You may want to know your test result before you have sex with a new partner. In some cases, test results may help diagnose a medical problem or guide your health care. There may be other benefits of testing that we don‘t know about now.

RISKS, STRESS, AND DISCOMFORT

Drawing blood may cause pain, and a bruise may form where the needle enters the vein.

Learning HIV-1 test results may cause you severe stress, anxiety, and depression. You might be tempted to have unsafe sex if the result is negative. This would increase your risk of getting HIV. Also, a negative antibody test result does not always mean that you are HIV uninfected, if you were infected with the virus in the last three weeks.

OTHER INFORMATION

Your identity and test results will remain confidential, known only to the investigators. Information will be kept for an indefinite period of time. You are free to refuse to participate and to withdraw at any time without penalty or loss of benefits to which you are otherwise entitled. Your care at this clinic will not be affected by whether you decide to participate or not. Participation in the study is entirely voluntary.

However, we will attempt to find you if you do not show up for a scheduled appointment to invite you to return to the clinic.

Once you enrol in the study, we will offer 3 strategies to improve your research participation.

Home visits

If you grant permission, clinic staff will make an appointment to visit you once at your home. During this visit, they will update your tracing locator form with landmarks in the vicinity of your home and take a GPS-way point. A GPS machine looks like a big mobile telephone and can take coordinates of any location with a precision of about 5 meters. These coordinates will be used by KEMRI-UW staff only if we need to trace you for an abnormal lab results or missed visit. We believe this technology will help us trace you more discretely, since tracing staff will need to ask fewer questions to locate your residence. Study staff will only come to your home with your permission. SSC-894-amended_6th rev, E Sanders, 21 April 2015 Study number |5|__|__|__|__|

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SMS-reminder message

If you provide us with a mobile phone number, we will send you a reminder message for your clinic appointment 1 day in advance. If you do not want to receive such a message, please let us know either now or at any time in the future.

Fingerprint scan

To help us keep track of who is enrolled in our clinic, we will ask you to put your right index finger on a small machine that can scan your finger print. This will be done at each clinic visit and will take less than 5 seconds. This scan will translate into a unique identification number that will be added to your study ID card, and will be accessible only to our staff and to staff of the IAVI Kangemi project in Nairobi. If you are in Nairobi and need emergency care, you are welcome at the Kangemi clinic.

Your finger scan will let the Kangemi clinic staff know that you are enrolled in the

KEMRI clinics in Mtwapa and Kilifi.

If you don‘t want to have your fingerprint taken, please let us know – you are free to refuse.

You are also free to ask questions of the investigator both before consenting to participate and at any time thereafter. Members of the research team are available to answer questions here in the clinic anytime during working hours. All laboratory tests and medical care conducted expressly for the purpose of this study will be provided at no cost to you.

Your specimens will be stored indefinitely in the research laboratories at KEMRI.

Your samples will be labelled only with your study identification number and the code linking this number to your name will be kept in a computer with restricted access. Future research on your specimens will involve research on HIV-1, other infectious diseases associated with HIV, common viruses and infections people in this community are exposed to, and the body‘s response to infection.

Your stored blood may also be sent to other expert laboratories that are not based in

KEMRI, for more specialized tests. It is not known at this time to which laboratories these samples will be shipped. However, we will only use the stored blood to do useful studies on HIV and permission will be obtained from the KEMRI/National ethics committee. In all cases your privacy is protected because blood is stored with a number not a name on it.

Your participation in the study is completely voluntary. You can withdraw from the study at any time without giving a reason. You may be removed from the study at any time for the following reasons:

Do you have any questions? Do you agree to participate?

Who should I contact with any questions about the study?

The coordinator is Dr. Eduard Sanders. If you have any questions you can reach him at KEMRI (telephone: 041-7522063/0725-242233/0735-522063).

If you have a medical problem related to your participation, please contact:

Dr. Norbert Peshu, KEMRI (telephone: 041-7522063/0725-242233/0735-522063.).

If you have a question about the ethics of this investigation you can contact: SSC-894- amended_6th rev, E Sanders, 21 April 2015 Study number |5|__|__|__|__|

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The secretary, KEMRI/National Ethical Review Committee, P. O. BOX 54840-

00200, Nairobi, Mobile phone: 0722205901 or 0733400003.

Subject’s Statement for Screening consent:

This study has been explained to me, and I voluntarily consent to be screened for the cohort study. I have understood that I will be given an appointment within 2 weeks to learn results from the syphilis test and learn more about HIV risk reduction. I may also be contacted if my screening sample is positive in the specialised HIV test (i.e.

RNA or p24 ag positive). At the result visit, I may be invited to the cohort study.

Screening consent signature box

Name of subject ______

Signature of Subject ______Date ______

Name and signature of witness

Name ______Signature ______Date ______

(if volunteer was not able to read and understand the informed consent document)

Name and Signature of Person obtaining consent

Name ______Signature ______Date ______

Copies to: investigator, subject SSC-894-amended_6th rev, E Sanders, 21 April 2015

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Subject’s Statement for Enrolment consent:

I have understood that I commit to monthly follow up visits over a period of one-year.

I have had an opportunity to ask questions. I understand that future questions I may have about the research will be answered by one of the investigators. I will receive a copy of this consent form.

Please initial or put thumb print if you have no objection to

____ being visited at home

____ receiving a SMS reminder message for my clinic appointment

____ having my fingerprint scanned each time I visit the clinic

Enrolment consent signature box

Name of subject ______

Signature of Subject ______Date ______

Name and signature of witness

Name ______Signature ______Date ______

(if volunteer was not able to read and understand the informed consent document)

Name and Signature of Person obtaining consent

Name ______Signature ______Date ______

Copies to: investigator, subject SSC-894-amended_6th rev, E Sanders, 21 April 2015

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CONSENT FOR SPECIMEN STORAGE AND SHIPMENT VOLUNTEER:

Please carefully read the statements below and think about your choice. No matter what you decide it will not affect whether you can be in the research study, or your routine health care. Please tick which statements you agree with.

Sample storage

______I agree to have a sample of my blood or specimen (e.g. bacterial isolate) stored at KEMRI and used for the research test to assess acute HIV-1 infection, and other tests related to HIV-1 infection (e.g. viral load and drug resistance testing)

______I do not agree to have a sample of my blood or specimen stored and used for future, testing related to HIV infection.

Sample shipment

______I agree to have a sample of my blood or specimen (e.g. bacterial isolate) to be transported outside Kenya and used for future testing related to HIV infection.

______I do not agree to have specimens to be transported outside Kenya and used for future testing related to HIV infection.

Participant Signature/Mark: ______Date: ______

Participant Name: ______Time: ______

INVESTIGATOR/ DESIGNEE:

I certify that I have followed the study SOP to obtain consent from the participant.

S/he apparently understood the nature and the purpose of the study and consents to the participation in the study. S/he has been given opportunity to ask questions which have been answered satisfactorily.

Investigator/ designee Signature: ______Date: ______

Investigator/ designee Name: ______Time: ______

WITNESS: IF THE VOLUNTEER CANNOT READ:

I affirm that the Informed Consent Document has been read to the volunteer and he/she understands the study, had his/her questions answered, and I have witnessed the volunteer‘s consent to study participation. (Only necessary if volunteer was not able to read and understand this informed Consent Document)

Impartial Witness signature: ______Date: ______

Impartial Witness Name:______Time: ______

(please print name)

Thumbprint of participant if they cannot write:

______

Appendix II: The map of Kenya showing the location of Kilifi County

Appendix III: Ethical Approval

Appendix IV: Risk Assessment Questionnaire

Unless otherwise specified, 0=No, VISIT VISIT VISIT __ VISIT VISIT VISIT _ VISIT 1=Yes; fill visit number (1, 2, etc) ______1a. Date (day/month/year) __/__ __/__/_ __/__/__ __/__/ __/__ __/__/_ __/__/ /__ _ __ /__ _ __ 1b. Time start interview ______BIRTH CONTROL 2. What method of birth control are you using? Condoms ______Vasectomy ______None (1 = Not using any) ______HYGIENE AND HEALTH (H&H) 3. How many times in the past week did you wash your genitals with? (Fill out #; 0=method not applied) Water alone ______Antiseptic (e.g., Dettol) ______Soap and water ______Detergent ______Lemon ______Didn‘t wash (1 = didn‘t ______wash) 4. How many times in the past ______week did you bathe? (Fill #) SEXUAL PRACTICES IN PAST WEEK 5. HAVE YOU HAD SEX IN ______PAST WEEK? (No = 0, if No skip to Q11) 6. With how many different people, including your spouse ______(s) have you had sex? (fill #) 7. How many regular sex partners, including spouse(s) ______have you had? (fill #, if no regular partner(s), fill 0 then skip to Q8) 7a. How many times did you ______have sex with your regular partner(s)? 7b. How many times did you have sex using a condom ______with your regular partner(s)? 7c. If you had sex with a regular partner, do you know his / her ______HIV status? (if no, skip to Q8) 7d. If yes, is he / she HIV ______positive?

8. How many casual sex partner(s) have you had? (Fill ______

#; if no casual partner(s), fill 0 then skip to Q11) 8a. How many times did you ______have sex with casual partner(s)? 8b. How many times did you have sex using a condom with your casual partner(s)? ______Unless otherwise specified, VISIT VISIT VISIT __ VISIT VISIT VISIT _ VISIT 0=no, 1=yes; fill visit number ______(1, 2, ..) 9. How many new sex partners have you had? (fill #, if no new sex partner (s), fill 0 then skip to Q11) ______10a How many times did you . have sex with new sexual partners? ______10b How many times did you . have sex using a condom with new sexual partners? ______SEXUAL PRACTICES IN PAST MONTH 11. With how many different people, including your spouse (s) have you had sex? (fill #; 0= no sex in past month skip to Q15) ______12. How many of your sex partners were male? (Fill #) ______12a Of these __ (Baker et al. . given in Q12) sex partners, how many were new male partners that is someone you never had sex with before? (Fill #) ______12b How frequently did you use condoms when having sex with these new male partners? (0=never; 1= sometimes; 2= frequently; 3=always; 9= Don‘t know) ______13 How many of your sex partners were female? (Fill #) ______13a Of these__ (Baker et al. . given in Q13) sex partners, how many were new female partners, that is someone you never had sex with before? (Fill #)

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13b How frequently did you use . condoms when having sex with these new female ______

partners? (0=never; 1= sometimes; 2= frequently; 3=always; 9= Don‘t know) 14. Did you know any of these __ (say total given in Q11) sex partners to be HIV infected? If no, skip to Q15 ______14a How many of these __ (say . total given in Q11) sex partners were HIV infected? ______14b How frequently did you use . condoms when having sex with these HIV infected partner(s)? (0=never; 1= sometimes; 2= frequently; 3=always; 9= Don‘t know) ______OTHER SEXUAL PRACTICES (In the past 3 months or since last visit) 15. Have you had sex during menses? (0=No; 1=Yes; 9=Don‘t know) ______16. Have you had sex with: 1 = Women only; 2 = Men only; 3 = Men and Women. ______

Unless otherwise specified, VISIT VISIT VISIT __ VISIT VISIT VISIT _ VISIT 0=no, 1=yes; fill visit number ______(1, 2, ..) 16a In the past three months, have . you had anal sex (with anal sex we mean the penis inserts the anus) If No skip to Q17a 16b How do you practice anal sex . (1= insertive; 2= receptive; 3= both) 16c When you practice anal sex, . how often do you use a condom (0= never; 1= sometimes; 3= always; 9= don‘t know) 17a If sex with women (Q16 = 1 . or 3): In the last week how many times have you had vaginal intercourse? (Fill #) ______17b If sex with women (Q16 = 1 . or 3): In the last week how many times have you had anal intercourse with a woman? (Fill #)

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18a If sex with men (Q16 = 2 or . 3): In the last week how many times have you done receptive anal intercourse (Bottom)? (Fill #) ______

18b If sex with men (Q16 = 2 or 3): In the last week how many times have you done insertive anal intercourse (Top) with a man? (Fill #) ______18c If Q16 =2 or 3 and Q18a > 1, . in the last week, how often do you use condoms for receptive anal sex/ going bottom? (0=never; 1= sometimes; 3= always; 9= Don‘t know) ______18d If (Q16= 1 or 3 & MALE) & . (Q17b > 1 or Q18b > 1), in the last week, how often did you use condoms for insertive anal sex/going top? (0=never; 1= sometimes; 3= always; 9= Don‘t know) ______19a In the last week, have you . given oral sex [i.e. ‗blow job‘] (0=No; 1=Yes). If No skip to Q20 ______19b If yes (19a): in the last week . how many times did you give a ‗blow job‘? (Fill #) ______19c If yes (19a): when you gave a . blow job, how often did your partner use a condom? (0=never; 1=sometimes; 3=always) ______19d If yes (19a): At the last time . you gave a blow job did he ejaculate in your mouth? (0=No; 1=Yes; 9= don‘t know) ______20 In past 3 months or since last visit: have you participated in group-sex? (by group sex we mean sex with more than one person at the same time) (if no skip Q 21) ______20a If participated in group sex, . was it with (1= women only; 2=men only; 3= Women and Men)

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TRANSACTIONAL SEX 21. In the last three months, have you been paid for sex with ______cash, living expenses, or goods? (If no, skip to Q26) 21a How did your last client pay . you for sex? (1 = cash, 2 = ______gift(s), 3 = other [specify] ______

Unless otherwise specified, VISIT VISIT VISIT __ VISIT VISIT VISIT _ VISIT 0=no, 1=yes; fill visit number ______(1, 2, ..) 21b If cash (Q21a = 1), how much . money did your last client ______pay you for sex? (#Fill) ______21c If Q21b = 3, specify other ______. ______22a By how many clients were . you paid for sex in the past one-week? (Fill #) ______22b By how many clients were . you paid for sex in the past one month? (Fill #) ______23. Who are most of your clients? (From the Coast: 1 = Indians; 2 = Arabs; 3 = Other Kenyans; 4 = Wazungus; 5 = Other Africans; From Elsewhere. 6 = Kenyans- upcountry; 7 = Wazungu- tourists; 8 = African-tourists) ______24. Who was your last paying client? (From the Coast: 1 = Indian; 2 = Arab; 3 = Other Kenyan; 4 = Mzungu; 5 = Other African; From Elsewhere. 6 = Kenyan- upcountry; 7 = Mzungu- tourist; 8 = African-tourist) ______25. How did your last client make contact with you? (1=on the street; 2=in a market; 3=in a bar; 4=in a nightclub; 5=at my home; 6=on the beach; 7=using the internet/email, 8 = on my cell-phone, 9 = other) ______25a If Q25 = 9, specify other ______. ______26. In the last three months, have you paid for sex with cash, living expenses, or goods? (If no, skip to Q27)

______26a How many sexual partners . did you pay in the last week? (Fill #) ______26b How many sexual partners . did you pay in the last month? (Fill #) ______SUBSTANCE ABUSE AND OTHER PRACTICES 27. In past 3 months: have you ______used intravenous drugs? (If no skip to Q28) 27a If yes, did you share needles ______

. at any time since your last visit? 28. In the past month have you used the following: (0=none; ______1=daily; 2=weekly; 3=1-3 times / month) Cigarettes ______Marijuana ______Miraa ______Other drug ______29. In the last month, how often have you had a drink ______containing alcohol? (0=none; 1=daily; 2=weekly; 3=1-3 times / month) – if none skip to Q33) 30. In the last month, how often did you get drunk? (0=never, ______1=daily; 2=weekly; 3=1-3 times / month) 31. In the last month how often do you have sex after ______alcohol? (0=never; 1=sometimes; 3 = always) Unless otherwise specified, VISI VISIT VISIT __ VISIT VISI VISIT _ VISIT 0=no, 1=yes; fill visit number T _ __ __ T _ _ (1, 2, ..) 32. In the last month, have you experienced any of the following after drinking alcohol? (no / yes) Gotten into a fight ______Had accident / Injured ______yourself Been arrested ______d. Been raped / sexually ______assaulted 33 During the last three months, ______have you been raped? 34 If yes, the last time you were raped, who did this to you? (1 = regular partner (e.g. boyfriend/spouse), 2 = new sex partner or client 3 = family member 4 = neighbour 5 = government official (e.g. police, army, prison guard) 6 = unknown (e.g. crowd/mob/members of public)) 8 = other ______35 If yes, the last time you were raped, how many people did this to you? (Fill #) ______36 During the last three months, have you been physically assaulted (e.g. beaten up, attacked). If No, skip to Q37 ______36a If yes, the last time you were ______

physically assaulted, who did this to you? (1 = regular partner (e.g. boyfriend/spouse), 2 = new sex partner or client 3 = family member 4 = neighbour 5 = government official (e.g. police, army, prison guard) 6 = unknown (e.g. crowd/mob/members of public)) 8 = other 36b If yes, the last time you were physically assaulted, how many people did this to you? (Fill #) ______37 During the last three months, have you been verbally abused because of your sexual behaviour, sexual orientation or occupation? If No, skip to Q38 ______37a If yes, the last time you were verbally abused because of your sexual behaviour, sexual orientation or occupation, who did this to you? (1 = regular partner (e.g. boyfriend/spouse), 2 = new sex partner or client 3 = family member 4 = neighbour 5 = government official (e.g. police, army, prison guard) 6 = unknown (e.g. crowd/mob/members of public)) 8 = other ______

37b If yes, the last time you were verbally abused because of your sexual behaviour, sexual orientation or occupation, where did this occur? (1 = at my own home, 2 = at my parent‘s home, 3= in a bar or nightclub 4 = in a public place (e.g. street, shop, beach) 5 = in a government building (e.g. police station, prison) 6 = in a health care building (e.g. hospital, clinic, pharmacy) 8 = other 9 = unknown ______38. Finish time interview ______Initials of person completing ______questionnaire