\\ ANTIRETROVIRAL RESISTANCE AND GENETIC DIVERSITY OF HUMAN IMMUNODEFICIE CY VIRUS AMONG ANTENATAL CLINIC RESPONDENTS ON NEVIRAPINE REGIMEN FROM NORTH-RIFT KENYA / I Michael Kibet iptoo B.Sc. (Moi University), M.Sc. (Kenyatta University) 1841S084/04 A thesis submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy (Immunology) in the School of Pure and Applied Sciences, Kenyatta University 2008 ii DECLARATION This is to certify that this thesis is my original work and has not been presented for a degree in another university or for another award Michael Kibet Kiptoo ~. \__~f2 _ Signature -L~_1I_~f..:~~~_ Date Department of Zoological Sciences, Kenyatta University This thesis has been submitted for examination with our approval as supervisors Prof. Jones M. Mueke ~ -:7!f_~~ Signature -----!-~-~-~~-~~-~ Date Department of Zoological Sciences, Kenyatta University Prof. Zipporah W. Ng'ang'a -------~~----Signa~~~----J Date Department of Medical Laboratory Sciences, Jomo Kenyatta University of Agriculture and Technology Dr. Elijah M. Songok--_ . r Signature LIJ!::__M!:?Y_:._!~~~ Date Centre for Virus Research, Kenya Medical Research Institute Date Centre for Biotechnology and Research Development, Kenya Medical Research Institute III DEDICATION This thesis is dedicated to my late father Barsulai who passed away on 4th May, 2008 and my late grandfather Tomno, who never lived to see me through my university education but always encouraged me to study hard. Their spirits of hard work lives on. IV ACKNOWLEDGEMENTS I would like to thank my supervisors, Prof. Z.W. Ng'ang'a, Prof. J. M. Mueke, Or. E.M. Songok and Dr. S. Mpoke for assuming the onerous task of my work. I owe this thesis to them for the support during the processes of conceptualisation, implementation of the fieldwork, bench work and data analysis. I am grateful for their stimulating discussions and valuable comments on the preparation of this thesis. I am greatly indebted to Prof. Hiroshi Ichimura of the Department of Viral Infection and International Health, Kanazawa University Graduate School of Medical Sciences, Japan, for the scholarship that enabled me to visit his laboratory to carry out the drug resistance component of the study. I would like to thank my colleague Or. R. Lwembe for providing accommodation and also supporting the running of the laboratory assays during my stay in Kanazawa University. Many people and organizations made the research work for this thesis possible. I would like to thank the then Director of NASCOP, Or. Kenneth Chebet, for the support of his office in the implementation of the research proposal. I would like to thank the Medical Officers of Health in Kitale, Kapsabet, and South Nandi Hills district hospitals for not only accepting their institutions to be the study sites but also for their dedication in supporting the study. I would like to thank all the hospital staff who contributed in one way or another to make the study a success. This work would not have been possible were it not for the financial support by a bilateral grant from the Japan International Co-operation Agency (JICA) to KEMRI vthrough the Research and Control of Infectious Diseases Program In Kenya, (KEMRIlnCA IDP project) Phase H. I would also like to thank my colleagues at the HIV/AIDS biosafety level 3 facility at the Centre for Virus Research, KEMRI, for the moral support and the spirit of dedication which saw us work to late hours and weekends. Finally, I would like to thank my wife, Janet, our daughter Memo, for their moral support during the course of the study especially when I had to spend a lot of time in the laboratory, at study sites, and for accepting to stay without me for the 3 months when I was away in Japan. VI TABLE OF CONTENTS Page Declarati0n--------------- ---------------------------------------------------- --------------------ii Dedication------------------------------------------------------------- --------------------------iii AcknowIedgements----------------------------------------------------------------------------- iV TabIe of contents-------------------------------------------------------------------------------- vi List of figures------------------------------------------------------------------------------------xi List 0f tabIes------------------------------------ ------------------------------------------------ xii List ofplates------------------------------------------------------------------------------------xiii List of abbreviations and acronyms---------------------------------------------------------xiv Abstract ---------------------------------------------------------------------------- ------------xviii CHAPTER ONE 1.0 ~TRO])1JCTION-----------------------------------------------------------1 1.1 Background Information------------------------------------------------------1 1.2 Statement of the Problem-----------------------------------------------------2 1.3 Rationale of the Study---------------------------------------------------------4 I .4 Null Hypothes is---------------------------------------------------------------- 5 1.5 Objectives of the Study--------------------------------------------------------5 1.5.1 General Objective-------------------------------------------------------------- 5 1.5.2 Specifie Objectives------------------------------------------------------------ 5 1.6 Ethical Considerations---------------------------------------------------------6 1.7 Limitations of the Study-------------------------------------------------------6 VII CHAPTER TWO 2.0 ~ITE~T~ RE~EW---------------------------------------------------7 2.1 The History and Origin of HIV----------------------------------------------7 2.2 Classification of HIV----------------------------------------------------------9 2.3 Diversity of HIV--------------------------------------------------------------12 2.4 Biology of HIV Infection----------------------------------------------------14 2.5 Genomic Organization of HIV----------------------------------------------15 2.6 The Structure of HIV---------------------------------------------------------18 2.7 Life Cycle of HIV-1----------------------------------------------------------20 2.8 Global Epidemiology of HIV-----------------------------------------------23 2.9 Natural History of HIV------------------------------------------------------24 2.10 Survival ofIndividuals Infected with HIV-1-----------------------------26 2.11 Prevention of HIV------------------------------------------------------------27 2.11.1 HIV Vaccines-----------------------------------------------------------------28 2.11.1.1 Challenges in the Development of HIV Vaccines------------------------28 2.11.1.2 Types of HIV Vaccines------------------------------------------------------ 30 2.11.1.2.1 Live Attenuated Vaccines---------------------------------------------------30 2.11.1.2.2 Inactivated Vaccines---------------------------------------------------------30 2.11.1.2.3 Virus-Like Particles (VLP) as Vaccine------------------------------------31 2.11.1.2.4 Subunit Vaccines-------------------------------------------------------------31 2.11.1.2.5 Naked DNA and Live Recombinant Vaccines---------------------------33 2.11.2 Microbicides to Prevent HIV Transmission-------------------------------35 2.11.3 Antiretroviral Therapy-------------------------------------------------------36 2.11.3.1 Reverse Transcriptase Inhibitors-------------------------------------------37 2.1 1.3.2 Protease Inhibitors------------------------------------------------------------ 38 2.11.3.3 2.12 2.13 2.14 2.14.1 2.14.2 2.14.3 2.14.3.1 2.14.3.2 2.14.3.3 2.14.3.4 viii Fusion Inhibitors-------------------------------------------------------------- 38 Mechanisms of HIV Drug Resistance-------------------------------------39 Drug Resistance and Viral Subtypes---------------------------------------40 Mother- To-Child Transmission of HIV (MTCT)------------------------42 Pathogenesis of MTCT------------------------------------------------------42 Risk Factors for MTCT------------------------------------------------------4 3 Strategies for Reduction of MTCT-----------------------------------------44 Behavioural Intervention----------------------------------------------------44 Nutritional Intervention------------------------------------------------------4 5 obstetri c Intervent ion--------------------------------------------------------45 Antiretroviral Prophylaxis---------------------------------------------------45 CHAPTER THREE 3.0 MATERIALS ANDMETHODS----------------------------------------48 3. 1 Study Area ----------------- ----------------------------------------------------4 8 3.2 Study Population--------------------------------------------------------------49 3.2.1 IncIusion Criteria-------------------------------------------------------------4 9 3.2.2 Exc Iusion Criteria------------------------------------------------------------4 9 3.3 Sample Size Determ ination-------------------------------------------------49 3.4 Study Design------------------------------------------------------------------50 3.5 Demographic Characteristics of the Antenatal Clinic Attendees------50 3.6 Blood CoIlection-------------------------------------------:------------------- 51 3.7 Enumeration of CD4/CD8 Cells--------------------------------------------51 3.8 Viral Load Determ ination--------------------------------------------------- 51 3.9 Diagnosis of HIV in Infants using PCR-----------------------------------52 IX 3.10 Polymerase Chain Reaction (PCR), Cloning, and Sequencing---------53 3.11 Genotypic Drug Resistance Testing, Phylogenetic Analysis, and Subtyping of the Sequenced Samples--------------------------------------56 3.12 Data Analys is----------------------------------------------------------------- 58 CHAPTER FOUR 4.0 RESULTS---------------------------------------------------------------------59 4.1 Demographic Characteristics of the Study Population------------------59 4.1.1 Background Characteristics of the Antenatal Clinic Attendees--------59 4.1.2 Age Distribution--------------------------------------------------------------59 4.1.3 Educational Status------------------------------------------------------------60 4.1.4 Marital Status-----------------------------------------------------------------60 4.2 HIV Prevalence---------------------------------------------------------------61 4.2.1 Background Characteristics of HIV Seropositive ANC attendees-----61 4.2.2 HIV Prevalence by Age------------------------------------------------------62 4.2.3 HIV Prevalence by Marital Status------------------------------------------62 4.2.4 HIV Prevalence by Level of Education------------------------------------63 4.3 Infant diagnosis, CD4 counts, Viral load, HIV subtypes, and Drug resistance-----------------------------------------------------------------------64 4.3.1 Infant Diagnosis Using PCR------------------------------------------------64 4.3.2 CD4 eounts--------------------------------------------------------------------6 5 4.3.3 Viral Load Profiles and PCR amplification-------------------------------66 4.3.4 HIV Subtypes Based on the HIV-1 envelope Region (C2V3)----------70 4.3.5 HIV Subtypes Based on the pol Region (RT)----------------------------72 4.3.6 Reverse Transcriptase Inhibitors Associated Mutations-----------------74 'KE Y - A UNIVERSITY u RAR' x4.3.7 Analysis of Mother-Child Pairs---------------------------------------------77 CHAPTER FIVE 5.0 DISCUSSION, CONCLUSIONS AND SUGGESTIONS-----------81 5.1 Discussion---------------------------------------------------------------------81 5.1.1 Demographic Characteristics of the Study Population------------------81 5.1.2 CD4 T-Iymphocyte Counts and Viral Load among ANC Attendees-82 5.1.3 Vertical Transm ission of HIV----------------------------------------------83 5.1.4 Genetic Diversity of HIV-1 in North-Rift Kenya------------------------84 5.1.5 Reverse Transcriptase Inhibitors Associated Mutations-----------------85 5.2 ConcIusions--------------------------------------------------------------------8 7 5.3 Recommendations and Suggestions for Future Work-------------------88 REFERENCES--------------------------------------------------------------------------------89 APPEND ICES--------------------------------------------------------------------------------1 09 Appendix I Appendix 2 Appendix 3 Map of Study Area----------------------------------------------------------109 Informed Consent-----------------------------------------------------------110 Drug Resistance Study Questionnaire------------------------------------112 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 XI LIST OF FIGURES Phylogenetic Tree of the SIV and HIV viruses---------------------------l 0 Diversity of HIV --------------------------------------------------------------14 Genomic Organization of HIV ----------------------------------------------16 The Human lmmunodeficieny Virus---------------------------------------20 The Life Cycle of HIV -1----------------------------------------------------21 Course of Hl V-1 lnfection--------------------------------------------------25 HIV Prevalence by age group-----------------------------------------------62 HfV Prevalence among ANC attendees by marital status---------------63 HIV Prevalence among ANC attendees by level of education---------64 Relationship between PCR amplification and viral load----------------70 Phylogenetic tree based on a part of the env-C2V3 gene (550bp) ----71 Phylogenetic tree based on a part ofthepol-RT gene (697bp) --------73 Phylogenetic relationships of the pal (RT) nucleotide sequences (clones) from the mother and child (KTL252-M/C)---------------------78 Phylogenetic relationships of the pal (RT) nucleotide sequences (clones) from the mother and child (SNH072-MlC)---------------------79 Phylogenetic relationships of the pal (RT) nucleotide sequences (clones) from the mother and child (KTL254-MlC)---------------------80 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 4.6 Table 4.7 Table 4.8 Table 4.9 Table 4.10 Table 4.11 Table 4.12 xii LIST OF TABLES Distribution of ANC attendees by study site------------------------------59 Distribution of ANC attendees by age-------------------------------------60 Distribution of ANC attendees by educational level---------------------60 Distribution of ANC attendees by marital status-------------------------61 Distribution ofHIV Seropositive ANC attendees by hospital----------61 Characteristics of HIV Seropositive ANC attendees--------------------62 T-Iymphocyte (CD4) Count among ANC attendees---------------------66 Viral load among ANC attendees------------------------ ------------------66 Demographic factors and HIV subtype data for 30 ANC attendees---72 Demographic factors and HIV subtypes for 36 ANC attendees and 3 infants--------------------------------------------------------------------------74 NRTI associated mutations and level of resistance----------------------75 NNRTI associated mutations and level of resistance--------------------76 Plate 4.1 Plate 4.2 Plate 4.3 Plate 4.4 xiii LIST OF PLATES A PCR gel showing amplification results of the pol-Integrase gene (29 7bP)-- ---------- ---- --- ------ ---- ----- -------------- ------ ----- ------ ---- --- -65 A PCR gel showing amplification results of the env (C2V3) gene (550 bp )-------------------------------------------------------------------------6 7 A PCR gel showing amplification results of the pol-RT gene (697pb )-------------------------------------------------------------------------68 A PCR gel showing the pol-RT gene (697bp) PCR product insert ---69 ABC AIDS ALVAC AMP APCs ARV ART ATV CBS CCR5 CD cDNA CDC CS CSF CRFs CTL CXCR4 ddI ddc d4T DNA DLV EFZ xiv LIST OF ABBREVIATIONS AND ACRONYMS Abacavir Acquired Immune Deficiency Syndrome A canarypox virus vector Amprenavir Antigen presenting cells Anti-retroviral Anti-retroviral therapy Atazanavir Central Bureau of Statistics Cystein-Cystein linked chemokine receptor 5 Cluster of Differentiation Complementary Deoxyribonucleic acid Centers for Disease Control and Prevention Caesarean section Cerebrospinal fluid Circulating recombinant forms Cytotoxic T lymphocyte Cystein-X-Cystein linked chemokine receptor 4 Didanosine Zalcitabine Stavudine Deoxyribonucleic acid Delaviridine Efavirenz Env FDA FTC gag GOK gp HAART Hb IllDI mV-1I2 HSV-2 HTL V-IIIIIIII IAV! mv KEMRI Kb LAV LPV ML J.LI MIIC IIII MOH mRNA MTCT MVA xv HIV -1 envelope gene US Food and Drug Administration Emtricitabine HIV -1 group antigen gene Government of Kenya glycoprotein Highly active antiretroviral therapy Hemoglobin High Density Formamide Human immunodeficiency virus type 1 or 2 Herpes simplex virus type 2 Human T-Celllymphotrophic virus type llIIIIII International AIDS Vaccine Initiative Indinavir Kenya Medical Research Institute Kilobase Lymphadenopathy Associated Virus Lopinavirritonavir millilitre microlitre Major Histocompatibility Complex IIII Ministry of Health Messenger ribonucleic acid Mother-to-child-transmission of HI V Modified Vaccinia Virus Vector XVI MV NACC NASBA NASCOP NFV NNRTI NRTI NSI NVP PBS PBMCs PCR PI PMTCT pol PR Measles virus National AIDS Control Council Nucleic Acid Sequence Based Amplification National AIDS and STDs Control Programme Nelfinavir Non-nucleoside reverse transcriptase inhibitors Nucleoside reverse transcriptase inhibitors Non-syncytium-inducing Nevirapine Phosphate buffered saline Peripheral blood mononuclear cells Polymerase chain reaction Protease inhibitors Prevention of mother- to-child transmission of HIV HIV -1 polymerase gene Protease Regulator of Virion Ribonucleic acid Rev Response Unit Reverse transcriptase Reverse transcriptase inhibitors Retonavir Sendai virus Syncytium-inducing Saquinavir Rev RNA RRE RT RTI RTV SeV SI SQV xvii Tat TDF UNAIDS VeT VEEV Vif VLP Vpr Vpu VSV WHO ZDV/AZT Trans-Activator of Transcription Tenofovir Joint United Nations Joint Programme on HIV/AIDS Voluntary Counselling and Testing Venezuelan equine encephalitis virus Viral Infectivity Factor Virus Like Particles Viral Protein R Viral Protein U Vesicular stromatitis virus World Health Organization Zidovudine XVIII ABSTRACT Mother-to-child transmission (MTCT) of HIV -1 is responsible for infection of hundreds of thousands of infants every year. It is estimated that 600,000 newborns are infected yearly worldwide, with MTCT accounting for 90% of these infections. Human immunodeficiency virus (HIV) can be transmitted from mother-to-child at various stages of pregnancy including in utero and intra partum. A number of feasible and effective interventions to reduce MTCT among women of child bearing age are available. These interventions include prevention of primary HIV infection, avoiding unwanted pregnancies among HIV positive women, reduction of transmission from infected mothers to infants during pregnancy, labour, delivery, and breastfeeding through provision of voluntary counselling and testing (VCT) services, antiretroviral therapy (ART), safe delivery practices, and breast milk substitutes. However, these approaches are not always possible in resource-poor countries. The use of antiretroviral (ARV) drugs, in particular nevirapine, zidovudine and zidovudine/lamivudine combination, has been studied in both developing and developed countries. Although these studies have shown reduction in transmission of HIV, concerns regarding the development of drug resistant strains have been raised. The Ministry of Health in Kenya has implemented nevirapine regimen to reduce MTCT in the public health facilities. This study aimed to investigate drug resistance in an MTCT setting in Kenya. A total of 309 HIV seropositive pregnant women taking part in the prevention of mother to child transmission of HIV (PMTCT) programme in three hospitals, namely, South Nandi Hills, Kapsabet, and Kitale district hospitals were enrolled in this study. A structured questionnaire was used to collect demographic information. Venous blood was collected into vacutainer tubes containing EDTA as anticoagulant. The enumeration of T-Iymphocytes was carried out by flow cytometry and viral load was determined by nucleic acid amplification. The proviral HIV DNA extracted from peripheral blood mononuclear cells (PBMCs) was sequenced to determine the drug resistance associated mutations and HIV-I subtypes. The significance of associations was investigated by chi-square test and odds ratios. The HIV prevalence among the pregnant women was 6.7% (309 of 4638). The majority (85%) of the women visiting the antenatal clinic were not aware of their HIV status. Sixty percent (60%) of the pregnant women had a CD4 count of more than 350cells/mm . The HIV transmission rate was 6% (4 of 59 infants). Drug resistance associated mutations were detected as minor populations except in one mother-child pair where major populations were found. Nevirapine drug resistance was detected in 19.4% (7 of 36) and 100% (3 of 3) of the women and infants tested respectively. Even though the women had not been exposed to nucleoside reverse transcriptase inhibitors (NRTls), drug resistance associated mutations were detected in 8 mothers (22.2%) as minor populations. The major circulating HIV -1 subtype in North-Rift Kenya was identified as Al (50% and 71.8%) based on the env (C2V3) and pol (RT) regions respectively. Human immunodeficiency virus type 1 subtypes 0 (12.8%), C (10.3%), A2 (2.6%) and G (2.6%) were also detected, based on sequencing of the pol region. Drug resistance outcomes in mothers and infants should be considered as an important secondary end point in PMTCT assessment. CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Information The pandemic of Human Immunodeficiency Virus (HIV), the cause of Acquired Immune Deficiency Syndrome (AIDS), is clearly the defining medical and public health issue of our generation and ranks among the greatest infectious disease scourges in history (Fauci, 1999). According to estimates by the Joint United Nations Programme on HIV and AIDS (UNAIDS) and World Health Organization (WHO) (UNAIDS/WHO), approximately 2.3 million children under 15 years were infected with HIV in 2006, with greater than 95% of these infections occurring in resource- poor nations (UNAlDS/WHO, 2006). The vast majority of children with HIV acquire the infection through mother-to-child transmission (MTCT), which occurs in utero, during labour, delivery and while breastfeeding. In the absence of any intervention, the risk of MTCT is 15% to 30% in non-breastfeeding populations. In the setting of breastfeeding, the risk is 20% to 45% (De Cock et al., 2000). In societies where breastfeeding is the norm, MTCT accounts for about one-third of all transmission. As a result, the proportion of infants infected through MTCT is higher in such societies than in those where mothers with HIV infection can safely avoid breastfeeding. In the developed countries, the risk of MTCT has been reduced to below 2% (Dorenbaum et al., 2002) by employing interventions that include antiretroviral therapy which achieve undetectable viral loads before delivery (Watts et al., 2004) 2when administered to HIV infected women during pregnancy and labour, as well as to exposed infants during the first weeks of life; avoidance of breastfeeding; and delivery by elective caesarean section (CS) (The International Perinatal HIV Group, 1999). There are several drug regimens in use for reduction of MTCT. These include the long and short courses; single and multi-drug regimens. In 1994, a drug regimen using zidovudine (ZDV) was shown to reduce MTCT by two thirds in the absence of breastfeeding. But at an average cost of US $ 1,000 per pregnancy, this regimen was far too expensive for use in resource poor countries (Connor et al., 1994). In 1998, a study in Cote d' Ivoire showed that a simpler drug regimen, a one-month course of zidovudine late in pregnancy, could half the rate of transmission so long as the women avoided breastfeeding (Wiktor et al., 1999). In 1999, a study in Uganda showed that one dose of nevirapine to the mother at the onset of labour and a dose given to the infant within 72 hours of delivery was highly effective in reducing MTCT. This short course regimen could only cost US$4. There are other short course regimens involving zidovudine, or combination of zidovudine and lamivudine (PETRA ®, 2002). Despite the reduction of MTCT using anti-retroviral (ARV) drugs, the development of drug resistance has been reported (Jacks on et al., 2000; Beckerman, 2002). 1.2 Statement of the Problem The survival of people diagnosed with HIV /AIDS dramatically improves with access to highly active antiretroviral therapy (HAART). Similarly, the use of short course antiretroviral monotherapies and/or combination prevents hundreds of thousands 3paediatric infections per year. Nevirapine remains central to PMTCT and to combination antiretroviral treatment throughout much of the developing world. The use of single dose nevirapine (NVP) is the cornerstone of regimen recommended by the WHO to PMTCT among women without access to ARV treatment and those not meeting treatment criteria (WHO, 2006a, b). However, NVP resistance is detected in 20-69% of women (Eshleman et al., 2001; Jackson et al., 2000; Lee et al., 2005; Eshleman et al., 2005a; Shapiro et al., 2006) and 33-87% of infants (Eshleman et al., 2005b) after exposure to a single, peripartum dose ofNVP. Though the use of short course antiretroviral monotherapies may prevent hundreds of thousands of paediatric infections per year, the use of these drugs may be inducing drug resistant viruses that may render follow up treatment options against HIV in Africa limited. There are reports, however that drug resistant mutations induced by nevirapine are short term and viruses revert to wild type in the absence of antiretroviral drug pressure (Eshleman et al., 2001). Evidence to support these claims is limited. This is especially so in African countries where multiple subtypes of HIV co-exist. In order to guide treatment options for people infected with HIV/AIDS, National AIDS Control programmes in African countries are taking it as a priority to gather data on genotype and phenotype patterns of HIV isolates in populations being exposed to any form of antiretroviral prophylaxis. In 2002, the Kenya National AIDS Control Programme (NASCOP) began a program to provide nevirapine prophylaxis to HIV infected antenatal clinic attendees in public hospitals. The program involves voluntary counselling and testing (VCT), provision of nevirapine to HIV infected mothers and follow up of mother and child for a short 4time after delivery. The monitoring of HIV-I strains and anti-Hl V drug resistance studies have however not been carried out. The present study was designed to determine the genetic diversity among HIV infected antenatal clinic attendees and to establish if the development of drug resistance would be detected after nevirapine use to reduce the chances ofMTCT. 1.3 Rationale of the Study The use of combination of antiretroviral therapy has resulted in considerable improvement in the management of HIV-I infection (Sow et aI., 2007). The major obstacle to the effective management of HIV /AIDS and reduction of mother-to-child transmission is the emergence of drug resistant virus strains (Jackson et al., 2000). The side effects and the development of drug resistance are not well studied in developing countries. Most of the studies have been carried out in developed countries where the HIV subtypes are different from the ones circulating in Africa. Recent studies have showed that the prevalence of antiretroviral-resistant HIV -1 in the primary infected pregnant mothers is 20 and 25% using nevirapine and zidovudine respectively (Leroy et al., 2002; Welles et al., 2000). In December 2003, the World Health Organization (WHO) and the joint United Nations Programme on HIV and AIDS (UNAIDS) launched the "3 by 5" initiative, with the goal of having 3 million people on antiretroviral therapy (ART) in developing countries by the end of 2005. By December 2005, an estimated 1.3 million people from low and middle income countries had started treatment, with 810,000 of these living in sub-Saharan Africa (WHO, 2006c). In line with this 5initiative, Government of Kenya (GOK) committed to progressively deliver effective therapy to 95,000 patients by the end of 2005 and 140,000 by 2008 (MOH, 2005a). The dramatic rise in global funding for HIV and AIDS, together with reduced drug costs, improves the feasibility of providing antiretroviral therapy in settings with limited resources (Yazdanpanah et ai, 2005; WHO, 2003). As the cost of the antiretrovirals have become affordable, there is need to establish the level of drug resistance of circulating HIV c1ades in Kenya. 1.4 Null Hypothesis Vertical transmission of HIV in the presence of antiretroviral therapy is not due to transmission of drug resistant strains. 1.5 Objectives of the Study 1.5.1 General Objective To determine antiretroviral drug resistance associated mutations and genetic diversity among the HIV infected antenatal clinic attendees in Kitale, South Nandi Hills, and Kapsabet district hospitals. 1.5.2 Specific Objectives (i) To determine the prevalence of HIV among antenatal clinic attendees in the study area. (ii) To determine the HIV subtypes/c1ades circulating in the study area. (iii) To determine the level resistance to nevirapine. (iv) To determine the T-Iymphocyte profiles among antenatal clinic attendees. 61.6 Ethical Considerations Clearance to carry out the study was obtained from the KEMRI Scientific Steering Committee (SSC) and the Ethical Review Committee (ERC). Informed consent was obtained from the antenatal clinic attendees. In the case of the infants, the consent was obtained from the mothers or legal guardians. 1. 7 Limitations of the Study Under programme settings in this study, follow-up of HIV infected mother and her infant was poor since the use of incentives and resources for home visits were not available. The study relied on the goodwill of the participants to obey their antenatal appointments. It was not possible to verify if the nevirapine doses dispensed were taken as indicated. The HIV status of only 59 infants was determined whereas there were 309 HIV seropositive pregnant women recruited. Due to financial constrains, the molecular aspect of the study was based principally on the env (C2V3) and pol (RT) regions of the HIV. 7CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 The History and Origin of HIV The origin of AIDS and HIV has puzzled scientists ever since the illness first came to light in the early 1980s. For over twenty years it has been the subject of fierce debate and the cause of countless arguments, with everything from a promiscuous flight attendant to a suspect vaccine programme being blamed. The first cases of AIDS were described in the United States in 1981 (CDC, 1981). At this point, the term AIDS (Acquired Immune Deficiency Syndrome) was not used to describe this new unexplained immune deficiency syndrome. The syndrome had several names, including "gay syndrome", due to it being initially identified in homosexuals. The immune defenses ofthe patients were considerably weakened. Various pathogens including bacteria, viruses and parasites, which were not highly infectious under normal circumstances, took advantage of the condition to proliferate and cause serious and normally rare illness such as pneumocystis carinii pneumonia and Kaposi's sarcoma. Kaposi's sarcoma had been recognized as a rare tumor of blood vessels associated with aging. The appearance of these conditions in young men and previously healthy men was alarming. The development of AIDS in hemophiliacs who had received injections of factor VIII from pooled and concentrated human plasma and intravenous drug users suggested that AIDS might be caused by a virus. Several viruses were considered but it was not long before a new retrovirus was identified by independent groups as being associated with AIDS (Sonnabend et al., 1983). 8The first description of the virus causing AIDS, called LA V or "Lymphadenopathy Associated Virus" was by French researchers from the Pasteur institute (Barre- Sinoussi et aI., 1983). From early 1983, research intensified on this newly identified virus. A long period focusing on the characterization of the virus and the development of serological tests began, in parallel to research aiming to prove the link between the virus discovered and the AIDS disease. The characterization of th~ proteins making up the virus also began in 1983. The analysis of the proteins of the virus showed that LA V was totally different from human T-Cell Iymphotrophic virus type IIII (HTL V-I and HTLV -11) viruses. The American team of Robert Gallo (National Cancer Institute, United States) who had described the only human retrovirus known at this time, human T-Celllymphotrophic virus type I (HTLV -I) also isolated the virus from patients with AIDS and at risk of AIDS and termed it human T-Celllymphotrophic virus type III (HTLV-III) (Gallo et al., 1984). Another group of researchers also isolated the virus and termed it AIDS- related virus (Levy et al., 1984). The three names were used for the same virus until 1986 when the new virus was renamed Human Immunodeficiency Virus (HIV) (Coffin et al., 1986). In 1986, an antigenic variant of the virus was isolated in West Africa and hence the original virus was named HIV -1 and the variant designated as HIV -2 (Clavel et al., 1986). It is now thought that HIV came from a similar virus found in chimpanzees. It is also generally accepted that HIV is a descendant of a Simian Immunodeficiency Virus (SIV) because certain strains of SIV bear a very close resemblance to HIV-I and HIV -2. Human Immunodeficiency Virus type 2 for example corresponds to SIVsm, a 9strain of the Simian Immunodeficiency Virus found in the sooty mangabey, which is indigenous to western Africa (Marx et al., 1991). The more virulent strain of HIV, namely HIV-1, was until recently more difficult to place. Until 1999, the closest counterpart that had been identified was SIVcpz, the SIV found in chimpanzees. However, SIVcpz had certain significant differences from HIV. In February 1999 a group of researchers from the University of Alabama (Gao et al., 1999) reported that they had found a type of SIVcpz that was almost identical to HIV- 1. This particular strain was identified in a frozen sample obtained from a sub-group of chimpanzees known as Pan troglodytes troglodytes, which were once common in west-central Africa. 2.2 Classification of HIV Human Immunodeficiency Virus is a lentivirus. Lentiviruses (Latin, lentus means slow) are complex viruses distinguished by the presence of a vase or cone-shaped nucleoid, absence of oncogenicity, and the lengthy and insidious onset of clinical signs. Lentiviruses constitute a genus of the retroviridae family. Retroviruses (retro- from Latin, turning back) are RNA viruses that replicate via DNA intermediates using reverse transcriptase (RT). Other lentiviruses include those infecting cats (feline immunodeficiency virus), sheep (visna virus), goats (caprine arthritis-encephalitis virus), horse (Equine infectious anemia virus), cow (Bovine immune deficiency virus) and non-human primates (simian immunodeficiency virus). These enveloped RNA viruses produce characteristically slow, progressive infections. Lentivirus replication depends on the presence of an active RT responsible for the 10 transformation of the viral RNA genome into a proviral DNA copy that intergrates into a set of mRNAs that encode the viral proteins and progeny genomic RNA. Human Immunodeficiency Virus type 1 is phylogenetically close to SIVcpz, a commensal virus in chimpanzees, and probably arose as the result of a single transmission event from chimpanzees to humans (Gao et al., 1999), whereas HIV-2 is closely related to SIVmac, the etiological agent of simian AIDS, and to SIVsm, a commensal virus in Sooty Mangabey monkeys. The relationship of HIV and SlV is shown in Figure 2.1. Q)tOUP ~...... F ~D H=r G JA __ •.• C SIVMM HIV-2. S\"'~ Figure 2.1 Phylogenetic tree of the SIV and mv viruses (Adapted from Kuiken et aI., 1999) 11 The viruses depicted in the HIV -SIV phylogenetic tree are as follows: (a) Human i. HIV-I M (Main) group, including reference strains from subtypes A-J. Group M is responsible for the pandemic. ii. HIV-I 0 (Outlier) group, most commonly found in West Africa iii. HIV -I N (Not-M, Not-O) group, found in small number of individuals in West Africa. iv. HIV-2 subtypes A and B (b) Simian i. SIVcpz from chimpanzee Pan troglodytes troglodytes (P.t.t.): ii. SIVcpz.GAB, SIVcpz.US, and SIVcpz.Cam3 iii. SIVcpz from chimpanzee Pan troglodytes shweinfuthii (P.t.s.): SIVcpz.ANT iv. SIV African Green Monkey (SIVagm): v. Tantalus (TAN): SIVagm.TANI vi. Vervet (VER): SIVagm.VERTYO, SIVagm.VERAGM3, SIVagm.VER9063, SIVagm.VER155 vii. Grivet (GRI): SrVagm.GRI677 viii. Sabaeus (SAB): SIVagm.SABIC ix. SIV Sooty Mangaby (SIVsm) (also found in macaques): SIVsm.mac251, SIVsm.smm9 x. SIV L'hoest: SIV.LHOEST xi. SIV Mandrill: SIV.MNDGBI 12 xii. SIV Sun: SIV.SUN 2.3 Diversity of HIV Human Immunodeficiency Virus is a highly variable virus due to rapid mutation. This results in many different strains of HIV, even within the body of a single infected person. There are two types of HIV: HIV-1 and HIV-2. Both types are transmitted by sexual contact, through blood, and from mother to child, and they cause clinically indistinguishable AIDS. However, HIV-2 is less easily transmitted, and the period between initial infection and illness is longer in the case of HIV-2. Worldwide, the predominant virus is HIV -1. The relatively uncommon HIV -2 type is concentrated in West Africa and is rarely found elsewhere (Peeters et al., 1991). The strains of HIV -1 are classified into three groups: the "major" group M, the "outlier" group 0 and the "new" group N. These three groups represent three separate introductions of SIV into humans. Members of HIV -1 group 0 have been recovered from individuals living in Cameroon, Gabon, and Equatorial Guinea with the genomes sharing less than 50% identity with group M viruses (Subbarao & Schochetman, 1996). The more recently discovered group N HIV -1· strains have been identified in infected Cameroonians (Simon et al., 1998). More than 90% of HIV-1 infections belong to HIV -1 group M. Within group M there are nine genetically distinct subtypes (or c1ades) ofHIV-l. These are subtypes A, B, C, D, F, G, H, J and K (Tatt et al., 2001). The HIV-1 population present within an individual can vary from 6% to 10% in nucleotide sequences. Human Immunodeficiency Virus type 1 isolates within a c1ade may exhibit nucleotide distances of 15% in gag and up to 30% in gp 120 coding sequences. 13 High rates of genetic recombination are a hallmark of retroviruses. A significant fraction of the HIV-I group M global diversity includes interclade viral recombinants. These HIV -1 recombinants are found in geographic areas such as Africa, South America and Southeast Asia where multiple subtypes co-exist and account for 10% of circulating HIV -1 strains. The HIV -1 recombinants are known as "circulating recombinant forms" or CRFs. Most HIV -1 recombinants have arisen from Africa and a majority contains segments originally derived from clade A viruses (Gao et al., 1998). In Thailand, for example, the predominant circulating strain consists of a clade A gag plus pol gene segment and a clade E env gene (CRFO 1_AE) (Carr et al., 1996). Further, the subtypes are classified into sub-subtypes such as AI, A2, A3, FI and F2. The classification of HIV strains into subtypes, sub-subtypes and CRFs is a complex issue and the definitions are subject to change as new discoveries are made. Figure 2.2 illustrates a proposed classification of HIV -1 and HIV -2 by Kiptoo, unpublished, 2008. 14 CRFOI-34 HIY 1__ 1_-\ my I mYI~1--1- I Subtypes (A-F)_1 N 0 ISubtypes (A-D, F-H, J&K) ISub-subtypes (A I, A2, A3, A4, Ft, F2) Figure 2.2 Diversity ofHIY (Kiptoo, unpublished) 2.4 Biology of HIV Infection A quintessential property of HIY -1 and the other primate lentiviruses is to sequentially use CD4 and a second cellular receptor during entry into susceptible cells. The main cellular targets for HIY -1 are the CD4+ T-helper/inducer subset of Iymphocytes, CD4+ cells of macrophage lineage and some populations of dendritic cells. Many of the early HIY -1 isolates were classified on the basis of their replicative, cytopathic and cell tropic properties (Collman et al., 1989). Primary HIY -I isolates have been classified into different phenotypic groups according to distinct in vitro properties. There are several classification systems which have been used to define phenotypes. The initial system defined primary isolates as macrophage (M)-tropic or T-cell-line tropic. The M-tropic HIY -I isolates are those that are able to replicate in cultures of monocyte-derived macrophage (Collman et al., 15 1989). These M-tropic isolates are frequently identified shortly after infection with HIV-l. The T-tropic isolates are obtained late in the disease course and do replicate in continuous human CD4+ T-cell lines. The second system categorizes isolates as either syncytium-inducing (SI) or non- syncytium-inducing (NSJ) on the basis of whether they form syncytia in MT-2 cells (Schuitemaker et aI., 1992). The third system defines viruses as slow/Iow or rapid/high depending on their growth kinetics in culture (Fenyo et al., 1988). These classifications are often used interchangeably, but they are not synonymous. The finding that chemokine receptors have a critical role in the cellular entry of HIV- 1 has led to a new classification of HIV -1 according to co-receptor usage. A major determinant of HIV -1 tropism (phenotype) lies at the level of virus entry into target cells, which in turn is governed by the expression of co-receptors in conjunction with CD4, either CCR5 or CXCR4, or both. The use of CXCR4 is the defining feature of viruses that form syncytia in T -cell lines and CCR5 is a property of NSI, M-tropic viruses and many T-tropic primary isolates can use both co-receptors. Those that use CCR5 are termed as R5 viruses and CXCR4 are termed X4. The isolates that use both co-receptors with comparable efficiency are referred to as R5X4 (Berger et al., 1998). 2.5 Genomic Organization of HIV The genome of HIV-l contains three major genes (flanked by long terminal repeats) that are essential for replication mechanisms. These genes are group antigen gene (gag), polymerase gene (pol) and envelope gene (env). The gag gene encodes the precursor protein 55 (p55), which is further cleaved by the viral protease to structural 16 proteins p24, p17, p7 and p6. The pol gene encodes precursor protein which is similarly cleaved by protease to three viral enzymes: protease (pl I), RT (P66/51), and intergrase (P32). In addition, the HIV-I genome has other six accessory genes, known as trans-activator of transcription (tat), regulator of virion (rev), negative regulatory factor (ne/), viral infectivity factor (vi/), viral protein R (vpr) and viral protein U (vpu) which codes for proteins that control the ability of HIV to infect a cell, produce new copies of virus, or cause disease (Figure 2.3). Gene Figure 2.3 Genomic organization ofHIV (Adapted from Larder et aI., 1998) The intergrase enzyme integrates the DNA produced by reverse transcriptase into the host's genome. The protease enzyme cleaves the proteins produced by the gag and pol genes into separate functional units. The tat consists of between 86 and 101 amino acids depending on the subtype (Jeang, 1996). Tat helps HIV reproduce by compensating for a defect in its genome: the HIV RNA initially has a hairpin- structured portion which prevents full transcription occurring. However, a small 17 number of RNA transcripts will be made, which allow the tat protein to be produced. tat then binds to and phosphorylates cellular factors, eliminating the effect of the hairpin RNA structure and allowing transcription of the HIV DNA (Kim & Sharp, 2001). The protein appears to play a role in the HIV disease. The protein is released by infected cells in culture and is found in the blood of HIV -1 infected patients (Xiao, 2000). Tat can be absorbed by cells that are not infected with HIV, and can act directly as a toxin producing cell death via apoptosis in un infected bystander T cells, assisting in progression towards AIDS (Campbell et al., 2004). The rev protein allows the fragments of HIV mRNA that contain a rev response unit (RRE) to be exported from the nucleus to the cytoplasm. This mechanism allows a positive feedback loop to allow HIV to overwhelm the host's defenses, and provides time-dependent regulation of replication (a common process in viral infections) (Strebel, 2003). The expression of ne! early in the viral life cycle ensures T cell activation and the establishment of a persistent state of infection. Ne! also promotes the survival of infected cells by downmodulating the expression of several surface molecules important in host immune function. These include major histocompatibility complex class I (MHC I) and MHC 11present on antigen presenting cells (APCs) and target cells, and CD4 and CD28 present on CD4+ T cells. One group of patients in Sydney was infected with a nefdeleted virus and took much longer than expected to progress to AIDS (Learmont et al., 1999). The vif, a 23-kilodalton protein, is important for viral replication, though its exact role is yet unclear. However, it is thought that vifhelps the virus to infect other cells after it leaves a host cell. Vif appears to be involved in determining how the RNA genome 18 and gag protein bind to each other, and inhibits a cellular protein that modifies the cDNA. Vif is involved in preventing the cellular protein, termed APOBEC3G, from entering the virion during budding from a host cell. In the absence of vif, APOBEC3G causes hypermutation of viral genome, rendering it dead-on-arrival at the next host cell. The APOBEC3G protein is thus a host defence to retroviral infection which HIV-l has overcome by the acquisition ofvif(Strebel, 2003). The vpr, a 10-kilodalton protein, regulates nuclear import of the HIV -I pre- integration complex, and is required for virus replication in non-dividing cells. Vpr also induces cell cycle arrest in proliferating cells, which may result in immune dysfunction (Bukrinsky & Adzhubei, 1999). Human Immunodeficiency Virus type 2 contains both a vpr protein and a related (by sequence homology) vpx protein (viral protein X). Two functions of vpr in HIV -1 are split between vpr and vpx in HIV -2, with the HIV -2 vpr protein inducing cell cyIce arrest and the vpx protein required for nuclear import. A distinguishing feature of HI V-I is the presence ofvpu which is absent in HIV-2 and SIV. Vpu is a multimeric, 81-am ino-acid, integral membrane phosphoprotein containing an N-terminal transmembrane anchor sequence and an approximately 50- amino-acid cytoplasmic domain (Maldarelli et al., 1993). Vpu is involved in viral budding, enhancing virion release from the cell (Callahan et al., 1998). 2.6 The Structure of HIV All members of the Lentivirus family ofretroviruses including HIV-l, HIV-2 and SIV share numerous structural and molecular features. These viruses have an RNA 19 genome and two associated molecules of RT that catalyze the reverse transcription of viral RNA into DNA (Wilhelm & Wilhelm, 2001). Human Immunodeficiency Virus is a retrovirus of a non-complementary pair of single coding or positive strands of RNA enclosed within an inner, nucleocapsid, and protein core surrounded by a lipid bilayer envelope. Projecting from this are around 72 little spikes, which are formed from the proteins gp120 and gp 41. Just below the viral envelope is a layer called the matrix, which is made from the protein p17 (Figure 2.4). The viral envelope contains cellular proteins acquired during virus budding, including intracellular adhesion molecule (ICAM), p2-microglobulin and the human major histocompatibility complex (MHC) class I and II molecules (Rizzuto & Sodroski, 1997; Fortin et al., 1997). The viral core (or capsid) is usually bullet-shaped and is made from the protein p24. Inside the core are three enzymes required for HIV replication called reverse transcriptase, integrase and protease. Also held within the core is HIV's genetic material, which consists of two identical strands of RNA. 20 Spike of envelope glycoprotein (gp120) Reverse transcriptase Figure The Human Immunodeficiency Virus (Adapted from Larder et al., 1998) 2.7 Life Cycle of HIV-l The HIV replication starts with attachment to the target cell. The attachment involves interaction between the glycoprotein gp120 and CD4 receptor on the target cell. This provokes conformational changes in gp120 which exposes a region of gp120, the V3 loop, which binds to a cytokine receptor on the target cell, such as CCR5 or CXCR4 depending on the strain ofHIV. The stages of the HIV-l life cycle are summarized in Figure 2.5. 21 HIVvirion Fusion of HIV membrane with cell membrane; entry of viral genome into cytoplasm Reverse transcriptase- mediated synthesis of proviral DNA I I I I I I I I I I I I I I:,--------, " Cytokine activation " of cell; transcription I of HIV genome; ~ transport of spliced and unspliced RNAs to cytoplasm genome Integration of provirus into cell genome • ~"" HIV core ~ \ "\. structure 0" Synthesis of HIV. proteins; assembly~ of virion core structure~-- .HIV proteins Expression of gp120/gp41 on cell surface; budding of mature virion Cytoplasm Nucleus Figure 2.5 The life cycle ofHIV-l (Adapted from Abbas et al., 2000) The conformational change in gp120 exposes a portion of the gp 41. A ~.~\4~ within gp41 causes the fusion of the viral envelope and the host~ envelope, allowing the capsid to enter the target cell. The exact mechanism by which gp41 causes the fusion is still unknown (Piatak et al., 1993; Kahn & Walker, 1998). Once HIV has bound to the target cell, the HIV RNA and various viral enzymes are injected into the cell. Once the viral capsid has entered the cell, the RT liberates the single-stranded (+) RNA from the attached viral proteins and copies it into a negatively sensed viral cDNA of 9 kb pairs (Figure 2.5). 22 The integration of the proviral DNA into the host genome is catalyzed by the integrase enzyme. The HIV provirus may remain transcriptionally inactive for months or years. This is the latent stage of HIV infection (Zheng et al., 2005). Initiation of HIV gene transcription in T cells is linked to physiological activation of the T cell by antigen or cytokines. In the presence of optimal signals to initiation transcription, few if any HIV messenger RNA (mRNA) molecules are actually synthesized because transcription by mammalian RNA polymerase is very inefficient and the polymerase complex usually stops before the mRNA is completed. The tat protein binds to the nascent mRNA and increases the process of RNA polymerase by several hundred-fold, which allows transcription to be completed to produce a functional viral mRNA. Then the messenger RNA is transported outside the nucleus, and is used as a blueprint for producing new HIV proteins and enzymes. The final step of the viral cycle, assembly of new HIV -1 virions, begins at the plasma membrane of the host cell. The env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by protease and processed into the two HIV envelope glycoproteins gp41 and gp120. These are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. The gag (p55) and gag-pol (p 160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell. Maturation either occurs in the forming bud or in the immature virion after it buds from the host cell. During maturation, HIV proteases (proteinases) cleave the polyproteins into individual functional HIV proteins and enzymes. The various structural components 23 then assemble to produce a mature HIV virion (Gelderblom, 1997). The newly matured HIV particles are ready to infect other healthy cells. 2.8 Global Epidemiology of HIV It was estimated that between 34.1 and 47.1 million people worldwide were living with HIV by December 2006 (UNAIDS/WHO, 2006). It was estimated that during 2006, between 3.6 and 6.6 million people were newly infected with HIV and between 2.5 and 3.5 million people with AIDS died. Sub-Saharan Africa remains the epicenter with 23.8 to 28.9 million people living with HIV at the end of 2006 (UNAIDS/WHO, 2006). The epidemic is not homogeneous within regions. Even at the country level there are wide variations in infection levels between different areas, sexes and socio-economic status. The steepest increases in infection have occurred in Eastern Europe and Central Asia (25% increase to 1.6 million) and East Asia (UNAIDS/WHO, 2006). In Kenya, the first AIDS case was recognized in 1984 and since that time, HIV and AIDS still remains a huge barrier to social and economic development (Obel et al., 1984). The National AIDS Control Council (NACC) was established in 1999 to spearhead the national response, and to serve as the Government of Kenya's coordinating body. With the establisment of this body, the national repsonse has accelerated rapidly. Across the country, prevention, treatment and mitigation programmes are being scaled up. 24 In 2003, the first national HIV prevalence survey was carried out, which estimated that 7% of adults aged 15-49 years in Kenya were infected (CBS, 2003). At the end of 2003, the country prevalence was estimated at 6.7% with arange of 4.7%-9.6%. Adults living with HIV were 1.1 million, range 0.76-1.6 million. It was estimated that 150,000 people (89,000-200,000) had died due to AIDS in Kenya (UNAIDS/WHO, 2004). 2.9 Natural History of HIV The pathogenesis of HIV is complex and multifactorial (Fauci, 1993). The probability of transmission with each sexual encounter with HIV -1 is small (0.0001) and is related to CD4+ T cells, in part and to the dose of HIV inoculum (Gray et al., 2001). Infection of CD4+ T cells is mediated by gp120 and gp41, the T-'cell receptor CD4 and the chemokine coreceptors CCR5 and CXCR4 (D'Souza & Harden, 1996). Primary infection with HIV may be accompanied by transient illness similar to glandular fever, malaise, muscle pains, swollen lymph nodes, sore throat and rash (Schacker et al., 1998). There is transient depletion of peripheral CD4+ T cells, expansion of CD8+ T cells and high plasma levels of HIV particles. From this acute stage until late stages of infection, the activated CD4+ T cells produce billions of virions daily. Once the acute infection is established, a cellular immune response to HIV -1 is generated that partially controls viral replication (Koup et al., 1994). In addition to the general loss of CD4+ T cells, the virus preferentially infects and eliminates HIV-I-CD4+ T cells, which compromises the virus specific immune response. In addition, the error-prone viral replication process generates mutants that can escape neutralizing antibodies and virus-specific cytotoxic T lymphocytes (CTLs) 25 in HIV-1 (Richman et al., 2003). This contributes to the continued difficulties that the immune system encounters in containing the infection. In advanced stages of HIV infection, non-specific constitutional symptoms such as fever, night sweats, diarrhoea, weight loss and skin infections such as oral candidiasis, shingles and recurrent anogenital herpes simplex occur. These conditions signal the development of serious opportunistic infections and tumours, which constitute AIDS when the CD4+ T-cell count is less than 200cells/mm3. The median period between infection with HIV and the onset of clinically apparent disease is approximately 10 years (pantaleo et aI., 1993) (Figure 2.6). !Acuto HIV syndt'ome Wide diSSemination of virus Seeding of lymphoid orqans er>E E 800 -~ 4V 700 -~•..c; 600 -~ 0Q 500III~ Q 4000.J: Q. 300E3- 200P- + ~ 0 opportuniSliC/, diseases I r. ConstitUtiOnalt • ••.• symptoms I ~ ';Aa.• t ~ '\ •... .•........It.~ ~ .•.. . .•...•.~~.~.~,.-.... . "'- Clinical Latemy S 9 10 111 2 3 <1 5 6 Years 0 1075-e :tQ.l l/I 0 1/256 .~ 01JCii' 11128 <; - 10S IJl=;- 1J 35 years. The majority (34.8%) were women aged 21-25 years (Table 4.2), while the minority (4.4%) were women aged above 35 years. 60 Table 4.2 Distribution of ANC attendees by age Age group (yrs) Number (N) Proportion (%) <21 1310 28.5 21-25 1598 34.8 26-30 990 21.5 31-35 495 10.8 >35 202 4.4 Total 4595 100 4.1.3 Educational Status Most (69.4%) of the respondents had primary school education with 3.8% having tertiary education (Table 4.3). Table 4.3 Distribution of ANC attendees by educational level Level of Education Number (N) Proportion (%) Primary 3157 69.4 Secondary 1219 26.8 Tertiary 173 3.8 Total 4549 lOO 4.1.4 Marital Status Of the 4,550 ANC attendees who had their marital status recorded, 81.9% were in a monogamous relationship, 7.3% in polygamous, 10.8% were single, while 0.3% were widowed (Table 4.4). 61 Table 4.4 Distribution of ANC attendees by marital status Marital status Number (N) Proportion (%) Single 491 10.8 Monogamous 3713 81.6 Polygamous 334 7.3 Widowed 12 0.3 Total 4550 100 4.2 mv Prevalence 4.2.1 Background Characteristics of HIV Seropositive ANC attendees A total of 309 ANC respondents were HIV seropositive in Kitale, Kapsabet and South Nandi Hills district hospitals. Among the HIV seropositive, Kitale had the highest proportion (68.6%) followed by Kapsabet (18.1 %), and 13.3% from South Nandi Hills (Table 4.5). Table 4.5 Distribution of HI V Seropositive ANC attendees by hospital Hospital Number (N) Proportion (%) Kapsabet (KAP) 56 I8.! Kitale (KTL) 212 68.6 South Nandi Hills (SNH) 41 13.3 Total 309 100 Majority ofthe ANC attendees (85.1 %) did not know their HIV status prior to visiting the clinic for antenatal care. Majority of the HIV seropostive ANC attendees (97.1 %) were not on anti-retroviral therapy (Table 4.6). 62 Table 4.6 Characteristics of HI V Seropositive ANC attendees Characteristics Category Number (N) Proportion (%) HIV status known No 263 85.1 Yes 46 14.9 Used antiretroviral drugs No 299 97.1 Yes 9 2.9 4.2.2 HIV Prevalence by Age Data on the age specific HIV prevalence among ANC attendees was analysed and the results are shown in Figure 4.1. The 31-35 age group had the highest (8.5%) HIV prevalence and the least (2.5%) was among mothers aged more than 35 years. <21 21-25 26-30 Age group (years) 31-35 >35 9 8 - 6.6 6.1 r----,..-- 4.9 r-- 2.5 r--;:o ,g 85 ~ ~7 Cl)g 6 Cl) iU 5>e 4Cl. ~ 3 III> 2:I: 1 o Figure 4.1 HIV Prevalence by age group 4.2.3 HIV Prevalence by Marital Status Widowed ANC attendees had the higher (16.7%) HIV prevalence followed by polygamous, monogamous and single with 14.1%, 5.3% and 4.1%, respectively 63 (Figure 4.2). There was a significant relationship between HIV status and marital status. Women in a polygamous relationship were therefore more likely to be HIV infected as compared to those in a monogamous relationship (pI!! 8c. ~ 6 1/1> 4~ 2 o Single Monogamous Polygamous Marital status Widowed Figure 4.2 HIV Prevalence among ANC attendees by marital status 4.2.4 HIV Prevalence by Level of Education The highest HIV prevalence (6.3%) was recorded among ANC attendees who had attended secondary schools followed by those with primary and tertiary level of education (6% and 5%, respectively) (Figure 4.3). However, there was no significant relationship between HIV seropositivity and the level of education (p=O.653 and p=0.469 for secondary and tertiary, respectively). FNVATTIIINIUI=R~ITV I tRDAD\ 64 7 6 6.3 5 10 .~ Figure 4.3 HIV Prevalence among ANC attendees by level of education o Primary Secondary Level of Education Tertiary 4.3 Infant diagnosis, CD4 counts, Viral load, mv subtypes, and Drug resistance 4.3.1 Infant Diagnosis Using PCR The HIV status of 59 infants was determined using an in-house PCR using primers specific for the HIV intergrase enzyme. Four infants (6.8%) were HIV infected. The PCR products were positive when a 297 base pair band size was visualized (Plate 4.1). 65 '- 100bp molecular marker KTL254M KTL254C SNH072M SNH072C KTL198M KTLl98C KTL270C KTL253C KTL202C KTL255C KTLl41C ~KTL252M ~KTL252C jKTL256C Negative control Positive control Key: M-mother, C-child, KTL-Kitale, SNH- South Nandi Hills, KAP-Kapsabet Plate 4.1 A PCR gel showing amplification of the pol-Intergrase gene (297bp) 4.3.2 CD4 counts The CD4 count was available for 278 ANC attendees. The mean CD4 count was 466 cells/mm" with a range of 9-2000 cells/mm3. The CD4 counts were further categorised into three groups «200, 200-349 and >350 cells per cubic millilitre) based on the WHO CD4/disease staging categories. The majority of the antenatal clinic attendees had >350 cells/mm' (60.07%) followed by 200-349 cells/mm" and <200 cells/mm' with 25.9 and 14.03% respectively (Table 4.7). 66 Table 4.7 T-Iymphocyte (CD4) count among ANC attendees CD4 (cells/mm3) Number (N) Proportion (%) <200 200-349 >350 Total 39 72 167 278 14.03 25.90 60.07 100 4.3.3 Viral Load Profiles and PCR amplification The viral load was available for 56 ANC attendees (Table 4.9). The mean viral load was 57,524 IU/m!. When the viral loads were transformed to 10gIO,the mean was 3.79 with a range of2.1-6.2. The majority of the women (69.64%) had a viral load ~1000 IU/ml (Table 4.8). Table 4.8 Viral load among ANC attendees Viral load (IU/ml) Number (N) Proportion (%) <50 ::;500 <1000 ~1000 Total 6 5 6 39 56 10.71 8.93 10.71 69.64 100 The PCR amplicons with bands of 550bp and 697bp for env and pol genes, respectively, were considered positive for HIV (Plates 4.2 and 4.3). After cloning, the presence of the insert was confirmed by PCR and gel eletrophoresis (Plate 4.4). 67 lOObp molecular marker SNH004M SNH003M SNH002M SNH005M SNH008M SNH009M SNHOIOM KAP003M KAP004M KAPOOIM KAP005M SNHOIIM SNH018M SNH017M Negative control Positive control "" Key: M-mother, C-child, KTL-Kitale, SNH- South Nandi Hills, KAP-Kapsabet Plate 4.2 A PCR gel showing amplification results of the env (C2V3) gene (550bp) 68 lOObp molecular marker KTL202C KTLl41M SNH072M SNH072C KTL198M KTLI98C KTL253M KTL253C KTL255M KTL255C KTL254M KTL254C KTL252M KTL252C Positive control Negative control Key: M-mother, C-child, KTL-Kitale, SNH- South Nandi Hills Plate 4.3 A peR gel showing amplification results of the pol-RT gene (697bp) 69 lOObp molecular marker SNHOO1M-Clone- I SNHOOIM-Clone-2 SNHOO1M-Clone-3 SNHOO1M-Clone-4 SNHOOIM-Clone-5 SNHOOIM-Clone-6 SNHOO1M-Clone-7 SNHOOIM-Clone-8 SNHOOIM-Clone-9 SNHOO1M-Clone-l 0 SNHOOIM-Clone-ll SNHOOIM-Clone-12 SNHOOIM-Clone-13 SNHOOIM-Clone-14 SNHOOIM-Clone-15· SNHOOIM-Clone-l 6 Key: M-mother, SNH- South Nandi Hills Plate 4.4 A PCR gel showing the pol-RT gene (697bp) PCR product insert Out of 45 samples from the mothers, 37 were successfully amplified using primers for the RT region. The relationship between amplification and viral load is shown in Figure 4.4. 70 la Q. 15EcaVI-o 10oz • Unsuccessful o Successful 25 20 15 <50 ~500 <1000 2:1000 Unknown Viral load (IUlml) Figure 4.4 Relationship between PCR amplification and viral load 4.3.4 HIV Subtypes Based on the HIV-l envelope Region (C2V3) ••• Thirty sequences based on the env region (C2V3) were successfully analysed. In order to deduce the subtypes, the sequences were aligned with the known HIV -1 subtypes in the Los Almos gene bank (Figure 4.5). Among these, 15 were subtype Al (50%),60 (20%) and 9 G (30%) (Table 4.9). 71 SIVcpzGAB.NUC SNH003M AF355330fAFJ553 1000 AJ4907491H1M4907491G 472 SNHOl5M SNI 793 •.... , S718 ,999 998 SN•.... 785 899 ~935 SNH SNH SNHOIIM - ~ m""""791 775 SNH052M 0 U61880j94TZI 6041DfTZ1 381 970 AY6697501AY6697501D1UG S HOl7M 1000 S H007M 965 SNH013M "-- KTL081M =79H· 701 SNHOl9M 908 SNH034M KAP007M I\.TL0b8M•••• 562 __.Jc,c S, H033M Lt530 SNH044M KTL069M ~ KAPOO6ML.- A 204 AYI753251AYI753251AllKEI 102 958 AI.KE.94.Q23 45 L........f498 KAP002M AF457068jKNHI207IA1 IKE J.......t351 KTL067M KAPOOIM KTL082M ~ SNH014M 165 AF457063jKNHI0881AllKEl 660 KAP003M 0.1 - Key: Msmother, KTL-Kitale; SNH-South Nandi Hills; KAP-Kapsabet Study sequences are in red font and references in black font JOtGICMI fTD ~012M NIIOJOM H026M SNflOO5M S, 'H002M 006 1 OOIM Figure 4.5 Phylogenetic tree based on a part of the env-C2V3 gene (550bp) G 72 Table 4.9 Demographic factors and HIV subtype data for 30 ANC attendees Study No. Age (yrs) Marital status mv Subtype SNH003M SNH015M SNH012M SNH010M SNH005M SNH026M SNH002M SNH006M SNH001M SNH011M SNH018M SNH052M SNH017M SNH007M SNH013M SNH019M SNH034M KTL081M KTL067M KAP001M KTL082M SNH014M KAP007M KAP002M KAP003M KTL068M KTL069M SNH033M SNH044M KAP006M Hospital SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH SNH KTL KTL KAP KTL SNH KAP KAP KAP KTL KTL SNH SNH KAP 17 23 27 20 26 17 17 28 N/A 22 18 23 35 32 30 16 25 21 18 25 16 22 32 23 N/A N/A 17 29 24 N/A Married Married Married Married Married Married Married Married Married Married Married Married Married Married Married Single Married Married Single Married Married Married Married Married Married Married Married Married Married Married G G G G G G G G Goooo oo A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 N/A: Not available; KAP: Kapsabet; SNH: South Nandi hills; KTL: Kitale 4.3.5 HIV Subtypes Based on the pol Region (RT) A total of 39 sequences (36 and 3 from mothers and infants respectively) based on the pol region (RT) were successfully analysed. In order to deduce the subtypes, one clone from each sample was aligned with the reference strains in the Los Almos gene data bank and analysed phylogenetically (Figure 4.6). Among these, 28 were subtype Al (71.8%),5 D (12.8%), 4 C (10.3%), 1 A2 (2.6%) and 1 G (2.6%) (Table 4.10). C]~-Tc CPZ.GA.-.CPZGABG.IN.95.95IN21068C.BW.96.96BW0502KTLl34M-3G.BR92.92BR025KTL255M-2 .----K:O::TL=-Ef!JI6.EfH2220 L----C KTL252M-11----- AC.TZ. AY734554 G.SE.93.SE6165 lt.-C===~G.BE.96.DRCBLG.NG.92.92NG083 [~~=02~AG.CM.97.97CM MP807 G02 AG.SE.94.SE7812 02 AG.FR91 DJ264 02 AG.NG.-.mNG .----- G.Fl.93.HH8793 12 1.•..----- KTL273M-2 '"'7'"'"1"-1--- KTL259M-17L.;.:~{ll'l~ A2.UG. AY444195.A2.KE.DQ136741rr===;::::~::K:T~LO~I~7M;-;2~~; KTL068M-2D.CD.84.84ZR085--t=>... D.CD.83.ELI 4~:;:::::::·C~D;i~~3 C;·W~:fz.96.96TZBF071 DKTLI88M-1KTL205M-1D.UG.94.94UGI14 '------ 10CD.TZ.96.96TZ BF061 D.UG.AF388105 r~~~~~~~~~ KTL264M-ISNHOOIM-IKTLI98M-2KTL260M-101 AE.CF.90.9OG.F11697 ~ ~~~~~O~I ~AE~'CF'90'9OG.F402KTL25 1M-IAI.UG.85.U455 KTL074M-I KTL073M-12 KTL088M-I ,----- KTLlI7M-1 '----- KTL265M-1 73 A2 AF457063 1I 1~~~~A~I~'SJE.94.SE7253AI.UG.92.92UG037KTL253M-1 KTL112M-7 A 1KTL268M'{) KTL202M-17KTL27IM-2 KTL141M-31 SNH03IM-1KTL067M-3KTL063M_4 ~ ===~KTL210M-17AI.KE.94.Q2317 KTL272M·I I'----{]Qi:&r~~~6if~LI P-----KTL249M-1 .---- KTL I85M-2L- __ -{l!~TL223M-4 T~~.gM-1'-----;w