i DRUG RESISTANCE MUTATIONS IN HIV PATIENTS ON ANTIRETROVIRAL THERAPY IN GHANA By EVELYN YAYRA AFUA BONNEY This thesis is submitted to the University of Ghana, Legon in partial fulfillment of the requirements for the award of PhD Biochemistry degree JUNE 2013 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION I hereby declare that this thesis is a result of my own research work carried out under the supervision of Professors Sammy Tawiah Sackey, William Kwabena Ampofo and Alexander Kwadwo Nyarko and that references made to other people’s work have been duly acknowledged. I further declare that this work has not been submitted in part or whole to any other Institution for the purpose of acquiring a degree. ----------------------------------------------------------- EVELYN YAYRA AFUA BONNEY (Author) ----------------------------------------------------------- PROFESSOR SAMMY TAWIAH SACKEY (Supervisor) ----------------------------------------------------------- PROFESSOR WILLIAM KWABENA AMPOFO (Supervisor) ------------------------------------------------------------- PROFESSOR ALEXANDER KWADWO NYARKO (Supervisor) University of Ghana http://ugspace.ug.edu.gh iii DEDICATION For all that she was to me; I dedicate this thesis to the memory of my late mother Letitia Afua Bertha Kwame University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENTS My first and most profound gratitude goes to the Almighty God for His love, grace, mercy and faithfulness to me. He made it possible in every way for me to undertake and complete this work. Glory to His Name! I am very grateful to my supervisors Prof. Sammy T. Sackey of the Department of Biochemistry, Cell and Molecular Biology; Prof. William K. Ampofo of the Virology Department, Noguchi Memorial Institute for Medical Research and Prof. Alexander K. Nyarko of the School of Pharmacy, all of the University of Ghana, for their immense support, expert guidance and co-operation throughout the work. Their invaluable contributions, including financial support, helped greatly to improve the work. I owe a lot of gratitude to my dear husband, Dr. Joseph Humphrey Kofi Bonney for his support, sacrifices, co-operation and guidance throughout the work. I also appreciate deeply the co-operation and sacrifices of my lovely daughters Josephine Ewurabena Mawulorm Bonney and Lydia Mawuyram Baaba Bonney. I am also grateful to my extended family; my father James H. K. Ugly-Kwame and mother Letitia A. B. Kwame (of blessed memory), my sister Comfort Akorfa Kwame and my nieces Dzorgbenyuie Kottoh and Edzesim Adatse, who were very supportive and helpful to me. I thank the patients who consented for me to use their blood samples for the study and the staff at Korle Bu, St. Martins de Porres, Atua Government and Kumasi South hospitals who helped me with sample collection. I am particularly grateful to Professor Margaret Lartey, Dr. Ernest Kenu and Adjoa Obo-Akwa of the Fevers Unit, Korle Bu Teaching Hospital. I am grateful to the Head, staff and students of the Department of Biochemistry, Cell and Molecular Biology, University of Ghana, for all their support and co-operation during the course of my studies. I thank the Director of Noguchi Memorial Institute for Medical Research (NMIMR) for allowing me to carry out my research in the Institute and the Transport Unit of NMIMR for their immense help during sample collection. I thank the Head (Prof. W. K. Ampofo) and members of the National Influenza Centre (NIC) for their support with transportation during sample collection. The Head and entire staff of the Virology Department deserve my deep appreciation for providing an enabling environment for me to work in. The following however deserve a special mention for their expert technical support: Ivy Asantewaa Asante, University of Ghana http://ugspace.ug.edu.gh v Nana Afia Asante Ntim, Yaw Owusu Amoah, Prince Kofi Parbie, Esinam Agbosu, Dzigbordi Aziati, Christopher Zaab-Yen Abana, Gifty Mawuli, Naa Dedei Aryeequaye, Afrakoma Obeng, and Alexander Martin-Odoom. I also thank James Aboagye and Naa Dedei Hammond for helping me with the statistical analyses. Miss Rosemary Ardayfio has been of great help, inspiration and encouragement to me since she joined Virology Department. I also thank Ewurabena Oduma Duker (my Princess) for believing in me, standing by me and helping me out with other things so I could finish this work. I am very grateful to Dr. Charles Addoquaye Brown, of School of Allied Health Sciences, University of Ghana, for finding time amidst his busy schedule to proofread my work and to Drs. James A. M. Brandful, John K. Odoom, Jonathan P. Adjimani, Michael Ofori, Anita Ghansah, Jewelna Osei-Poku, Prof. Michael D. Wilson, Richard H. Asmah, Rita N. D. Obeng, Shirley C. Nimo-Paintsil, Helena Baidoo and Naiki Puplampu for their encouragement. For giving me a push whenever I needed it and reminding me that ‘this one too shall pass’, I thank my dear sister-friends Mrs. Sena Adzoa Matrevi, Mrs. Helena Naa Nuerki Lamptey and Miss Ivy Asantewaa Asante for being there for me. I appreciate the mentorship of Drs. Lucia Perez-Alvarez and Elena Delgado, of the Department of HIV Biology and Variability, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain, who hosted, trained and equipped me with skills to conduct this study. I am grateful to the HIV Research Trust, UK, for sponsoring this training. Dr. Nii Akwei Addo and the National AIDS Control Program (Ghana) also provided some reagents towards my work, Professor Eiji Ido of the Tokyo Medical and Dental University under the TMDU-NMIMR HIV Projects supported me with some reagents for the work and Professor Koichi Ishikawa gave me financial support. The University of Ghana, my employer, granted me study leave with pay and academic user fee waiver. I am greatly thankful for all these important support. Finally, I thank all other family members, friends and colleagues who prayed for me, believed in me, wished me well and contributed to the completion of this work in any and every way. God bless you ALL!! University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENTS DECLARATION ii DEDICATION iii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS vi LIST OF FIGURES xi LIST OF TABLES xii LIST OF ABBREVIATIONS AND ACRONYMS xiii ABSTRACT xvi CHAPTER ONE 1 1.0 INTRODUCTION 1 1.1 AIM 6 CHAPTER TWO 7 2.0 LITERATURE REVIEW 7 2.1 Human Immunodeficiency Virus (HIV) 7 2.1.1 General introduction 7 2.1.2 Life cycle 7 2.1.3 Genome organization 9 2.1.4 Genetic variability 12 2.1.5 Pathogenesis 14 2.1.5.1 Rapid replication of HIV in CD4+ cells 17 2.1.5.2 Establishment and maintenance of HIV reservoirs 17 2.1.5.3 Host restriction factors 18 2.1.6 The course of HIV infection and disease 19 2.1.7 Diagnosis of HIV infection 22 2.1.7.1 Antibody tests 23 2.1.7.1.1 Rapid HIV tests 24 2.1.7.1.2 Western blot 24 University of Ghana http://ugspace.ug.edu.gh vii 2.1.7.2 Antigen tests 25 2.1.7.3 Qualitative PCR 25 2.1.8 Diagnosis of HIV infection in Ghana 26 2.2 Antiretroviral drugs and therapy 27 2.3 Mechanism of resistance to antiretroviral drugs 32 2.3.1 Resistance to nucleoside and nucleotide reverse transcriptase inhibitors 32 2.3.1.1 Impairment of analogue incorporation 32 2.3.1.2 Removal of the analogue from the terminated DNA chain 33 2.3.2 Resistance to non-nucleoside reverse transcriptase Inhibitors 36 2.3.3 Resistance to protease inhibitors 39 2.3.4 Resistance to fusion inhibitors 42 2.4 Development of HIV drug resistance 44 2.5 Markers for monitoring patients on ART 46 2.5.1 Using immunologic and virologic markers 46 2.5.2 HIV Drug resistance testing 48 2.5.2.1 Genotypic assays 48 2.5.2.1.1 Genotypic DR testing using peripheral blood mononuclear cells 51 2.5.2.2 Phenotypic assays 52 2.6 The HIV and AIDS epidemic 53 2.6.1 HIV and AIDS in Ghana 54 2.7 Nucleic acid extraction/isolation methods 58 2.7.1 Column-based nucleic acid purification 59 2.8 Polymerase chain reaction (PCR) 59 2.9 Gel electrophoresis 61 2.9.1 Agarose gel electrophoresis 61 2.10 Real time (Quantitative) PCR 62 2.11 DNA sequencing 63 University of Ghana http://ugspace.ug.edu.gh viii 2.11.1 Sanger chain termination method 64 CHAPTER THREE 66 3.0 MATERIALS AND METHODS 66 3.1 General introduction 66 3.2 Materials 66 3.3 Methods 66 3.3.1 Study population 66 3.3.2 Blood sample collection from study participants 67 3.3.3 Separation of blood into plasma and peripheral blood mononuclear cells (PBMC) 67 3.3.4 Determination of HIV type 68 3.3.5 Extraction and purification of nucleic acids from plasma and PBMC 70 3.3.5.1 Extraction and purification of viral RNA from plasma using QIAamp viral RNA mini kit 70 3.3.5.2 Extraction and purification of viral RNA from plasma using nucleic acid purification kit 70 3.3.5.3 Extraction and purification of proviral DNA from PBMC using QIAamp DNA blood mini kit 71 3.3.5.3 Extraction and purification of proviral DNA from PBMC using nucleic acid purification kit 71 3.3.6 Determination of HIV-1 viral load 71 3.3.7 Reverse transcriptase polymerase chain reaction for PR and RT genes from RNA 72 3.3.8 Polymerase chain reaction for DNA extracts from PBMC 72 3.3.9 Amplification of RT-PCR and PCR products by nested PCR 73 3.3.10 Agarose gel electrophoresis 75 3.3.11 Purification of PCR products 75 3.3.12 Direct sequencing of purified PR and RT PCR products 75 3.3.13 Purification of cycle sequenced products 76 3.3.14 Sequence Analysis on the 3130 ABI Genetic Analyzer 76 University of Ghana http://ugspace.ug.edu.gh ix 3.3.15 Sequence data analysis 77 3.3.16 Data Analyses 78 3.4 Staff training and capacity building at NMIMR 78 CHAPTER FOUR 79 4.0 RESULTS 79 4.1 Study population 79 4.2 Patient information 79 4.3 History of anti-retroviral therapy in patients 84 4.4 Adherence to ART and herbal medicine use 86 4.5 Physicians’ assessment of the patients 86 4.6 Nested PCR 88 4.7 Sequencing 92 4.8 Prevalence of HIV drug resistance mutations 94 4.8.1 Types of drug resistance mutations found among persons on first-line regimen 96 4.8.2 Types of drug resistance mutations found among persons on second -line regimen 99 4.8.3 Occurrence of thymidine analog mutations (TAMs) 101 4.8.4 Drug resistance mutations found in plasma and PBMC pairs 101 4.9 Relationship between change in CD4 counts, viral load, duration on first-line ART and the presence of drug resistance mutations 106 4.10 Relationship between adherence to ART, herbal medicine use and the presence of drug resistance 106 4.11 Polymorphisms at drug resistance–associated positions in PR sequences 107 4.12 Prevalence of HIV-1 subtypes 107 4.13 Capacity building at Virology Department at NMIMR 113 CHAPTER FIVE 114 5.0 DISCUSSION AND CONCLUSIONS 114 5.1 Discussion 114 University of Ghana http://ugspace.ug.edu.gh x 5.1.1 General introduction 114 5.1.2 HIV serotype, immunologic and virologic markers 114 5.1.3 PCR amplification of reverse transcriptase and protease genes 116 5.1.4 Sequencing of reverse transcriptase and protease genes 117 5.1.5 Prevalence and characteristics of drug-resistance mutations 117 5.1.5.1 Drug resistance mutations among patients on first-line regimen 118 5.1.5.2 Drug resistance mutations among patients on second-line regimen 124 5.1.5.3 Drug resistance mutations in paired plasma and PBMC 126 5.1.6 Effect of change in CD4 counts, viral load at sampling and duration on first-line ART on presence of drug resistance mutations 128 5.1.7 Effect of adherence to antiretrovirals or herbal medicine use on the presence of drug resistance mutations 128 5.1.8 Polymorphisms at DR sites in the PR sequences 128 5.1.9 HIV-1 subtype information 129 5.2 Conclusions 130 5.3 Recommendations 132 REFERENCES 133 APPENDICES 165 University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES Figure 1 Life cycle of HIV 8 Figure 2 A genome map of HIV-1 11 Figure 3 Phases of infection following exposure to HIV 16 Figure 4 The natural course of HIV infection 21 Figure 5 Chemical structure of antiretroviral drugs 30 Figure 6 An illustration of mechanism of resistance to NRTIs 35 Figure 7 An illustration of mechanism of resistance to NNRTIs 37 Figure 8 Ribbon structure of HIV-1 reverse transcriptase showing drug resistance positions 38 Figure 9 Structural model of HIV-1 protease showing positions of drug resistance 41 Figure 10 An illustration of mechanism of resistance to fusion inhibitors 43 Figure 11 The map of West Africa showing the location of Ghana 55 Figure 12 An Inno-lia strip showing bands after the assay 69 Figure 13 Summary of mean CD4 counts of patients over treatment period 81 Figure 14 Percentages of patients with viral loads within categories indicated 82 Figure 15 Prevalence of HIV types in the study population 83 Figure 16 Classification of patients based on clinical assessment 87 Figure 17 Representative gel photograph of RT amplification products 90 Figure 18 Representative gel photograph of PR amplification products 91 Figure 19 Occurrence of NRTI and NRTI resistance mutations in patients on first- and second-line regimens 95 Figure 20 Occurrence of thymidine analogue mutations in patients on first- and second-line regimens 102 Figure 21 Phylogenetic tree of RT sequences with subtype reference sequences 110 Figure 22 Phylogenetic tree of RT sequences from paired plasma and PBMC samples 111 Figure 23 Dot plot of alignment of protease sequences from study with a subtype B reference sequence HXB2 112 University of Ghana http://ugspace.ug.edu.gh xii LIST OF TABLES Table 1 Details of the primers used for PCR and sequencing and location of primers 74 Table 2 Summary of the characteristics of study patients 80 Table 3 ARV therapy histories of patients showing drugs taken as first-line or second-line regimen 85 Table 4 Summary of PCR results showing numbers of patients with amplified genes of interest by type of sample and study site 89 Table 5 Summary data showing number of patients with successfully sequenced genes of interest and the number of sequences with drug resistance mutations 93 Table 6 Types of NRTI resistance mutations in patients on first- line regimen 97 Table 7 Types of NNRTI resistance mutations in patients on first- line regimen 98 Table 8 Types of NRTI resistance mutations in patients on second-line regimen 100 Table 9 Types of NNRTI resistance mutations in patients on second-line regimen 103 Table 10 Types of PI resistance mutations in patients on second- line regimen 104 Table 11 Comparison of NRTI and NNRTI mutations in paired plasma and PBMC samples 105 Table 12 Relationship between presence of drug resistance mutations and change in CD4 counts, viral load at sampling and duration on first-line therapy 108 Table 13 Relationship between adherence to ART, herbal medicine use and the presence of drug resistance mutations, 109 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF ABBREVIATIONS AND ACRONYMS µg Microgram µl Microlitre 3TC Lamivudine A Adenine ABC Abacavir ABI Applied Biosystems Inc AIDS Acquired immune deficiency syndrome APOBEC Apolipoprotein B editing complex APV Amprenavir ART Antiretroviral therapy ARVs Antiretrovirals ATP Adenosine triphosphate ATV Atazanavir AZT Azidothymidine BLAST Basic local alignment search tool Bp Base pair C Cytosine CA Capsid CCR5 Chemokine coreceptor 5 CD4 Cluster of differentiation 4 CD8 Cluster of differentiation 8 CDC Centers for Disease Control cDNA Complementary DNA CRFs Circulating recombinant forms CT Cut-off threshold CXC4 Chemokine coreceptor 4 D4T Stavudine dA Deoxyadenosine DC Dendritic cells DDI Didanosine ddNTPs Dideoxynucleotide triphosphates dG Deoxyguanosine DLV Delavirdine DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotide triphosphates DR Drug resistance DRM Drug resistance mutations Ds Double-stranded EDTA Ethylenediaminetetraacetic acid EFV Efavirenz EIA Enzyme immune assay ELISA Enzyme linked immunosorbent assay Env Envelope EtBr Ethidium bromide ETR Etravirine University of Ghana http://ugspace.ug.edu.gh xiv FDA Food and Drugs Administration FI Fusion inhibitor FOS-APV Fosamprenavir FRET Fluorescent resonance energy transfer FTC Emtricitabine G Guanine Gag Group-specific antigen gene GALT Gut-associated lymphoid tissue Gp Glycoprotein HAART Highly active antiretroviral therapy HIV Human immunodeficiency virus HIV-1 Human immunodeficiency virus type 1 HIV-2 Human immunodeficiency virus type 2 HIVDB HIV database HIVDR HIV drug resistance HLA Human leukocyte antigen HR 1 Hydrophobic region 1 HR 2 Hydrophobic region 2 HSS HIV sentinel survey IC Inhibitory concentration IDs Identification numbers IDV Indinavir IFN Interferon IgG Immunoglobulin G IgM Immunoglobulin M IN Integrase LPV/r Lopinavir/Ritonavir LTR Long terminal repeat M Molar MA Matrix MEGA Molecular evolutionary genetics analysis MgCl2 Magnesium chloride Ml Millilitre mRNA Messenger ribonucleic acid NACP National AIDS/STI Control Programme NBT Blue tetrazolium NC Nucleoprotein Nef Negative regulatory factor NFV Nelfinavir NK Natural killer NMIMR Noguchi Memorial Institute for Medical Research NNRTI Non-nucleoside reverse transcriptase inhibitor NRTI Nucleoside reverse transcriptase inhibitor nRTIs Nucleoside and nucleotide reverse transcriptase inhibitors NVP Nevirapine PAGE Polyacrylamide gel electrophoresis PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline PCR Polymerase chain reaction University of Ghana http://ugspace.ug.edu.gh xv PHI Primary HIV-1 infection PI Protease inhibitor PIC Pre-integration complex Pol Polymerase PPi Pyrophosphate PR Protease pVL Plasma viral load qPCR Quantitative polymerase chain reaction rDNA Recombinant DNA Rev Regulatory factor RLS Resource-limited setting RNA Ribonucleic acid RPV Rilpivirine RT Reverse transcriptase RT-PCR Reverse transcription polymerase chain reaction RTV Rotonavir SIV Simian immunodeficiency virus SP1 Spacer peptide 1 SQV Saquinavir Ss Single-stranded ssDNA Single-stranded deoxyribonucleic acid SU Structural unit T Thymine T-20 Enfuvirtide TAE Tris acetate EDTA TAMs Thymidine analogue mutations Tat Transactivator Tev Tat env rev TBE Tris borate EDTA TDF Tenofovir TE Tris-EDTA TLR Toll-like receptor TM Transmembrane TPV Tipranavir TRIM Tripartite motif Tris Trisaminomethane U Uracil U.S United States UNAIDS United Nations joint programme on HIV/AIDS URFs Unique recombinant forms USA United States of America UV Ultra violet V Voltage Vif Viral infectivity factor VL Viral load Vpr Viral protein r Vpu Viral protein u WHO World Health Organization University of Ghana http://ugspace.ug.edu.gh xvi ABSTRACT Highly active antiretroviral therapy (HAART) is known to improve treatment in Human Immunodeficiency Virus (HIV)-infected patients but the emergence of drug resistance is an obstacle in the effective management of HIV infection and Acquired Immune Deficiency Syndrome (AIDS). Plasma viral load monitoring is the gold standard used in high-income countries for monitoring treatment but it is not available in many resource-limited settings. Although limited viral load testing is now available in Ghana, viral load is not routinely used for monitoring majority of patients on HAART. Physicians in Ghana therefore depend mainly on CD4 counts and clinical symptoms to monitor treatment. Thus, a significant proportion of patients may suffer virologic failure while continuing to take first-line antiretroviral therapy (ART). This may encourage the development and accumulation of drug resistance mutations and compromise future treatment efforts. The aim of this study was to investigate the presence of HIV drug resistance mutations in patients on ART in Ghana, relate these mutations to treatment regimens in order to inform policy on ART monitoring and patient management in the country. Venous blood was obtained from 338 patients on ART from the Korle-Bu Teaching, St. Martin de Porres, Atua Government and Kumasi South hospitals in Ghana. Personal information and ART history of the patients were also collected using a sample collection form. The CD4 counts and viral loads of patients were determined. HIV ribonucleic acid (RNA) and proviral deoxyribonucleic acid (DNA) were extracted from the plasma and peripheral blood mononuclear cells (PBMC), respectively. The HIV protease and reverse transcriptase genes were amplified from the RNA and DNA by polymerase chain reaction. University of Ghana http://ugspace.ug.edu.gh xvii The positive amplification products were sequenced and analyzed for drug resistant mutations using the Stanford HIV Drug Resistance Database. The mean age of patients was 42 years and 72% of patients were female. The mean CD4 counts increased from 161cells/µl at start of therapy to 454cells/µl at time of sampling. Only 7% of patients had detectable viral loads at time of sampling. Most of the patients (87%) were on first-line regimen. Physicians rating showed that 86% of patients were doing well and the rest were either not doing so well (11%) or were failing (3%). The reverse transcriptase gene was successfully sequenced from 65 (19%) and 99 (29%) of plasma and PBMC respectively while protease gene was successfully sequenced from 54 (16%) of plasma and 76 (23%) of PBMC. Out of these, 46 % and 49% of the plasma sequences had nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations respectively. From PBMC, 25% and 26%, respectively had NRTI and NNRTI mutations. Protease inhibitor (PI) resistance mutations were found in 28% of plasma and 9% of PBMC sequences. The most common NRTI mutation found was M184V and that of NNRTI was K103N. Thymidine analogue mutations (TAMs) including M41L, D67N, T215Y/F, L210W and K219E/Q were mostly found in patients on second-line regimen. Protease inhibitor mutations were mainly found in patients on second-line regimen and they included M46I, V82I and N88S. Similar resistance profiles were observed in paired sequences from plasma and PBMC of the same patients. The results showed that even patients who were doing well, based on their CD4 counts, viral load and physicians assessment, were harbouring drug resistance mutations including TAMs that could render the NRTIs ineffective. The results suggest that CD4 count is an insufficient marker for monitoring treatment since it continues to increase even in the presence of drug University of Ghana http://ugspace.ug.edu.gh xviii resistance mutations. Patients on ART in Ghana may therefore not be deriving optimal benefits from treatment because of the current monitoring system. Therefore drug resistance testing will be useful before switching regimens in order to decide drugs in the new regimen. This will reduce the accumulation of multi-nucleoside and thymidine analog mutations in patients and preserve future drug options. The study has provided vital drug resistance data to guide policy on ART monitoring in Ghana, improved protocols for HIV genotyping at the Virology Department of the NMIMR and built capacity for drug resistance analyses of patients that fail ART in Ghana. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION The human immunodeficiency virus (HIV) is the causative agent of the acquired immune deficiency syndrome (AIDS). Since 1981 when the first cases of AIDS were reported, the pandemic has claimed the lives of many adults and children throughout the world. The biggest disease burden is in sub-Saharan Africa where majority (about 68%) of HIV-infected people live. AIDS is known to have killed 30 million people since 1981while about 34 million people are currently living with HIV in the world (WHO, 2012; amfAR 2012). Since its approval for use in humans, about three decades ago, antiretroviral drugs have significantly improved the life of HIV-infected persons by slowing disease progression and reducing morbidity and mortality. Five distinct classes of antiretroviral drugs are currently used to treat HIV infection. These agents act at different stages in the replication cycle of the virus (Clavel and Hance, 2004). The entry or fusion inhibitors block the penetration of HIV into the target cells. The nucleos(t)ide reverse transcriptase inhibitors (NRTIs) act as DNA- chain terminators and inhibit reverse transcription of the viral RNA genome into DNA. The non-nucleoside reverse-transcriptase inhibitors (NNRTIs) bind in a hydrophobic pocket close to the active site of reverse transcriptase, and restrict the conformational change needed for the catalytic activity of the RT. The integrase inhibitors inhibit the action of integrase, the enzyme responsible for incorporating HIV DNA into the host cell DNA for transcription of the viral mRNA. The protease inhibitors (PIs) target and inhibit the viral protease, the enzyme required for the cleavage of precursor proteins (gag and gag–pol) into functional proteins, allowing the final assembly of the inner core of viral particle (Clavel and Hance, 2004). University of Ghana http://ugspace.ug.edu.gh 2 Unfortunately, the effectiveness of antiretroviral therapy can be markedly reduced by the emergence of drug resistance. Clinical experience with all the drugs has shown that the virus is able to easily evade the antiviral effects of the drugs, when administered as monotherapy, through the rapid accumulation of amino acid mutations in the target proteins (Hartman and Buckheit, 2012). These mutations arise due to the high rate of replication of the virus coupled with the error- prone nature of the reverse transcriptase enzyme (Shafer et al, 2000). These lead to the generation of quasispecies of the virus in an infected person. The drug-resistant variants are thought to exist prior to the use of the drugs and are selected for due to the drug pressure. Some HIV variants are known to exhibit intrinsic or “primary” resistance to some antiretroviral agents, but most drug resistance develops as a result of exposure to the drugs (Shafer et al, 2000; Arts and Hazuda, 2012). Even in the presence of virologic suppression, during therapy of HIV infection, drug resistance can still occur due to residual virus replication. HIV may replicate in body sites lacking adequate exposure to the antiretrovirals (ARVs) resulting in selection of the drug-resistant mutants (Martinez-Picado et al. 2000). Combinations of three drugs from at least two classes, called highly active antiretroviral therapy (HAART), are now used for the treatment of HIV infection. HAART regimens generally comprise three antiretroviral drugs usually two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI) [Yeni et al, 2002]. The use of drugs from different classes with different mechanisms of action helps to control the development of drug resistance. However, drug resistance can develop during HAART and against more than one drug within a class. In such cases, the emergence of drug-resistant variants complicates treatment by rendering current ARVs ineffective and potentially limiting future remedies (Kantor et al, 2004; Sigaloff et al, 2011). As treatment efforts intensify, drug resistance has become common as University of Ghana http://ugspace.ug.edu.gh 3 both a cause and a result of virologic treatment failure and incomplete virus suppression (Kantor et al, 2005). HIV drug resistance testing is therefore recommended for persons on treatment based on the mounting evidence that it improves the pharmacotherapy of HIV infection (Cohen et al, 2002). Most of the current knowledge of HIV drug susceptibility and resistance, and interpretations of genotypic changes in HIV reverse transcriptase (RT) and protease (PR), are based on data obtained from HIV-1 subtype B viruses prevalent in North America, Western Europe, and Australia. Worldwide, however, the majority of people with HIV are infected with non-B subtypes, which differ from subtype B by as much as 30% in envelope gene and 15% in polymerase gene (Osmanov et al, 2000). Thus, there is increasing need to generate data on sustained viral suppression and the evolution of drug resistance with antiretroviral drugs for the non-B subtypes prevalent in sub-Saharan Africa which bears the greatest disease burden. Antiretroviral therapy (ART) was introduced in Ghana in 2003 on a pilot basis. The programme has been scaled up from just 2 sites in 2003 to 160 sites at the end of 2011 (NACP, 2011). Ideally, all patients receiving antiretroviral therapy (ART) should undergo regular viral load testing in order to assess treatment failure or success. This is however not the case in Ghana since viral load testing is expensive and requires dedicated laboratory with highly trained staff. Immune status determined via CD4 counts are easier to measure and has been used in combination with clinical symptoms to inform decisions on switching ART regimen. The World Health Organization (WHO) guidelines recommend a CD4 count decline of 50% from the previous peak, or a one-third decline in the previous six months, as a trigger for changing treatment in resource limited settings where viral load testing is not routinely done (WHO, 2006). The use of mainly CD4 counts and clinical symptoms to monitor University of Ghana http://ugspace.ug.edu.gh 4 treatment outcomes is a limitation to Ghana’s ART program since these markers do not provide enough information on drug resistance mutations and other causes of treatment failure. Despite using the WHO recommendations (WHO, 2006), it is likely that persons who have not yet attained the 50% drop in CD4 counts may still harbor drug resistance mutations selected for by the ARVs used over the period. Maintaining such persons on the same ARVs in the presence of the drug resistance mutations eventually renders the treatment ineffective. Furthermore, future therapy with drug combinations containing some of the drugs that the virus is resistant to are compromised and this limits the choice of alternate regimens (Sigaloff et al, 2011). Also, limited ARVs from the reverse transcription and protease inhibitor drug classes are procured and used in Ghana and their use can be optimized if HIV drug resistance data is available. In Ghana, the current practice for HIV-infected patients who have failed first-line therapy (consisting two NRTI and one NNRTI) is to switch them to a second-line therapy, consisting two NNRTI and one PI (NACP/MoH/GHS, 2010). This decision to switch the drug regimen is mostly based on clinical symptoms and significant drops in CD4 count. The NRTIs applied in the second-line regimen are usually different from those used in the first-line regimen. However, some of the NRTI drug resistance mutations acquired during first-line therapy may have cross-resistance to some of the drugs in the second-line regimen (Shafer et al, 2000).There is therefore a need to analyze the RT and PR genes in persons on treatment in order to characterize mutations associated with resistance to antiretroviral drugs. HIV drug resistant testing protocols usually depend on plasma for the amplification of viral genes (Sarmati et al, 2002; Kabamba- Mukadi et al, 2010). However, this approach provides information mainly on circulating major viral population at the time of analysis. Analyzing proviral DNA from the peripheral blood mononuclear cells (PBMC) is an alternative approach (Kabamba-Mukadi et al, 2010). This could provide additional information on other University of Ghana http://ugspace.ug.edu.gh 5 HIV variants that the persons might have haboured over time and which are likely to resurface in immunological failure or drug interruption (Noe et al, 2009 and Rangel et al, 2009). This study therefore looked at drug resistance mutations from both plasma and peripheral blood mononuclear cells of persons on ART. It sought to provide data on the drug-resistance mutations at the time of sampling and over the treatment period. This information serves to provide a means to review progress of the patients on ART, redesign treatment regimens and enable appraisal of the policy on ARV use in Ghana. University of Ghana http://ugspace.ug.edu.gh 6 1.1 AIM The main aim of this study was to investigate the occurrence and type of HIV drug resistance mutations in patients on ART in Ghana and relate these mutations to treatment regimens in order to inform policy on ART monitoring and patient management in the country. The Specific Objectives were:  To identify HIV-infected persons on ART for a minimum of 6 months and document their drug histories  To determine the HIV serotype, and measure the immunologic and virologic markers  To amplify and sequence the protease and reverse transcriptase genes to find drug resistance mutations  To relate the mutation patterns observed to the drug histories of the patients by comparing data for patients on first-line to those on second-line regimens  To compare drug resistance mutations in plasma to those in peripheral blood mononuclear cells University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 The Human Immunodeficiency Virus (HIV) 2.1.1 General Introduction The human immunodeficiency virus (HIV) is a lentivirus and belongs to the family Retroviridae. HIV is an enveloped virus and has two positive sense RNA strands as its genetic material (Santos and Soares, 2010). Retroviruses are characterized by the possession of the enzyme reverse transcriptase, which allows viral RNA to be transcribed into DNA and incorporated into the host cell genome by the help of another enzyme, integrase. Glycoproteins on the surface of HIV bind to the target cell and help the virus to enter the cell. The virus encodes multiple regulatory proteins, which control the life cycle and viral expression (Frankel and Young, 1998). 2.1.2 Life Cycle As shown in Fig. 1, when the virus attaches itself to the host cell membrane, there is fusion of the viral envelope with the cell membrane resulting in the virus entering the cell. Once inside the cell, there is uncoating and the viral core undergoes a slow dissolution process (Arts and Hazuda, 2012). The process ensures the protection of the viral RNA but permits access to deoxyribonucleotide triphosphates (dNTPs) necessary for reverse transcription and proviral DNA synthesis. The single-stranded RNA genome is converted into double-stranded DNA by the help of reverse transcriptase (RT). Reverse transcriptase is a multifunctional enzyme with RNA-dependent DNA polymerase, RNase-H, and DNA-dependent DNA polymerase activities (Hughes and Hu, 2011). These enzyme activities are all required to convert the single-stranded HIV RNA into a double-stranded DNA (Hughes and Hu, 2011). University of Ghana http://ugspace.ug.edu.gh 8 Figure 1: A schematic diagram of the life cycle of HIV showing the various steps involved in the replication of the virus in a human host (Source: http://pathomicro.med.sc.edu/lecture/hivstage.gif) University of Ghana http://ugspace.ug.edu.gh 9 However, reverse transcriptase does not have a proofreading activity and so makes errors when copying the viral RNA into DNA. At the end of reverse transcription, a viral pre- integration complex (PIC) is formed. The PIC, which is made of viral as well as cellular components, is transported to the nucleus where the second essential HIV enzyme, integrase, catalyzes the integration of the viral DNA into the host DNA (Craigie and Bushman, 2011). Integration of the HIV DNA is required to maintain the viral DNA in the infected cell and is essential for expression of HIV mRNA and viral RNA (Arts and Hazuda, 2012) to ensure the replication of the virus within the host. Thus, the virus hijacks the cellular machinery for its own replication. In the final step of the replication cycle, the viral polypeptide is cleaved by the viral protease during proteolysis to produce the viral proteins needed to make an infectious viral particle (Sundquist and Kra¨usslich 2011). The viral RNA and proteins are assembled and packaged into a new virion that buds off the host cell membrane to infect a new cell and continue the process of replication. 2.1.3 Genome Organization The HIV genome (Fig. 2), which is approximately 9.7Kbp, has several major genes that are common to all retroviruses and some accessory genes that are unique to HIV (Mushahwar, 2007). The gag (group-specific antigen) gene encodes the nucleocapsid, the pol (polymerase) gene encodes the viral enzymes and the env (envelope) gene encodes the envelop proteins. The functions of these genes are further explained below: i. gag: codes for the gag polyprotein, which is processed during maturation to MA (matrix protein, p17); CA (capsid protein, p24); SP1 (spacer peptide 1, p2); NC (nucleocapsid protein, p7); SP2 (spacer peptide 2, p1) and p6. ii. pol: codes for viral enzymes, reverse transcriptase, integrase, and HIV protease. University of Ghana http://ugspace.ug.edu.gh 10 iii. env: codes for gp160, the precursor to gp120 and gp41, proteins found as spikes on the viral envelope, which enables the virus to attach to and fuse with target cells. iv. tat (transactivator), rev (regulatory factor) and vpr (viral protein r) are transactivators of gene expression. v. vif (viral infectivity factor), nef (negative regulatory factor) and vpu (viral protein u) are regulatory proteins. vi. tev: has been described in a few HIV isolates. It is a fusion of parts of the tat, env, and rev genes, and codes for a protein with some of the properties of tat, but little or none of the properties of rev. All these genes may be altered by mutation and with exception of tev, they all exist in all known variants of HIV. The pol gene is of particular interest to this study since it encodes the viral enzymes; reverse transcriptase, integrase and protease. These enzymes are the targets of most of the current antiretroviral agents (Arts and Hazuda, 2012). The pol gene is therefore analyzed to determine HIV- related drug resistance mutations. University of Ghana http://ugspace.ug.edu.gh 11 Figure 2: A genome map of HIV-1 showing the various genes that make up the virus and their corresponding proteins. MA-matrix; CA- capsid; NC-nucleocapsid; SU-structural unit; TM-transmembrane; PR-protease; RT-reverse transcriptase; IN-integrase; LTR-long terminal repeat (Source: http://www.stanford.edu/group/virus/retro/2005gongishmail/HIV-1b.jpg). University of Ghana http://ugspace.ug.edu.gh 12 2.1.4 Genetic variability HIV is known to have a very high genetic variability (Kantor et al, 2005; Buonaguro et al, 2007). This diversity is due to its extremely high replication rate and the error-prone nature of its reverse transcriptase. These two factors result in the generation of many variants of HIV even within a single infected person per day. In addition, different strains of HIV are able to infect one cell and this may lead to recombination between strains and the generation of recombinant forms. Two main types of HIV have been characterized and known to cause AIDS. Human immunodeficiency virus type I (HIV-1) was first recorded in the United States and subsequently in Europe and the eastern and central parts of Africa (CDC, 1981). Human immunodeficiency virus type 2 (HIV-2) was isolated in 1986 from two HIV-1 seronegative individuals from West Africa with AIDS (Clavel et al., 1986). Whereas HIV-1 infection is worldwide, that of HIV-2 appears to be restricted to West Africa (Temin, 1993; Ho et al., 1995). Both HIV-1 and HIV-2 cause AIDS in West Africa but HIV-2 has been shown to be sexually and perinatally less transmissible than HIV-1. Also, the rate of disease progression of HIV-2 is much slower than HIV-1 (Pepin et al., 1991; Whittle et al., 1994; Marlink et al., 1994). The basis of these differences in the natural history of both infections remains unclear and may result from the host alone or in association with viral factors (Barin et al., 1985; Kanki et al., 1986; Clavel et al., 1986; Schim van der Loeff et al., 1999). Both HIV types are distinct; HIV-1 is known to be related to simian immunodeficiency virus (SIV) from chimpanzees (Gao et al, 1999) while HIV-2 is closely related to SIV from sooty mangabeys (Gao et al, 1992). University of Ghana http://ugspace.ug.edu.gh 13 HIV-1 has been characterized into four groups (Gao et al, 1999; Van Heuverswyn et al, 2006). These are groups M, O, N and P. Group M is the major HIV-1 group and is further divided into subtypes A, B, C, D, F, G, H, J, K and circulating recombinant forms (CRFs). These subtypes are different from each other by 10-12% in nucleotides and 5-6% in their amino acids (Kantor et al, 2005). Group N (new, non-M, non-O) was identified in a few individuals in Cameroon (Simon et al, 1998; Ayouba et al, 2000). Group O (outlier) contain diverse viruses but it is not commonly found. Viruses belonging to this group are thought to be transmitted to humans by gorillas (Van Heuverswyn et al, 2006). The virus classified as belonging to HIV-1 group P was isolated from a Cameroonian woman and it is closely related to SIV gor (Plantier et al, 2009. The group M viruses are responsible for most of the HIV-1 epidemic in the world (Buonaguro et al, 2007; Santos and Soares, 2010). HIV-2 has been characterized into 7 main clades now called groups (www.hiv.lanl.gov, 2013). These groups (A, B, C, D, E, F and G) used to be known as subtypes but are now called groups because it is thought that each of these types resulted from separate introductions of SIV into the human populations. HIV-2 Groups A and B are known to be responsible for most of the HIV-2 infections (Lemey et al, 2002; Trevin et al, 2011). Recombination is rare in HIV-2; the first CRF of HIV-2 (CRF01_AB) was described in 2010 (Ibe et al, 2010). The differences among HIV types and subtypes could have implications for clinical management. Most of our current knowledge about antiretroviral drugs, their development and mechanisms of action and the mechanisms of viral resistance to these drugs are gained from studies using HIV-1 subtype B virus. University of Ghana http://ugspace.ug.edu.gh 14 This virus subtype occurs in North America, Western Europe and Australia and is responsible for only 10% of the global infections (Kantor et al., 2005). Majority of HIV infections are known to occur in sub Saharan Africa where other HIV-1 subtypes are predominant. Some previous studies have looked at drug resistance in HIV subtypes besides subtype B (Kantor et al, 2005; Soares et al, 2007; Martinez-Cajas et al, 2008; Martinez-Cajas et al, 2009). Studies in Ghana are however limited and involved mainly patients naïve to ART (Kinomoto et al, 2005; Sagoe et al, 2007; Delgado et al, 2008), thus there is a need to study viruses occurring in Ghana and in ART-experienced patients. 2.1.5 Pathogenesis Human immunodeficiency virus (HIV) infection is generally characterized by an acute phase of intense viral replication and dissemination to lymphoid tissues; a chronic, often asymptomatic phase of sustained immune activation, viral replication and establishment of stable tissue reservoirs; and an advanced phase of marked depletion of CD4+ T helper cells that leads to acquired immune deficiency syndrome (AIDS). Figure 3 shows the different phases of HIV infection, the cell types involved and the timelines associated with the various events. Major insight into HIV transmission and each phase of infection have been gained from studies on blood and tissue specimens obtained from HIV-infected individuals, as well as from studies using the simian immunodeficiency virus (SIV)-infected rhesus monkey model of AIDS (Moir et al, 2011). Immediately after infection, there is an early burst of viremia and rapid dissemination of the virus to lymphoid organs, particularly the gut-associated lymphoid tissue (Moir et al, 2011). Although there are vigorous cellular and humoral immune responses during primary HIV infection, the virus succeeds in escaping immune-mediated clearance. University of Ghana http://ugspace.ug.edu.gh 15 Therefore, once infection once established, is never eliminated completely from the body (Fauci and Lane, 2005). The course of disease and its deleterious effect on the human host are influenced by the ability of the virus to rapidly replicate in CD4+ cells, maintain HIV reservoirs in tissues and cells and the action of restriction host factors. University of Ghana http://ugspace.ug.edu.gh 16 Figure 3: Phases of infection following exposure to human immunodeficiency virus (HIV). Infection begins with transmission across a mucosal barrier. Early propagation occurs in partially activated CD4+ T cells, followed by massive propagation in activated CD4+ T cells of the gut-associated lymphoid tissue lamina propria. The virus then spreads to other secondary lymphoid tissues where stable tissue viral reservoirs are established. Immune response is usually delayed and provides only partial control of viral replication. (Adapted from Moir et al, 2011) University of Ghana http://ugspace.ug.edu.gh 17 2.1.5.1 Rapid Replication of HIV in CD4+ cells It is difficult to identify infected individuals soon after exposure therefore the early events leading to HIV infection in the rectal mucosa or genital tract are not well understood (Moir et al, 2011). In vivo models of simian immunodeficiency virus (SIV), epidemiological studies and ex vivo models have provided some clues into the transmission of HIV (Zhang, 1999; Haase 2005; Hladik and McElrath, 2008). Partially activated CD4+ T cells of the genital mucosa are the first targets of productive viral replication within a one week after exposure (Fauci, 2007). This is followed by the local propagation of SIV in the less abundant but more susceptible activated CD4+ T cells. As shown in Figure 3, the virus then migrates to the gut- associated lymphoid tissue (GALT) and causes massive depletion of memory CD4+ T cells in the intestinal lamina propria (Guadalape et al., 2003; Brenchley et al., 2004; Mehandru et al, 2004). 2.1.5.2 Establishment and maintenance of HIV reservoirs Human immunodeficiency virus tends to ‘hide’ and persist in certain tissues and cells of an infected person and remain there until there is a trigger for replication (Moir et al, 2011). Whilst in hiding, the virus continues to replicate, although at a slower pace, and mutates during the process. Therefore the viruses that are produced during reactivation of the reservoirs are mostly the drug-resistant viruses (Richman et al, 2009). The rapid establishment and persistence of various HIV reservoirs remain two of the most important impediments to achieving complete eradication of the virus in infected individuals, even in an era of clinically effective ART (Richman et al, 2009). Latent reservoirs can be divided into two main categories (Moir et al, 2011): (a) lymphoid tissues that provide HIV with an abundance of target cells, close contacts for efficient cell-to- University of Ghana http://ugspace.ug.edu.gh 18 cell propagation, and reduced drug penetration; and (b) cellular reservoirs that consist mainly of CD4+ T cells that are highly susceptible to HIV replication when activated but can also carry latent virus. 2.1.5.3 Host restriction factors Replication of HIV and other retroviruses may be controlled at the intracellular level by intrinsic host factors that function in a virus-specific and, in many cases, species-specific manner (Strebel et al 2009). Such restriction factors include members of the apolipoprotein B editing complex (APOBEC) cellular deaminase family such as APOBEC3G (A3G) and APOBEC3F (A3F). These proteins modify cytidine by removing the amine group thus converting it to uridine in the transient (-) ssDNA replication intermediate. This change is later reflected as G-to-A changes in the (+) strand (Mangeat et al, 2003; Zhang et al, 2003; Amoedo et al., 2011). Apolipoprotein B editing complex group of proteins are packaged within viral particles and, upon subsequent entry into a new target cell, induces dG-to-dA hypermutations in the nascent proviral DNA. They (A3G and A3F) target dinucleotide motifs within DNA and causing GG to AG and GA to AA substitutions in proviral DNA respectively. These proteins (A3G and A3F) exhibit potent anti-HIV-1 activity (Sheehy et al., 2002; Liddament et al., 2004; Bishop et al., 2006) and are expressed in lymphocytes, the major target cells for HIV-1 infection (Liddament et al., 2004; Wiegand et al., 2004). The HIV accessory protein vif is known to counter the crippling effect of APOBEC3G/3F on HIV DNA (Chiu et al., 2005). A second intrinsic cellular restriction factor is Trim-5α, a member of the tripartite protein family. Trim-5α targets the retrovirus capsid protein for degradation (Strebel et al., 2009). University of Ghana http://ugspace.ug.edu.gh 19 The activity of Trim-5α is highly dependent on species-specific compatibility. HIV Gag escapes the effects of human Trim-5α and SIV Gag escapes the effects of most simian Trim- 5α (Moir et al, 2011. The inability of HIV to infect non human primates is in part due to the inhibitory effects of simian Trim-5α on HIV (Moir et al., 2011). The two types of restriction factors (APOBEC and Trim-5α) are considered to be part of the innate immune response. Their effect could be harnessed to reduce the early deleterious effects of HIV infection and promote a more effective adaptive immune response against HIV. Receptors and HLA molecules associated with natural killer cell function and members of the toll-like receptor family associated with the various antiviral IFN-α-based pathways have also been associated with slower disease progression (Moir et al., 2011). 2.1.6 The Course of HIV Infection and Disease Majority of HIV-infected individuals experience an acute HIV syndrome approximately two to four weeks following the transmission of the virus (Fig. 4). This is defined as flu-like clinical manifestations associated with high plasma viremia and often fever and lymphadenopathy. Other symptoms have been reported but their severity varies from person to person (Gurunathan et al., 2009). During this early phase, HIV often replicates extremely aggressively due to the lack of an immune response and viral loads reach levels as high as 10 million copies per milliliter (Piatak et al., 1993; Little et al., 1999). Without ART, plasma viremia typically peaks at three to four weeks post exposure (Little et al., 1999; Fiebig et al., 2003), then declines spontaneously for several months before reaching a steady state or viral set point. The level of the viral set point is an important determinant of the rate of disease progression in HIV-infected individuals who are not treated with ART (Mellors et al., 1996). University of Ghana http://ugspace.ug.edu.gh 20 Approximately 12 weeks after transmission, neutralizing antibodies begin to rise and evolve. However, this result of immune response is inadequate, too late and too narrow. The neutralization-sensitive virus is quickly replaced in succession by the neutralization-resistant variants (Wei et al., 2003; Richman et al., 2003). The envelope of the escaping population of HIV tend to be more highly glycosylated, preventing the binding of neutralizing antibodies and promoting viral persistence (Wei et al., 2003). University of Ghana http://ugspace.ug.edu.gh 21 Figure 4: The natural course of HIV infection in the absence of antiretroviral therapy. (Source: Modified from Fauci et al., 1996). University of Ghana http://ugspace.ug.edu.gh 22 Early CD8+ T cell response also contributes to the decline in HIV plasma viremia during the acute phase of infection (McMichael et al., 2010). However, there is rapid evolution characterized by mutations in epitopes recognized by CD8+ T cells leading to escape from CD8+ T cell epitopes similar to that of neutralizing antibodies (Goonetilleke et al., 2009). This occurs earlier than the appearance of neutralizing antibodies, continues throughout the course of disease and contributes to viral persistence. HIV targets and destroys CD4+ T cells, a major constituent of the immune system. It also induces immunologic dysfunction of CD8+ T cells, B cells, natural killer (NK) cells, and non- lymphoid cells through mechanisms that include increased cell turnover, activation, differentiation, and homeostatic responses (Moir et al, 2011). All these factors lead to qualitative changes within each immune cell population and ultimately affect the strength of the immune system. A progressive depletion of CD4+ T cells occurs in the majority of HIV- infected individuals who remain untreated and can also occur in individuals receiving ART. In most patients on ART, there is dramatic increase in CD4+ T cell counts. However, some individuals have low CD4+ T cell counts throughout treatment even on virologically- suppressive ART (Moir et al, 2011). 2.1.7 Diagnosis of HIV Infection Laboratory tests to detect the presence of HIV in infected persons use blood, serum, plasma or mucosal swabs. The tests may detect viral antibodies, antigens, viral nucleic acids by PCR or grow the virus in culture (Fearon, 2005). The tests could be further grouped into serological and molecular methods. University of Ghana http://ugspace.ug.edu.gh 23 2.1.7.1 Antibody tests The enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA) are the most commonly used assays because they have high sensitivity and can easily be automated and used for bulk testing (Fearon, 2005). Enzyme immunoassays have evolved from first- generation EIAs (e.g. Vironostika HIV-1 Microelisa System) and second-generation EIAs (e.g. Genetic Systems rLAV EIA; Bio-Rad Laboratories) that detect IgG antibodies against HIV-1 to third-generation EIAs. The third-generation EIAs use “antigen sandwich” techniques and detect both IgG and IgM antibodies against HIV-1. There are kits that detect antibodies against HIV-1 and HIV-2 e.g. HIVAB HIV-1/HIV-2 (rDNA) EIA (Abbott Laboratories) and others that detect HIV-1/HIV- 2 Plus O antibodies. Fourth-generation EIA, identify HIV infection even earlier because they detect both HIV antibody and p24 antigen (Branson, 2007). In an ELISA test, a person's serum is diluted and applied to a plate with wells pre-coated with HIV antigens. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to human antibodies - is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate contains enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as absorbance of the colour produced at a specific wavelength. University of Ghana http://ugspace.ug.edu.gh 24 2.1.7.1.1 Rapid HIV tests Rapid HIV tests are single-use EIAs that contain all necessary reagents and yield results in less than 30 minutes. They usually contain antigens made from intact viruses and therefore detect antibodies to most types of HIV (Bulterys et al, 2004). These tests are useful to screen people since results are quickly available to enhance treatment decisions. Rapid tests can be used to determine the HIV status of pregnant women in labour to decide on antiretroviral therapy initiation to prevent mother-to-child transmission (Bulterys et al, 2004; Jamieson et al, 2007). An index patient or sample can also be tested after an occupational exposure, to allow prompt initiation of antiretroviral prophylaxis to the exposed health care worker or researcher (Bulterys et al, 2004; Panlilio et al, 2005). In high-volume, high-prevalence settings, such as emergency departments, rapid tests can make testing more feasible and generate results quickly enough to influence clinical management (Lyss et al, 2007). All samples tested positive for HIV antibody on EIA must be repeated on another EIA and confirmed on a Western blot (Dodd and Feng 1990; Fearon, 2005). 2.1.7.1.2 Western Blot Western blot assays detect antibodies to HIV but unlike the ELISA, the antibodies are detected to individual proteins (core and envelope) of the virus that have been purified and fixed onto a membrane (Nuwayhid, 1995; Jackson et al, 1997). Some commercially prepared Western blot test kits contain the HIV proteins already on a cellulose acetate strip. The Inno- lia HIV1/II Score (Innogenetics, Belgium), for example, detects antibodies to specific recombinant proteins and synthetic peptides from HIV-1 and HIV-2 and a synthetic peptide from HIV group O coated as discrete lines on a nylon strip with plastic backing. Five HIV-1 antigens: sgp120 and gp 41, which detect specific antibodies to HIV-1 and p31, p24 and p17, University of Ghana http://ugspace.ug.edu.gh 25 which may cross react with antibodies to HIV-2 and HIV-1 group O peptides were included. HIV-2 specific antigens gp 36 and sgp 105 are also applied to the strip to detect HIV-2. During western blot assays, the diluted serum is applied to the membrane and antibodies in the serum bind to the HIV proteins. Coloured bands are developed on the strip when an alkaline phosphatase labeled antihuman IgG is added followed by a colour developing solution. The bands, representing the antigens present in the person’s blood are visualized and judged for positivity based on the manufacturer’s protocol (Jackson et al, 1997; Fearon, 2005). The interpretation of western blot results varies from one manufacturer to the other but it is generally determined by the number and type of viral bands that are present on the strip after running the assay (Dodd and Fang, 1990; Nuwayhid, 1995; Fearon, 2005). If no viral bands are detected, the result is negative. If at least one viral band for each of the gag, pol, and env gene-product groups is present, the result is positive. Tests in which less than the required number or type of viral bands is detected are reported as indeterminate (Nuwayhid,, 1995). 2.1.7.2 Antigen tests The viral capsid or core antigen (p24 antigen) test detects the presence of p24, the capsid protein of HIV. The assay uses p24 antibody to capture the p24 antigen in the person’s blood sample (Darr et al, 1991). The test involves adding p24-specific monoclonal antibodies to the blood sample to be tested. If p24 protein is found in the blood, it will bind to the monoclonal antibody. An enzyme-linked antibody will then bind to the antigen-antibody complex and cause a color change. The p24 antigen test is not recommended for general diagnostics due to low sensitivity and its ability to work only during the short period after infection before antibodies are produced by the body (Allain et al, 1986). University of Ghana http://ugspace.ug.edu.gh 26 2.1.7.3 Qualitative PCR Polymerase chain reaction (PCR) assays amplify viral nucleic acid from a person’s blood. These assays are sensitive and specific and can pick very small numbers of viral particles (Jackson et al, 1993). The assay uses specific primers that target specific HIV genes such as gag, env or pol. Polymerase chain reaction is particularly useful in diagnosing babies born to HIV-infected mothers in whom maternal antibodies can persist till 15 months (European Collaborative Study Group, 1988; De Rossi et al, 1992; Fearon, 2005). It may also be used in clarifying indeterminate western blot results. The primers used in PCR may be HIV type and subtype specific therefore negative PCR results could be due to subtype variations in specimens and must be carefully interpreted (Jackson et al, 1993; Jackson et al, 1997; Fearon, 2005). Polymerase chain reaction can also be used to amplify HIV genes from plasma, peripheral blood cells and whole blood for genotyping and identification of drug resistance mutations (Sarmati et al, 2002; Shafer 2002; Steegena et al, 2006; Kabamba- Mukadi et al, 2010). In such cases, the HIV gene of interest (protease, reverse transcriptase, integrase or envelope) is amplified by PCR and sequenced to identify the genotype and drug resistance mutations if present in the patient’s sample (Steegena et al, 2006). 2.1.8 Diagnosis of HIV infection in Ghana Rapid antibody assays such as First Response (Premier Medical Corporation Ltd, India) and OraQuick (OraSure Technologies Inc., USA) are used to screen individuals for their HIV status. The algorithm requires that results are declared when the outcome of two rapid assays are consistent. Inconsistent results are clarified on a line immunoblot assay such as Inno-lia HIV I/II Score (Innogenetics, Belgium), Inno-lia is also used to determine the type of HIV infection: HIV-1 or HIV-2. For large scale screening, during surveillance and after a blood donation exercise, ELISAs are used. Fourth generation ELISAs are used for detection of both University of Ghana http://ugspace.ug.edu.gh 27 antigen and antibody (Branson 2007). When there are occupational exposures to infectious agents, qualitative PCR is used to determine HIV infection status within 2 weeks of exposure. Qualitative PCR is also used to clarify the HIV status of babies born to HIV infected mothers. 2.2 Antiretroviral drugs and therapy The development of inhibitors of the reverse transcriptase and protease, two of three essential enzymes of HIV-1 in the mid-1990s revolutionized the treatment of HIV-1 infection (Arts and Hazuda, 2012). The introduction of drug regimens that combined these agents further enhanced the overall efficacy and durability of antiretroviral therapy (Collier et al, 1996; D'Aquila et al, 1996; Staszewski et al, 1996). Drugs from five distinct classes are available to treat HIV infection (Arts and Hazuda, 2012). The drug classes are: 1. Nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs) 2. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) 3. Protease inhibitors (PIs) 4. Entry inhibitors or fusion blockers 5. Integrase inhibitors Reverse transcriptase (RT) was the first HIV enzyme to be exploited for antiretroviral drug discovery (Young 1988). This enzyme is the target for two distinct classes of antiretroviral agents; the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleoside reverse transcriptase inhibitors (NNRTIs). Nucleoside reverse transcriptase inhibitors were the first class of drugs to be approved by the FDA (Young, 1988). Nucleoside reverse transcriptase inhibitors act as DNA-chain terminators and inhibit reverse transcription of the viral RNA University of Ghana http://ugspace.ug.edu.gh 28 genome into DNA, a crucial event occurring at an early stage of the viral life cycle. They are administered as pro-drugs and require host cell entry and phosphorylation by cellular kinases into their functional forms before enacting an antiviral effect (Mitsuya et al, 1985; Furman et al, 1986; Mitsuya and Broder, 1986; St Clair et al, 1987; Hart et al, 1992). Non-nucleoside reverse transcriptase inhibitors bind near the active site of RT, causing conformational change in the enzyme’s active site and inhibiting reverse transcription of the viral RNA (Kohlstaedt et al, 1992; Tantillo et al, 1994; Spence et al, 1995). Protease inhibitors target the viral protease, an enzyme required for the cleavage of precursor proteins (gag and gag–pol), and inhibit the final assembly of the inner core of viral particles (Hartman and Buckheit, 2012). The integrase inhibitors prevent the incorporation of the viral DNA into the host genome by the enzyme integrase while the entry inhibitors block the fusion and penetration of HIV virions into their target cells (Arts and Hazuda, 2012). Twenty-three additional therapeutic agents have been approved for use in humans since the approval of zidovudine or azidothymidine (AZT) for the treatment of HIV-1 infection (US Food and Drug Administration, 2011; Arts and Hazuda, 2012). These drugs are listed below and the chemical structures of some of them are shown in figure 5. 1. NRTIs - abacavir (ABC, Ziagen), didanosine (DDI, Videx), emtricitabine (FTC, Emtriva), lamivudine (3TC, Epivir), stavudine (d4T, Zerit), zidovudine (AZT, Retrovir), and Tenofovir disoprovil fumarate (TDF, Viread), a nucleotide RT inhibitor; 2. NNRTIs - Rilpivirine (Edurant), Etravirine (Intelence), Delavirdine (DLV, Rescriptor), Efavirenz (EFV, Sustiva), Nevirapine (NVP, Viramune); University of Ghana http://ugspace.ug.edu.gh 29 3. Protease Inhibitors - Amprenavir (APV, Agenerase), Tipranavir (TPV, Aptivus), Indinavir (IDV, Crixivan), Saquinavir mesylate (SQV, Invirase), Lopinavir/Ritonavir (LPV/r, Kaletra), Fosamprevir calcium (FOS-APV, Lexiva), Ritonavir (RTV, Novir), Darunavir (Prezista), Atazanavir sulfate (ATV, Reyataz) and Nelfinavir mesylate (NFV, Viracept); 4. Fusion inhibitor: Enfuvirtide (T-20, Fuseon); entry inhibitors: Maraviroc (Selzentry) and 5. HIV integrase strand transfer inhibitors - Raltegravir (Isentress). University of Ghana http://ugspace.ug.edu.gh 30 Figure 5: Chemical structures of some antiretroviral drugs (Source: Izzedine et al., 2001) University of Ghana http://ugspace.ug.edu.gh 31 Clinical experience with all HIV agents has shown that HIV is able to clearly evade the antiviral effect of any drug used as mono-therapy through the rapid accumulation of amino acid changes in the targeted proteins - reverse transcriptase, protease, envelope, and integrase (Larder, 1995). This is due to the error-prone nature of the reverse transcriptase enzyme that catalyses the synthesis of a DNA copy of the viral RNA genome resulting in many quasispecies of the virus within an infected person. Some of the mutant are escape variant and are therefore become resistant to the antiviral in use as a monotherapy. Highly active antiretroviral therapy (HAART), which involves the combination of antiretroviral drugs from different drug classes, has improved the outcome of treatment of HIV infection. HAART regimens generally comprise three antiretroviral drugs; usually two nucleoside analogues and either a protease inhibitor or a non-nucleoside reverse-transcriptase inhibitor (Yeni et al., 2002). The use of agents from different classes is instrumental in controlling the development of resistance. 2.3 Mechanism of resistance to antiretroviral drugs Various mechanisms of resistance have been identified that differ for different drug classes and in some cases for drugs in a given class (Clavel and Hance, 2004). Drug resistance is a result of amino acid changes that occur in the genes of the HIV proteins that the drugs target (Larder et al, 1995). These mutations are presented as a letter (the name of amino acid in the drug sensitive HIV), followed by a number (the amino acid codon at which the change has occurred) and another letter (the amino acid in the drug-resistant HIV). For example the mutation K103N in the RT gene represents the substitution of Lysine (K) with Asparagine (N) at codon 103 in the reverse transcriptase gene. Appendix VI has the one-letter codes and acronyms of the amino acids. University of Ghana http://ugspace.ug.edu.gh 32 2.3.1. Resistance to nucleoside and nucleotide reverse-transcriptase inhibitors Nucleoside and nucleotide reverse transcriptase inhibitors (NRTI) arrest the synthesis of viral DNA by reverse transcriptase. These drugs resemble the natural nucleoside used for DNA polymerization except they lack a 3' OH group. They are incorporated into the growing viral DNA chain instead of the normal nucleoside and because they lack a 3' hydroxyl group needed to form the phosphodiester bond, there is chain termination, and the synthesis of viral DNA is aborted. Chain termination can occur during RNA-dependent DNA or DNA- dependent DNA synthesis, inhibiting the production of either the minus or plus strands of the HIV-1 proviral DNA (Cheng et al., 1987; Balzarini et al., 1989; Richman, 2001). Two distinct mechanisms are involved in the resistance by HIV to NRTIs (Zdanowicz, 2006: (1) impairment of the incorporation of the analogue into DNA and (2) removal of the analogue from the prematurely terminated DNA chain. These two mechanisms are illustrated in Figure 6. 2.3.1.1 Impairment of Analogue Incorporation Several mutations or groups of mutations in reverse transcriptase can promote resistance by selectively impairing the ability of reverse transcriptase to incorporate an analogue into DNA. The substitution of methionine by valine at position 184 in the reverse transcriptase, which is described as M184V, is the main mutation that confers resistance to lamivudine. Methionine 184 is found at the heart of the catalytic site of reverse transcriptase. A valine substitution at this position interferes with the proper positioning of lamivudine triphosphate within the catalytic site because valine has a different side chain and this affects the folding of the protein in the active site (Sarafianos et al., 1999). University of Ghana http://ugspace.ug.edu.gh 33 Other mutations include the Q151M complex of mutations and the K65R mutation (Zdanowicz, 2006). The group of mutations referred to as the Q151M complex is most often selected in the course of the failure of regimens containing stavudine and didanosine (Iversen et al., 1996). This pathway always starts with the Q151M substitution, located in the immediate vicinity of the nucleotide binding site of reverse transcriptase. Once Q151M develops, other secondary mutations that enhance resistance follow and increase the activity of the enzyme (Kosalaraksa et al., 1999). The Q151M complex is relatively rare (less than 5 percent of all HIV-1 strains with resistance to nucleoside analogues) but can confer high-level resistance to all NRTI except lamivudine and tenofovir (Iversen et al., 1996, Shafer et al, 2000). The Q151M mutation complex is more commonly found in HIV-2 drug resistance (Trevin et al, 2011). The K65R mutation is often associated with treatment failure after tenofovir or abacavir use. This mutation appears to confer resistance to most analogues, with the exception of zidovudine (Zdanowicz, 2006). 2.3.1.2 Removal of the Analogue from the Terminated DNA Chain Removal of the nucleoside analogue from the terminated DNA chain is associated with a group of mutations commonly called “thymidine analogue mutations” (TAMs). Mutations from this group most frequently arise during treatment with drug combinations that include thymidine analogues, such as zidovudine and stavudine. These mutations can however promote resistance to almost all nucleoside and nucleotide analogues, including tenofovir (Larder and Kemp, 1989; Shafer et al., 1996; Picard et al., 2001). These mutations occur gradually, and their order of emergence can vary (Boucher et al., 1992). Thymidine analogue mutations (M41L, D67N, K70R, L210W, 215Y/F and K219Q/E) promote resistance by promoting adenosine triphosphate (ATP)- or pyrophosphate (PPi) - University of Ghana http://ugspace.ug.edu.gh 34 mediated removal of nucleoside analogues from the 3' end of the terminated DNA strand (Arion et al., 1998; Meyer et al., 1999). Adenosine triphosphate and pyrophosphate are abundant in normal lymphocytes but are usually not involved in the DNA-polymerization reaction. However, when reverse transcriptase expresses TAMs, its acquired structure allows entry of ATP and pyrophosphate into a site close to the incorporated analogue (Boyer et al, 2001; Chamberlain et al., 2002). In this position, ATP or pyrophosphate can attack the phosphodiester bond that links the analogue to the growing DNA chain leading to the removal of the analogue. This promotes the building of the growing DNA chain and enhances viral replication. University of Ghana http://ugspace.ug.edu.gh 35 Figure 6: An illustration of two mechanisms of resistance to nucleoside reverse transcriptase inhibitors (NRTIs). (A) Mutations in the RT gene, such as thymidine analogue mutations, aid the ATP- or PPi- mediated removal of the incorporated NRTI such as zidovudine (AZT) from the growing DNA chain and allows polymerization to continue. (B) Mutations in the RT gene cause stearic hindrance in the active site of the enzyme and prevent certain NRTI such as lamivudine from binding and being incorporated during reverse transcription thus permits polymerization in the presence of the drug. RNA is shown with white circles and DNA with black circles. Red ‘Z’ stands for AZT and Black ‘A’ stands for ATP. (Source: www.openi.nlm.nih.gov) University of Ghana http://ugspace.ug.edu.gh 36 2.3.2 Resistance to non-nucleoside reverse-transcriptase inhibitors Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) are small molecules that have a strong affinity for a hydrophobic pocket located close to the catalytic domain of the reverse transcriptase. The binding of the inhibitors prevents the conformation change needed at the active site of the enzyme, thereby inhibiting the enzyme’s ability to synthesize DNA (Esnouf et al., 1997). Figure 7 is an illustration of the mechanism of action and resistance to non- nucleoside reverse transcriptase inhibitors. The mutations that are selected for after the failure of treatment with NNRTIs are all located in the pocket targeted by these compounds, and they reduce the affinity of the drug (Boyer et al., 1993; Richman et al., 1994; Bacheler et al., 2000; Ren et al., 2001; Hsiou et al., 2001). The mutations that emerge are however drug-dependent due to some differences in the interaction of the different NNRTIs with the hydrophobic pocket (Boyer et al., 1993). For instance, nevirapine resistance is often associated with Y181C but other mutations, such as Y188C, K103N, G190A, and V106A also occur (Shafer et al, 2000). Initial resistance to efavirenz is generally characterized by the K103N mutation, but the Y188L mutation is also seen (Boyer et al., 1993). Cross resistance usually occurs with NNRTI because of the similarity in their mechanism of action. Therefore when resistance develops from the use of one drug, it is highly likely that other drugs in the same class may be affected (Zdanowicz, 2006). A crystal structure of HIV- 1 reverse transcriptase showing positions of drug resistance mutations is presented in Figure 8. University of Ghana http://ugspace.ug.edu.gh 37 Figure 7: An illustration of the mechanism of action of and resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs). The NNRTIs bind in a hydrophobic pocket adjacent to the active site of the reverse transcriptase enzyme as shown in (A) and inhibit the binding of substrates at the active site for polymerization to occur. In a drug-sensitive HIV-1 strain (B) polymerization is blocked. In a drug-resistant HIV-1 strain (C), mutations in the hydrophobic pocket prevent NNRTI from binding therefore polymerization continues in the presence of the drug. (Source: www.depts.washington.edu) A C B University of Ghana http://ugspace.ug.edu.gh 38 Figure 8: Structure of HIV-1 reverse transcriptase coupled with HIV RNA template (pink ribbon) and primer (Brown ribbon) showing positions of drug resistance mutations. Light blue balls indicate positions of NNRTI resistance mutations and yellow balls indicate positions of NRTI resistance mutations. The active is shown by a white arrow. (Source: http://home.ncifcrf.gov) University of Ghana http://ugspace.ug.edu.gh 39 2.3.3 Resistance to protease inhibitors Viral proteins are first synthesized as large polypeptides and then processed by the viral protease into functional proteins. The protease inhibitor (PI) group of drugs is designed to block this step in the replication of the virus (Roberts et al, 1990; Erickson and Kempf, 1994). The HIV protease cleaves large polyprotein precursors at specific sites, releasing the structural proteins and enzymes necessary for the assembly of infectious viral particles. When the protease action is inhibited, viral particles are produced, but they are immature and are not infectious (Clavel and Hance 2004). The protease of HIV is composed of non-covalently associated structurally identical monomers, each 99 amino acids long (Shafer et al, 2000). It has a central, symmetric, substrate-binding cavity (Zdanowicz, 2006) covered by a mobile flap that moves to allow substrate polypeptides in and functional protein products out (Shafer et al, 2000). Protease inhibitors are similar in structure to the viral peptides that are normally recognized and cleaved by the protease and so compete with the normal substrates for the binding site of the enzyme (Roberts et al., 1990; Erickson and Kempf, 1994). These compounds display a stronger affinity for the active site of the HIV protease and inhibit the catalytic activity of the enzyme in a highly selective manner. Resistance to protease inhibitors is the consequence of amino acid substitutions that emerge either inside the substrate-binding domain of the enzyme or at distant sites and affects the folding of the protease (Kaplan et al., 1994; Condra et al., 1995; Molla et al., 1996; Zhang et al, 1997). These amino acid changes affect the binding of the inhibitors to the active site of the protease, thereby reducing their affinity for the enzyme compared to the natural substrate (Chen et al., 1995, Ridky et al., 1998, Hong et al., 2000; Prabu-Jeyabalan et al., 2002). University of Ghana http://ugspace.ug.edu.gh 40 Protease inhibitor mutations can be classified into three: those occurring in the substrate binding site (protease substrate cleft mutations), those occurring in the flap region that covers the substrate binding cleft and those occurring at other sites close to the active site of the enzyme (Shafer et al, 2000). Mutations occurring at positions 23, 30, 32, 47, 48, 50, 82 and 84 are substrate binding cleft mutations (Shafer et al, 2000; Shafer and Schapiro, 2008). These mutations alter the size of the substrate binding domain and enhance the preferred binding of the natural viral protein substrate in the presence of the most inhibitors (Prabu- Jeyabalan et al., 2002). The protease flap mutations occur at codon 46 and 54. This region is known to extend over the substrate binding cleft and must be flexible enough to allow for the polypeptide substrate to enter and the functional proteins to exit the substrate binding domain. Changes in amino acids in this region therefore inhibit the flexibility required for the protease activity. Mutations at codons 76, 88 and 90 are known to be close to the active site of the protease but their mechanism of resistance is not known (Shafer et al, 2000). Figure 9 is a crystal structure of HIV-1 protease indicating positions of drug resistance mutations. Due to differences in the chemical structure of the inhibitors and their peculiar interaction with the substrate-binding domain of the enzyme, some mutations are selected for only specific protease inhibitors. However, there is considerable overlap between the combinations of mutations and this is responsible for the wide cross-resistance that is generally observed within this drug class (Schapiro et al., 1999; Hertogs et al., 2000). University of Ghana http://ugspace.ug.edu.gh 41 Figure 9: The structural model of HIV-1 protease homodimer showing positions of protease inhibitor resistance mutations in one subunit. The polypeptide backbone of both protease subunits (positions 1 to 99) is shown. The active site (codons 25 to 27 from both subunits), is displayed in ball and stick mode. The protease was co-crystallized with indinavir, which is displayed in space-fill mode. This model is based on a structure published by Chen et al, 1994 University of Ghana http://ugspace.ug.edu.gh 42 2.3.4. Resistance to fusion inhibitors Human immunodeficiency virus enters target cells through an intricate sequence of interactions between the HIV envelope glycoprotein (gp) complex (gp120–gp41) and specific cell-surface receptors CD4, CCR5 and CXC4 (Kilby and Eron, 2003). First, gp41 interacts with the cell membrane and draws the virus closer to its target. The distal hydrophobic region of gp41, HR2, folds onto a more proximal hydrophobic region, HR1. This brings the membranes of the virus and target cell into close proximity and enhances the fusion of the membranes to allow for viral entry. Enfuvirtide, a 36-aminoacid peptide derived from HR2, destabilizes this process by binding to HR1, preventing the folding of gp41 and blocking the fusion of the target cell and viral membranes and consequently viral entry (Fig. 10). Viral resistance to enfuvirtide usually results from mutations located in a stretch of 10 amino acids within HR1 (Rimsky et al., 1998; Wei et al., 2002). These mutations prevent enfuvirtide from binding to HR1 and thus allow the fusion of the virus to the cell membrane and enhance viral entry (Fig. 10). Changes in amino acids in gp41 outside HR1 — and even changes in gp120 — appear to be associated with significant differences in the susceptibility of the virus to enfuvirtide (Derdeyn et al., 2001; Reeves et al., 2002). University of Ghana http://ugspace.ug.edu.gh 43 Figure 10: Illustration of mechanisms of action of and resistance to a fusion inhibitor Enfuvirtide (T-20). A) The inhibitor disrupts the interaction between the HR1 and HR2 domains of gp41 and prevents the fusion of the viral envelop with the host cell membrane. B) Drug resistance mutations in the envelop gene of HIV-1 (gp 41) reduce the ability of the inhibitor to bind therefore permits the fusion of the viral envelope to the host cell membrane. (Image Source: www.immunopaedia.org.za) A B University of Ghana http://ugspace.ug.edu.gh 44 2.4 Development of HIV Drug Resistance Many factors influence the rapid and widespread emergence of drug resistance that is seen in HIV. Key among them are the extremely high replication rate of HIV and the lack of proof reading activity by the viral RT (Clavel and Hance, 2004). In an untreated individual, there are 104–105 or more HIV-1 particles per ml of plasma, which turn over at a rate of ~1010 per day (Ho et al., 1995; Wei et al., 1995; Perelson et al., 1996) due to the exceptionally high rate of replication. Owing to the error-prone reverse transcription process, it is estimated that one mutation is introduced for every 1000–10,000 nucleotides synthesized (Mansky and Temin, 1995; O’Neil et al., 2002; Abram et al., 2010). With a genome size of ~10,000 nucleotides in length, 1 to10 mutations may be generated in each viral genome with every replication cycle. Majority of these mutations are base substitutions but insertions and duplications can also occur (Zdanowicz, 2006). This means that numerous HIV ‘‘variants’’ or quasi species are rapidly formed and propagated within an infected person. With this enormous potential for generating genetic diversity, HIV-1 variants with reduced susceptibility to any one or two drugs will often preexist in the viral quasi species before initiation of therapy (Coffin, 1995). While some HIV variants may exhibit intrinsic or ‘‘primary’’ resistance to antiretroviral agents, most drug resistance develops as a result of exposure to the drugs. The presence of the drug pressure is known to select for the drug resistance variants. Even during successful therapy, when there is viral suppression, drug resistance can still occur due to residual viral replication (Martinez-Picado et al, 2000). Any mutations that confer a selective advantage to a particular viral variant will allow that particular viral variant to predominate. Thus, the very use of antiviral drugs exerts a ‘‘selective pressure’’ that favors propagation of resistant viruses. The emergence of resistant single mutants can occur in a matter of weeks (Clavel and Hance, 2004). However some single mutations can only induce low-level resistance to some University of Ghana http://ugspace.ug.edu.gh 45 drugs. In such cases, high levels of resistance or complete resistance requires the gradual accumulation of additional mutations. Combination therapy can block this selection process for two reasons. First, multiple mechanisms are required for resistance to occur to all drugs in the regimen. Even if a small number of variants with the potential for resistance to individual agents exist before treatment, they may not be able to resist all the drugs in the regimen. Secondly, multiple drugs suppress viral replication more effectively than single agents (Gulick et al., 1997; Hammer et al., 1997). In patients who receive a triple combination of antiretrovirals from the onset of therapy, emergence of drug resistance results only if HIV continues to replicate in the presence of drugs. In these cases, drug levels are insufficient to block viral replication completely but sufficient to exert positive selective pressure on variants with decreased drug susceptibility. Under these conditions, viruses with resistance to all the components of the regimen will gradually emerge. Thus during HAART, drug resistance is most often the consequence of initial treatment failure. The resistance subsequently leads to increased treatment failure (Clavel and Hance, 2004). Additional factors that may contribute to the development of HIV drug resistance include poor patient adherence, sub-therapeutic blood levels of antiretroviral agents, and inappropriate choice of antiretroviral agent(s) (Zdanowics, 2006). Poor oral absorption, alteration of drug metabolizing enzymes by other agents, and various drug-drug interactions could affect blood levels of the drugs (Zdanowicz, 2006). Highly active antiretroviral therapy becomes successful when drug combinations that decrease the probability of selecting virus variants with multiple mutations and conferring resistance to a three-antiretroviral-drug regimen are used. University of Ghana http://ugspace.ug.edu.gh 46 Cross resistance of HIV is also of great concern. This phenomenon involves development of HIV resistance to drugs within a particular class or with similar mechanism of action, but to which the patient has not been exposed (Zdanowicz, 2006). The emergence of cross-resistant viruses during treatment tends to limit future drug options. 2.5 Markers for monitoring patients on ART As antiretroviral treatment (ART) for HIV infection became increasingly available in resource-limited settings, there was great concern regarding the development of drug resistance (Spacek et al., 2006). While there was general agreement that potential drug resistance should not deter treatment efforts, it was necessary to initiate the monitoring of the development and prevalence of drug resistance (W.H.O. Draft Guidelines for Surveillance of HIV Drug Resistance, 2003). Individual drug resistance testing was recommended for treatment monitoring but is largely unavailable in resource-limited settings. CD4 cell count is available in many such settings and HIV viral load in some but genotyping tests are rarely performed (Badri et al, 2008; WHO Draft Guidelines for Surveillance of HIV Drug Resistance, 2003). 2.5.1 Using immunologic and virologic markers In 2003, the vast majority of Africans treated with ART were not monitored with viral load testing. This was due to the cost and complexity of providing a reliable quantitative HIV RNA viral load service in resource-limited settings (Crowe et al., 2003; Fiscus et al., 2006). Early detection of virologic failure is however important for optimal management of HIV-infected patients receiving ART. University of Ghana http://ugspace.ug.edu.gh 47 In the absence of viral load monitoring however patients continue to take first-line ART for as long as their CD4 counts are increasing or stable. During this period some may experience undetected virologic failure which may lead to accumulation of drug resistance mutations. Moreover, accumulation of multiple antiretroviral drug resistance mutations may compromise the response to future drugs and fuel the spread of primary drug resistance within communities (Boucher et al., 1993; Kantor et al., 2004; Napravnik et al., 2005) even in patients whose viral loads are being monitored. Martinez-Picado et al (2000) also reported that antiretroviral-resistant HIV can be selected from residual virus replication during HAART in the absence of sustained rebound of plasma HIV-1 RNA (Martinez-Picado, et al., 2000). The World Health Organization (WHO) has recommended the use of CD4 cell count measurements and clinical outcomes for monitoring ART in the absence of viral load (WHO, 2006). However, the clinical outcomes and CD4 cell count changes that are able to predict virologic failure have not been identified. It is not clear whether the variability in CD4 cell count measurements adequately reflects the variability in viral load (Badri et al., 2008). Jevtovic et al (2005) reported that HAART may allow for the reconstitution of immune functions in most treated HIV patients but cause discrepant responses in some patients. These responses may include failure to achieve a significant increase in circulating CD4+ T cells despite undetectable plasma viral loads or a good immunological response while not reaching undetectable viremia (Jevtovic et al, 2005). Some other researchers (Florence et al., 2004; Bisson et al., 2006; Moore et al., 2006) found that a gain in CD4 cell count was useful to detect viral suppression in patients on ART. However, Badri et al. (2008) have shown that although changes in CD4 cell count correlated significantly with viral load at a group level, they had very poor predictive value when being used to assess individual patients. Meya et al. University of Ghana http://ugspace.ug.edu.gh 48 (2009) also found no significant difference in CD4 lymphocyte count gain between those with and without viral failure and concluded that CD4 cell count gain from baseline was not associated with viral outcome. Thus, CD4 cell count measurements cannot be used to substitute viral loads for measuring virologic failure. 2.5.2 HIV Drug resistance testing The use of drug resistance testing has become an integral part of HIV clinical care. The first clinical description of HIV resistance to antiretroviral drugs was published in 1989, in patients taking zidovudine monotherapy (Larder et al., 1989). Accumulation of mutations within the reverse transcriptase gene resulted in a marked increase in drug resistance (Larder et al., 1989). Human immunodeficiency virus variants resistant to every available antiretroviral agent have been identified in viral culture in the presence of drug and in treated HIV-infected patients (Shafer and Schapiro, 2008). The evolution of drug resistance has significant clinical implications for choosing effective antiretroviral regimens (Hirsch et al., 2008). Drug resistance testing is recommended by the International Society of AIDS, USA panel of experts for the management of HIV/AIDS patients. Two types of assays, phenotypic or genotypic, are used for resistance testing. These assays detect resistance in fundamentally different ways, although the results generally correlate with each other (Zdanowicz, 2006). 2.5.2.1 Genotypic Assays Genotypic testing is the most commonly used method of detecting resistant HIV-1 strains and is one of the earliest applications of gene sequencing for clinical purposes (Shafer, 2002). Genotypic testing has the ability to detect mutations present as mixtures, even if the mutation is present at a level too low to affect drug susceptibility in a phenotypic assay. Genotypic assays also provide insight into the potential for resistance to emerge and are advantageous University of Ghana http://ugspace.ug.edu.gh 49 because they can detect transitional mutations that indicate the presence of selective drug pressure but do not cause drug resistance by themselves (Shafer, 2002). Genotypic assays detect drug-resistance mutations present in relevant viral genes. Most genotypic assays involve sequencing of the pol gene that encodes the reverse transcriptase (RT) and protease (PR) genes to detect mutations that are known to confer drug resistance (Shafer, 2002). Genotypic assays that assess mutations in the integrase and gp41 (envelope) genes are also commercially available. Genotypic assays can be performed rapidly with results available within 1–2 weeks of sample collection. Plasma is the main source of virus used for tests of HIV-1 drug resistance in clinical settings. HIV-1 genotyping requires extraction, reverse transcription and amplification of viral gene. The sensitivities of most genotypic assays is generally reduced to 1000 RNA copies/ml (Shafer 2002). Direct sequencing of PCR products is usually done in clinical settings since it is faster and more affordable than clonal sequencing, which is mostly used in research settings. Direct sequencing is able to detect nucleotide mixtures when the least common nucleotide is present in at least 20% of the total virus population (Larder et al, 1993; D’Aquila et al, 2000). Dideoxynucleotide sequencing is the most commonly used method for genotyping (Shafer 2002). Commercial HIV-1 reverse transcriptase and protease genotyping kits such as ViroSeq and TRUGENE are available but are more expensive than in-house methods. The commercially available kits may have stronger quality control and validation profiles but they have similar performances when compared to in-house assays based on the dideoxynucleotide sequencing method (Erali et al, 2001; Shafer 2002). University of Ghana http://ugspace.ug.edu.gh 50 Previous studies that compared results from different laboratories showed that the dideoxynucleotide sequencing method was intrinsically reliable and results were reproducible for HIV-1 genotyping of cultured isolates (Husumi et al, 1992). The reproducibility of the dideoxynucleotide method was further attested by results from two clinical laboratories that sequenced protease and reverse transcriptase genes using plasma aliquots of 46 heavily treated patients. Although these laboratories used different in-house protocols with reagents from Applied Biosystems (Foster City, California), there was 99% sequence concordance between them (Shafer 2002). Quality control is required for genotyping assays and aims at preventing PCR contamination and sample mix-up. Physical separation of pre-amplification and post-amplification areas is usually recommended (Kwok and Higuchi, 1989). Inclusion of negative controls in each stage of the PCR process is also advised. Construction of phylogenetic trees with newly generated sequences and other sequences generated earlier from the same laboratory can also help to detect high levels of similarity between different sequences due to cross- contamination (Shafer 2002). Interpretation of test results requires knowledge of the mutations that different antiretrovirals select for and of the potential for cross resistance to other drugs conferred by certain mutations (Garcia-Lerma and Heneine 2002). The International AIDS Society-USA (IAS- USA) maintains a list of updated significant resistance-associated mutations in the reverse transcriptase, protease, integrase, and envelope genes. The latest of these lists was published in 2011 (Johnson et al, 2011). The Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu) also provides helpful guidance for interpreting genotypic resistance test results (Liu and Shafer, 2006). Other tools such as RegaDB (Rega Instituut, KU Leuven, University of Ghana http://ugspace.ug.edu.gh 51 French ‘Agence Nationale de Recherche sur le Sida’- ANRS program (France), Visible Genetics Inc. (Toronto) are also available to assist in interpreting genotypic test results (Torti et al., 2005; Flandre and Costagliola, 2006; Vercauteren and Vandamme, 2006; Gianotti et al., 2006). Clinical trials have demonstrated the benefit of consultation with specialists in HIV drug resistance to improve virologic outcomes (Tural et al., 2002). 2.5.2.1.1 Genotypic DR testing using peripheral blood mononuclear cells The majority of HIV-1 genotyping assays analyze viral RNA from plasma. Proviral DNA from peripheral blood mononuclear cells is an alternative marker for studying drug resistance (Chew et al, 2005). Proviral DNA is known to persist in infected cells, even after prolonged highly active antiretroviral therapy (HAART) has reduced plasma RNA viral load to undetectable levels (Chew et al, 2005). Data regarding detection of HIV-1 drug resistance mutations in proviral DNA has increased. Some researchers found key mutations in proviral DNA that were not present in plasma viral RNA (Riva et al., 2001; Chew et al., 2005; Bona et al., 2007; Wang et al., 2007). Using direct sequencing, these researchers observed that key mutations conferring resistance to reverse transcriptase inhibitors were found more frequently in proviral DNA than in plasma viral RNA. Major mutations in the protease region were only found in PBMCs. Wang et al. (2007) observed concordance between HIV strains in plasma and PBMC of treatment-naïve patients. Early establishment of the viral reservoir in patients acquiring resistant strains at primary HIV-1 infection has been previously described (Ghosn et al., 2006). They also showed that resistance associated mutations with similar profiles in paired plasma RNA and PBMC DNA persisted in such patients. In a case study, Usuku et al (2006) followed the changes in drug resistance mutations in a patient receiving HAART. They found out that the mutations University of Ghana http://ugspace.ug.edu.gh 52 detected in the plasma were infrequently detected in the proviral DNA. While plasma contains population of actively replicating viruses, the peripheral blood mononuclear cells contain a mix of newly synthesized HIV RNA, integrated and un-integrated HIV DNA. Thus the turnover in plasma is much higher than that in the peripheral blood mononuclear cells (Perelson et al., 1996; Kaye et al., 1995). Patterns of drug resistance mutations showed that the viral populations in PBMCs are more heterogeneous; that PBMCs included archival species that reflected the treatment history of the patient, with all the changes accumulated over the treatment period as well as the wild type viruses (Shafer 2002; Quan et al., 2008). The plasma viruses are related to the most recent treatment and represent a more homogeneous virus population during treatment. Using archival HIV DNA present in PBMCs will provide additional information on drug resistance mutations that could emerge after a failing therapy is changed based on genotypes detected in plasma. Also, sequencing HIV DNA from PBMCs may be useful in patients with undetectable viral loads (Sarmati et al. 2003). 2.5.2.2 Phenotypic Assays Phenotypic assays measure the extent to which an antiretroviral drug inhibits virus replication in vitro (Garcia-Lerma and Heneine, 2002). Reverse transcriptase and protease genes sequences and, more recently, integrase and envelope sequences derived from patient plasma HIV RNA are inserted into the backbone of a laboratory clone of HIV or used to generate pseudotyped viruses that express the patient-derived HIV genes of interest. These viruses are cultured at different drug concentrations, monitored by expression of a reporter gene and are compared with the growth of a reference HIV strain. The drug concentration that inhibits viral replication by 50% (IC50) is calculated, and the ratio of the IC50 of test and reference viruses University of Ghana http://ugspace.ug.edu.gh 53 is reported as the fold increase in IC50 (i.e., fold resistance) [Garcia-Lerma and Heneine, 2002]. Automated commercial phenotypic assays are commercially available with results reported in 2–3 weeks (Garcia-Lerma and Heneine, 2002). These include Antivirogram (Tibotec-Virco, Belgium), PhenoSense HIV-1 Drug Resistance Assay (ViroLogic Inc., South San Francisco, USA) and PhenoScript (VIRalliance, Paris, France). However, phenotypic assays cost more to perform than genotypic assays. Although clinically significant fold cut-offs are now available for some drugs, the interpretation of phenotypic assay results is complicated by incomplete information on specific resistance level (i.e. fold increase in IC50) that is associated with drug failure (Lanier et al., 2004; Miller et al., 2004; Naeger and Struble, 2006, 2007; Flandre et al., 2007). 2.6 The HIV/AIDS Epidemic Human Immunodeficiency Virus (HIV) was identified as the aetiologic agent of Acquired Immune Deficiency Syndrome (AIDS) in the early 1980s (Barre-Sinoussi et al., 1983, Popovic et al., 1984; Gallo et al., 1984). Since then the disease has continued to spread throughout the world claiming the lives of many adults and children and in some cases families and households. According to the statistics of the global HIV and AIDS epidemic published by UNAIDS, WHO and UNICEF in November 2011, 30 million people have died of AIDS since the beginning of the epidemic and 34 million people were living with HIV/AIDS by the end of 2010. Of these, 22.9 million live in sub-Saharan Africa. With around 68 % of all people living with HIV residing in sub-Saharan Africa, the region carries the greatest burden of the epidemic. Epidemics in Asia have remained relatively stable and are still largely concentrated among high-risk groups. Conversely, the number of people living University of Ghana http://ugspace.ug.edu.gh 54 with HIV in Eastern Europe and Central Asia has more than tripled since 2000 (UNAIDS, 2011). HIV/AIDS has had a great impact on society, both as an illness and as a source of discrimination. The disease also has significant economic impacts. 2.6.1 HIV/AIDS in Ghana Ghana is a West African country located on the coast with a land mass of 238,537 sq. km. (www.ghana.gov.gh, 2013). It is an English-speaking country that shares boarders with three francophone countries: Togo to the East, La Cote D’Ivoire to the West and Burkina Faso to the North. The south of the country is bounded by the Gulf of Guinea. The map of West Africa showing the location of Ghana is presented in Fig. 10. Ghana’s population was estimated at 25 million in 2010 (www.ghana.gov.gh, 2013). The country has 10 administrative regions namely Greater Accra, Western, Eastern, Volta, Central, Ashanti, Brong Ahafo, Northern, Upper West and Upper East regions. The country is further divided into 212 districts (ghana.gov.gh, 2013). University of Ghana http://ugspace.ug.edu.gh 55 Figure 11: The map of West Africa showing Ghana (arrowed). Source: http://maps.google.com University of Ghana http://ugspace.ug.edu.gh 56 The first case of HIV in Ghana was reported in 1986 (Neequaye et al, 1987; Hishida et al, 1994). In that year, 1100 persons were screened and 10% of them were seropositive. The positive cases were skewed towards the females in a ratio of 9:1. These earlier infections with HIV were due mainly to HIV-2 (Kawamura et al, 1989; Hishida et al, 1994, Takehisa et al, 1997). Later, Brandful et al. (1997) found increasing numbers of HIV-1 infections in Ghana in the mid 1990s (Brandful et al, 1997; Ampofo et al, 1999). Since then, the number of reported cases has steadily increased and HIV infections in the country have been predominantly due to HIV-1 (Brandful et al, 1997, Brandful et al, 1998), with HIV-2 and HIV-1/HIV-2 co-infections contributing to approximately 5% of the infections (Bonney et al, 2008). The HIV Sentinel Survey (HSS) by the National AIDS/STI Control Programme of the Ghana Health Service, which was started in 1992, has consistently provided epidemiological data on HIV trends in Ghana. An estimated 217,428 persons, representing 1.42% of the adult populations, were infected with the virus in 2011 (National AIDS Control Programme, Ghana, 2011). This prevalence is known to be on a decline from the estimated 1.57% in 2009 and 1.49% in 2010. HIV-1 contributed 98% of the infection while 0.7% was due to HIV-2. HIV-1/HIV-2 co-infections made up 1.3% (National AIDS Control Programme, Ghana, 2011) is The predominant HIV-1 subtype is CRF02_AG circulating in Ghana with unique recombinant forms (URFs) [Delgado et al., 2008; Brandful et al., 2012]. The number of adults receiving ART in Ghana has greatly increased from 1,804 in 2004 to 73,339 in 2012 (NACP, 2013). However, this figure represents 69% of adults needing ART in the country. Antiretroviral drugs available in Ghana include the NRTIs: stavudine, lamivudine, abacavir, didnosine, tenofovir, zidovudine, emtricitabineNNRTIs: efavirenz and nevirapine and PIs: lopinavir/ritonavir and nelfinavir (NACP/MoH/GHS, 2010). With the scale up in ART, there is a need to monitor the emergence of drug resistance which is an University of Ghana http://ugspace.ug.edu.gh 57 inevitable outcome of antiretroviral use. A survey for transmitted drug resistance, conducted by the National AIDS/STI Control Programme, at the two sites in the Eastern region where ART was first started in 2003. This survey showed that the level of transmitted drug resistance at the sites was low (<5%) according to the WHO guidelines for such surveys (HIVDR Threshold Survey, unpublished data). Previous studies have found low levels of drug resistance among drug-naïve persons in Ghana (Kinomoto et al., 2005; Sagoe et al., 2007; Delgado et al., 2008; Brandful et al., 2012). Thus transmitted drug resistance appears not to be a problem in Ghana. However, persons on treatment need to be monitored for drug resistance in order to guide their clinical management and inform the country’s ART policy. Ghana’s National ART Guidelines are based on the World Health Organization’s ART guidelines for resource-poor countries. The national guidelines recommend the use of two NRTI and one NNRTI in the first-line regimen and two NRTI and one PI in the second-line regimen (NACP/MoH/GHS, 2010). The national guidelines recommend the switch from first- line to second line when there is evidence of treatment failure confirmed by CD4 monitoring and viral load where available (NACP/MoH/GHS, 2010). However, due to unavailability of viral load to most patients, the change in regimen is determined by physicians based on CD4 counts and clinical symptoms. In the absence of routine viral load and drug resistance testing, drugs in the new regimen are decided without any information on the resistance profile of the virus circulating in the individual. The lack of virologic and genotypic tests leads to the accumulation of resistance mutations even in the presence of increased or sustained CD4 counts and may limit future treatment options (Kantor et al., 2004; Gupta et al., 2007). Patients on treatment may habour drug resistance mutations to NRTIs at the time of the switch to second- line. Some of these mutations may confer cross-resistance to some of the NRTIs in the second-line regimen and render them ineffective (Sigaloff et al., 2012). University of Ghana http://ugspace.ug.edu.gh 58 2.7 Nucleic acid extraction/isolation methods The extraction or isolation and purification of nucleic acid are key steps for most molecular biology protocols and all recombinant DNA techniques (Brown, 2003). The extraction of nucleic acids from biological material requires cell lysis, inactivation of cellular nucleases, and separation of the desired nucleic acid from cellular debris. Conventional methods usually employ a lysis procedure which is rigorous enough to fragment the complex starting material (e.g. blood or tissue) and inactivate nucleases; yet gentle enough to preserve the target nucleic acid (Roche Applied Science, 2012). Traditional methods for purifying nucleic acids from cell extracts are often combinations of extraction, precipitation, chromatography, centrifugation, electrophoresis, and affinity separation. Solvent extraction is usually used to eliminate contaminants from nucleic acids. There are several methods for removing the proteins from the lysed cell suspension and these include shaking with phenol (Kirby, 1968), shaking with a mixture of phenol, chroroform and isoamyl alcohol (Marmur, 1963) and enzymatic degradation with Pronase or Proteinase K (Hotta and Bassel, 1965). The nucleic acid is selectively precipitated by the addition of absolute ethanol or isopropanol. Precipitation with alcohol concentrates the high molecular weight nucleic acids whilst eliminating the small nucleic acid fragments, detergent and organic solvents used in the removal of proteins (Rodriguez and Tait, 1983). A subsequent wash with 70% ethanol and a brief centrifugation removes salt and moisture (Brown, 2003). Unfortunately, most of these methods require extensive handling of toxic chemicals (e.g. phenol or ethidium bromide), need expensive equipment (e.g. ultracentrifuges), and are time consuming. To minimize these problems, mini columns and other products were develop for the purification of nucleic acids (Roche Applied Science, 2012). University of Ghana http://ugspace.ug.edu.gh 59 2.7.1 Column-based nucleic acid purification Column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that the nucleic acid may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt content of the buffer, which may be a Tris-EDTA (TE) buffer or phosphate buffer (used in DNA microarray experiments due to the reactive amine groups in tris). The Quick Spin columns may also contain gel filtration matrices (either G-25 or G-50 Sephadex) which allow large molecules (e.g. DNA or RNA) to pass through quickly while retaining small molecules (e.g. nucleotides). The Quick Spin format improves the molecular sieving concept by using centrifugation to separate DNA or RNA rapidly and cleanly from small contaminants (Roche Applied Science, 2012). Prior to the major techniques employed today it was known that DNA binds to silica, glass particles or to diatoms which shield their cell walls with silica in the presence of chaotropic agents, such as sodium iodide or sodium perchlorate. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions (Marko et al., 1982). This was later improved to guanidinium thiocyanate or guanidinium hydrochloride as the chaotropic agent (Boom et al., 1990). The use of beads was later changed to mini columns. 2.8 Polymerase Chain Reaction (PCR) Polymerase chain reaction was developed for the in vitro amplification of the DNA or RNA of an organism or gene defect. This technique is versatile, specific and sensitive and has been widely used in molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, food science, hereditary studies, paternity testing, and many other applications (Horizon Press, 2012). Polymerase chain reaction takes University of Ghana http://ugspace.ug.edu.gh 60 advantage of an enzyme ‘polymerase’ that uses defined segment in a strand of DNA as a template for assembling complementary strand. PCR requires three-step cycling process 1) denaturing of a double-stranded DNA 2) annealing of primers and 3) strand extension. If an RNA sequence is to be amplified, a DNA copy of the RNA (cDNA) must first be synthesized using the reverse transcriptase enzyme before the PCR is begun. The PCR reaction contains a mixture of buffers, nucleotides, primers, enzyme and the nucleic acid from the specimen of interest. Denaturation is achieved by heating at 92oC-100oC and separates the complementary strands by breaking the hydrogen bonds that holds them together. In the annealing process, primers are attached to the denatured DNA strand when the temperature is reduced. Once annealing has occurred, the enzyme catalyzes the synthesis of a new strand of DNA at the extension step. The enzyme, DNA polymerase, adds on nucleotides complementary to those in the unpaired DNA strand onto the annealed primers. The reaction is achieved in a thermal cycler that is programmed to cycle the appropriate temperatures required for each step of the reaction process. The original molecules of DNA doubled in the first cycle of denaturation, annealing and extension. The cycle is then repeated a number of times in a chain reaction leading to the exponential amplification of the original DNA (Sambrook and Russel, 2001). Polymerase chain reaction is simple, robust, speedy and flexible. An enormous number of variations to the method have been described and entire journals and books have been devoted to the technique (Sambook and Russel, 2001). University of Ghana http://ugspace.ug.edu.gh 61 2.9 Gel Electrophoresis Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size. In biochemistry and molecular biology, it is used to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge (Kryndushkin et al, 2003). Electrophoresis can be done using either agarose or polyacrylamide gels to separate, identify and purify DNA fragments. The technique is simple, rapid to perform and capable of resolving fragments of DNA that cannot be separated adequately by other procedure such as density gradient centrifugation. The most common buffers used for electrophoresis are Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE) for nucleic acids. Borate can polymerize, and/or interact with cis diols such as those found in RNA. TAE has the lowest buffering capacity but provides the best resolution for larger DNA (Brody and Kern, 2004). After electrophoresis is complete, DNA may be visualized using ethidium bromide (EtBr) which, when intercalated into DNA, fluoresce under ultraviolet light. Polyacrylamide gels are most effective for separating small fragments of DNA (5-500bp). Their resolving power is extremely high and DNA fragments that differ by 1bp in size or 0.1% in mass can be separated from one another (Sambrook and Russel, 2001). 2.9.1 Agarose gel electrophoresis Agarose is a linear polymer composed of alternating residues of D- and L- galactose joined by α-(1-3) and β – (1-4) glycosidic linkages. The L-galactose residue has an anhydrous bridge between the three and six positions (Sambrook and Russel, 2001). Agarose is extracted from seaweed. Purified agarose is in powdered form, and is insoluble in water (or buffer) at room University of Ghana http://ugspace.ug.edu.gh 62 temperature but it dissolve on heating. When it starts to cool, it polymerises; the sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid matrix. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose. Molecules of double-stranded DNA migrate through the gel matrices at rates that are inversely proportional to the log10 of the number of base pairs (Helling et al, 1974). Thus, larger molecules migrate more slowly because of greater frictional drag and because they move less efficiently through the pores of the gel than smaller molecules (Thorne, 1966, 1967). Agarose gels have lower resolving power compared to that of polyacrylamide gels but they have a greater range of separation from 50bp to several mega bases in length. 2.10 Real-Time (Quantitative) PCR Traditionally, PCR is performed in a tube and when the reaction is complete the products of the reaction (the amplified DNA fragments) are analyzed and visualised by gel electrophoresis. However, real-time PCR permits the analysis of the products while the reaction is actually in progress. This is achieved by using various fluorescent dyes which react with the amplified product and can be measured by an instrument. This also facilitates the quantification of the DNA (Arya et al, 2005). Quantitative PCR (qPCR) is used to measure the quantity of a PCR product to extrapolate the starting amounts of DNA, cDNA or RNA. Real-time PCR is rapid since it is not necessary to perform electrophoresis or other procedure after the DNA amplification reaction. The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation (Horizon Press, 2012). There are also a number of strand-specific University of Ghana http://ugspace.ug.edu.gh 63 probes that use the phenomenon of fluorescent resonance energy transfer (Bernard and Wittwer, 2000). The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance in PCR technology. The technology is very flexible and many alternative instruments and fluorescent probe systems are currently available. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels (Ririe et al, 1997). Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Sequence variants including single base mutations could be detected by further analysis (Horizon Press, 2012). Quantitative PCR is now established as the method of choice for the detection of nucleic acids (Arya at al., 2005). 2.11 DNA Sequencing DNA sequencing is the process of reading the nucleotide bases in a DNA molecule. It includes any method or technology that is used to determine the order of the four bases- adenine, guanine, cytosine, and thymine-in a strand of DNA. Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as diagnostic, biotechnology, forensic biology, archaeology and anthropology. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA genomes of numerous types and species of life, including the human genome and other animal, plant, and microbial species. DNA sequencing is promoting new discoveries that are revolutionizing the conceptual foundations of many fields (Franca et al, 2002). University of Ghana http://ugspace.ug.edu.gh 64 2.11.1 Sanger Chain Termination Method The DNA sequencing method, described by Sanger and his co-workers in 1977 revolutionized the field of genomics (Sanger et al, 1977). The method known as the chain termination method or the dideoxynucleotide method consisted of a catalyzed enzymatic reaction that polymerizes the DNA fragments complementary to the template DNA of interest (unknown DNA). A 32P-labelled primer is annealed to a specific known region on the template DNA, which provides a starting point for DNA synthesis. In the presence of DNA polymerases, catalytic polymerization of deoxynucleoside triphosphates (dNTPs) onto the DNA occurs until the enzyme incorporates a modified nucleoside called a terminator or dideoxynucleoside triphosphate (ddNTP). The growing chain is then terminated because there is no free –OH group available for forming the phosphodiester bond with the next nucleoside triphosphate (Sanger et al, 1977). This method was performed in four different tubes, each containing the appropriate amount of one of the four terminators. All the generated fragments had the same 5’-end, whereas the residue at the 3’-end was determined by the dideoxynucleotide used in the reaction. After all four reactions were completed; the mixture of different-sized DNA fragments was resolved by electrophoresis on a denaturing polyacrylamide gel, in four parallel lanes. The pattern of bands showed the distribution of the termination in the synthesized strand of DNA and the unknown sequence was read by autoradiography (Sanger et al. 1977). Although other sequencing methods, such as the chemical method, were later described (Maxam and Gilbert, 1977), the chain-termination method developed by Frederick Sanger and coworkers became the method of choice due to its relative ease and reliability (Sanger et al, 1977; Sanger and Coulson, 1975). Various modifications have been made to the Sanger University of Ghana http://ugspace.ug.edu.gh 65 method to develop the current line of sequencing methods that are fully automated, require one-tube reactions and ready-to-use reaction mixtures. The overwhelming majority of current DNA sequencing assays are based on variations of the dideoxynucleotide method. University of Ghana http://ugspace.ug.edu.gh 66 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 General Introduction This section outlines all the materials and procedures used in the study. It includes the procedures used to collect and process blood samples into component plasma and peripheral blood mononuclear cells (PBMC), analyze plasma for HIV serotypes and extract nucleic acids from the plasma and PBMC. The section also describes procedures used to determine the plasma viral load for HIV-1, amplify protease and reverse transcriptase genes by polymerase chain reaction (PCR), sequence the genes and analyze for HIV-1 drug resistance mutations. The protocols used for PCR and sequencing were optimized for HIV-1 as the predominant type. 3.2 Materials The details of reagents, equipment, consumables, software and their sources and/or manufacturers, used to collect and process the venous blood from HIV-infected persons on ART to obtain drug resistance mutation data are shown in appendix IV. 3.3 Methods 3.3.1 Study Population HIV-infected persons who had been on ART for a minimum of 6 months, from St. Martin de Porres Hospital, Agomanya, Atua Government Hospital Atua, Korle-Bu Teaching Hospital, Accra and Kumasi South Hospital, Kumasi in the Eastern, Greater Accra and Ashanti Regions respectively were enrolled in the study. After informed consent was obtained, blood samples were collected from 338 persons (98 from Korle-Bu, 101 from St. Martin de Porres, 96 from Atua Government and 43 from Kumasi South). University of Ghana http://ugspace.ug.edu.gh 67 This study was approved by the Scientific and Technical Committee of Noguchi Memorial Institute for Medical Research (NMIMR). Ethical clearance for this study was also obtained from the Institutional Review Board of NMIMR in March 2010 (Certificate No. NMIMR-IRB CPN 036/09-10). The clearance was reviewed annually for each year of the study. Copies of the ethical clearance certificates can be found in Appendix V. 3.3.2 Blood sample collection from study participants Seven millilitres of venous blood samples were collected into EDTA vacutainer tubes from eligible persons after obtaining their informed consent. A sample collection form (Appendix I), detailing date of collection and other personal data, CD4 counts, drug history, adherence information, herbal medicine use and Physician’s comment, was completed for each participant. The samples were labeled with hospital identification numbers, packaged in cold boxes and transported by road to the Virology Department of the Noguchi Memorial Institute for Medical Research (NMIMR) for laboratory analyses. Samples were received at NMIMR within 48 hours of collection. Upon receipt, blood samples were checked with the sample collection and consent forms (Appendix II and III). The adequacy of the sample was ascertained and the hospital identity number was cross-checked with the information on the accompanying form. The samples were given laboratory identification and the accompanying information was entered into a Microsoft Excel sample collection database. 3.3.3 Separation of blood into plasma and peripheral blood mononuclear cells Blood samples were processed into plasma and peripheral blood mononuclear cells as follows: the blood was mixed and layered onto 3 ml of lymphocyte separation medium (Histopaque®-1077) in a 15ml centrifuge tube. This was centrifuged (Centrifuge H-900 (Kokusan, Japan) at 2500 rpm for 15 minutes to separate the various components into four University of Ghana http://ugspace.ug.edu.gh 68 layers. The upper layer (plasma) was carefully taken into new sterile and labeled cryovials and stored frozen in 1ml aliquots at minus 35oC until use. The next layer (buffy coat) containing the peripheral blood mononuclear cells (PBMCs) was carefully removed into another sterile 15ml centrifuge tube and the rest (lymphocyte separation medium and red blood cells) discarded. Five milliliters of PBS was added to the PBMCs, mixed and centrifuged at 2500 rpm for 5 minutes. The supernatant was discarded and the pellet re- suspended in 5ml of PBS. This washing step was repeated. The final pellet was re-suspended in 1.0ml of freezing medium, consisting of 1% DMSO in FBS, and stored in two aliquots of 0.5 ml each at minus 35oC. 3.3.4 Determination of the HIV type A line immuno-assay [Inno-lia HIV-1/II Score (Innogenetics, Belgium)] was used to confirm the presence of HIV antibody and also determine the specific type as HIV-1 or HIV-2. The test was performed according to the manufacturer’s protocol in test troughs provided with the kit (User Manual, Inno-liaTM HIV I/II Score, 2011). The presence and intensity of bands, representing gp120, gp41, P24, P17, gp105 and gp36 (Fig. 12) were used to interpret the results and based on the manufacturer’s instructions, a sample was declared as HIV-1 positive, HIV-2 positive or HIV-1/HIV-2 dual positive. University of Ghana http://ugspace.ug.edu.gh 69 Figure 12: A photo of Inno-lia strip (taken after the test) showing the various antigen bands coated onto it. The control band (3+, 1+, +/-) were used to grade the intensity of bands that developed during the test and this is used in result interpretation (Source: www.innogenetics.com). University of Ghana http://ugspace.ug.edu.gh 70 3.3.5 Extraction and purification of nucleic acids from plasma and PBMC QIAamp Viral RNA extraction kit (QIAGEN, USA) or QIAamp Blood DNA kit (QIAGEN, USA) or Nucleic Acid Purification kit (Roche Diagnostics, Germany) was used to extract and purify nucleic acids from the plasma and peripheral blood mononuclear cell samples. 3.3.5.1 Extraction and purification of viral RNA from plasma by QIAamp Viral RNA mini kit (QIAGEN, USA) Ribonucleic acid (RNA) was extracted from thawed plasma samples using the QIAamp viral RNA mini kit according to the manufacturer’s instructions (User Manual, QIAamp viral RNA mini kit, 2007) and with the following modifications. A starting volume of 200µl of plasma was used instead of the recommended 140µl. The elution volume was also changed to 50µl from the recommended 60-80µl. These modifications were made to synchronize sample working volumes of the QIAGEN and Roche kits. The eluted RNA was stored at -35oC until use. 3.3.5.2 Extraction and purification of viral RNA from plasma using Nucleic Acid Purification kit (Roche Diagnostics, Germany) The manufacturer’s instructions (User manual, Nucleic Acid Purification Kit, 2010) were followed to extract and purify RNA from plasma by the nucleic acid purification kit. In brief, lysis/binding buffer supplemented with poly (A) was added to 200µl of plasma and mixed. The mixture was carefully transferred to the labeled high pure filter tubes and centrifuged at 10,000rpm for 15 seconds. The collection tube with the flow through was discarded and the filter tube transferred into a new collection tube. To wash the membranes, inhibitor removal buffer (500 µl) was added to the filter tubes and spun at 10,000rpm for 1min. This was followed by the addition of wash buffers. The filter tubes were again spun at 10,000rpm for University of Ghana http://ugspace.ug.edu.gh 71 1min and 13,000rpm for 10sec with wash buffers 1 and 2 respectively and transferred into fresh RNase-free 1.5ml tube. Fifty microliters (50µl) of elution buffer was added to the filter tube and centrifuged at 10,000rpm for 1 minute to elute the purified RNA. The RNA was stored at -35o C until use. 3.3.5.3 Extraction and purification of proviral DNA from PBMC using QIAamp DNA blood mini kit (QIAGEN, USA) A starting volume of 200µl of peripheral blood mononuclear cells was taken through the DNA extraction procedure as instructed by the manufacturer of the kit. The lysis, separation, washing and elution were all done according to the manufacturer’s protocol (User manual, QIAamp DNA Blood mini kit, 2007). The DNA was eluted in 50µl of elution buffer. 3.3.5.3 Extraction and purification of proviral DNA from PBMC using Nucleic acid purification kit (Roche, Germany) The manufacturer’s protocol (User manual, Nucleic acid purification kit, 2010) was followed to extract and purify proviral DNA from 200 µl of peripheral blood mononuclear cells. The DNA was eluted in 50 µl of elution buffer. 3.3.6 Determination of HIV-1 viral load A quantitative real-time PCR (qPCR) assay was used for plasma viral load determination. The purpose was to quantify the amount of virus in the patient’s plasma. The LTR region of HIV- 1 was amplified in a real time PCR assay using primers F1: 5’GCCTCAATAAAGCTTGCCTTGA-3’ (sense) and R1: 5’GGCGCCACTGCTAGAGATTTT3’ (anti sense). University of Ghana http://ugspace.ug.edu.gh 72 A probe (pILNA) 5’FAM - CAGTACATGCAGGGCCTATTCCACCAG–TAMRA 3’ and Taqman® One-step RT-PCR reagents (ABI, USA) were used for the assay. The RT-PCR reaction was prepared according to the manufacturer’s instructions (Kit insert, Taqman® One- step RT-PCR Master Mix Kit, 2001) Amplification plots were analyzed real time and cut-off threshold (CT) values were obtained and used in a formula below to estimate the viral load. RNA copy number = 2^(plasmid Ct – Sample Ct) * (Plasmid copy number) * (volume of RNA extracted / volume of RNA used for PCR) * (1000 µL / volume of plasma used for extraction) Plasmid concentration 5 µg/µl =1.14X104 copies/ml 3.3.7 Reverse-Transcription Polymerase Chain Reaction (RT-PCR) for protease and reverse transcriptase genes from RNA extracts This procedure was used to amplify the protease and reverse transcriptase genes from the RNA that was previously purified from the plasma samples. The QIAGEN One-step® RT- PCR kit was used for the amplification according to the manufacturer’s protocol (Handbook, QIAGEN One-Step RT-PCR, 2010). Briefly, a total of 25µl reaction mix was prepared. The reaction mix consisted of 0.4mM dNTPs, 15µl of RT-PCR mix, 1µl of enzyme mix by and 10µl of RNA. Primers, DRRT1L/DRRT4L for RT gene and DRPRO5/DRPRO2L for the PR gene were used as previously described (Fujisaki et al, 2007).The primer sequences can be found in Table 1. The thermal cycling conditions were as previously described (Villahermosa et al, 2000). 3.3.8 Polymerase Chain Reaction (PCR) for DNA extracts from PBMC Peripheral blood mononuclear cells of an HIV-infected person contain the viral DNA that had been integrated into the human DNA (proviral DNA). This procedure was therefore to University of Ghana http://ugspace.ug.edu.gh 73 amplify the PR and RT genes from the proviral DNA. Proviral DNA samples from PBMC were amplified using AmpliTaq Gold® PCR Reagents (ABI, USA). Primers, DRRT1L/DRRT4L for RT gene and DRPRO5/DRPRO2L for the PR gene were used as previously described (Fujisaki et al, 2007). A total reaction of 25µl consisting 1X reaction buffer, 1.5mM MgCl2, 0.2mM dNTPs, 0.25µl AmpliTaq Gold® and 10µl of DNA extract was used. The sequences of the primers can be found in Table 1. The thermal cycling conditions used were modified from Villahermosa et al, 2000 as follows: 94oC for 2 minutes followed by 40 cycles of 94oC for 30 seconds, 55oC for 1 minute and 72oC for 60 seconds plus 72oC extension for 5 minutes. 3.3.9 Amplification of RT-PCR and PCR products by nested PCR AmpliTaq Gold® reagents (ABI) were used to re-amplify PCR products from the first round (RT-PCR and PCR) in a nested PCR reaction. A total reaction of 25µl consisting 1X reaction buffer, 1.5mM MgCl2, 0.2mM dNTPs, 0.25µl AmpliTaq Gold® and 5µl of RT-PCR or round one PCR products was used. Primers DRRT7L/DRRT6L and DRPRO1M/DRPRO6 were used for the RT and protease genes respectively (Fujisaki et al, 2007). The sequences of the primers are presented in Table 1. The thermal cycling conditions were as previously published (Villahermosa et al, 2000). University of Ghana http://ugspace.ug.edu.gh 74 Table 1: Details of the primers used for PCR and sequencing Name Position (HXB2) Sequence (5’-3’) Purpose DRPRO5 2074-2095 AGACAGGYTAATTTTTTAGGGA Round 1 PCR (PR GENE) DRPRO2L 2716-2691 TATGGATTTTCAGGCCCAATTTTTGA DRPRO1M 2148-2167 AGAGCCAACAGCCCCACCAG Nested PCR (PR gene) DRPRO6 2611-2592 ACTTTTGGGCCATCCATTCC DRRT1L 2388-2410 ATGATAGGGGGAATTGGAGGTTT Round 1 PCR (RT gene) DRRT4L 3425-3402 TACTTCTGTTAGTGCTTTGGTTCC DRRT7L 2485-2509 GACCTACACCTGTCAACATAATTGG Nested PCR (RT gene) DRRT6L 3372-3348 TAATCCCTGCATAAATCTGACTTGC PRTS 2157-2177 AGC CCC ACC AGA AGA GAG CTT Sequencing (PR gene) P3G 2198-2217 CAACTCCCTCTCAGAAGCAG Sequencing (PR gene) A2 2583-2601 TTAAAGCCAGGAATGGATG Sequencing (RT gene) RT-sec-1-S 2692-2716 CAA AAA TTG GGC CTG AAA ATC CAT A Sequencing (RT gene) PRSec2A 2811-2838 TGGGAAGTTCAATTAGGAATACCACATC Sequencing (PR and RT gene) The name, location on the HXB2 sequence, the primer sequence from 5’to 3’ and the purpose of each primer is shown. HXB2 is a reference HIV-1 subtype B sequence. All the PCR primers and two sequencing primers (P3G and A2) were previously published by Fujisaki et al (2007). PRTS, RT-sec-1-s and PRSec2A were previously published by Villahermosa et al (2000). University of Ghana http://ugspace.ug.edu.gh 75 3.3.10 Agarose Gel Electrophoresis Two gel concentrations (1.5% and 2.0%) were used to analyze the reverse transcriptase and the protease gene products respectively. This was to cater for the difference in expected product sizes of 463bp and 887bp respectively of the protease and RT genes. A 100bp ladder molecular weight marker (Invitrogen, USA) was used to estimate the size of the PCR products. The gel was viewed with a Gel Logic 100 Imaging system (Eastman Kodak Company, USA) and a photograph taken. A product of size 887bp and 463bp for the reverse transcriptase and protease genes respectively, indicated positive amplification of corresponding genes. 3.3.11 Purification of PCR products The purpose of this procedure was to separate the amplification products from left- over reaction components such as dNTPs and enzymes. All PCR products with expected sizes were purified using QIAquick PCR purification kit (QIAGEN) according to the manufacturer’s protocol (User manual, QIAquick PCR purification kit, 2010). The purified DNA was eluted in 50µl of elution buffer and at minus 20oC and used for sequencing. 3.3.12 Direct sequencing of purified PR and RT PCR products The sequencing method used was a variant of the Sanger dideoxynucleotide method. Cycle sequencing was done using Big Dye Terminator cycle sequencing kit version 3.1 (ABI, USA). The reaction mixture consisted of 1µl of purified PCR products, 1.6µl of sequencing primer (2µM), 3.4µl of nuclease-free water, 2µl each of the Big Dye Terminator mix and 5X Sequencing buffer in a total reaction mixture of 10µl (Villahermosa et al, 2000). The thermal conditions were 94oC for 2 minutes followed by 25 cycles of 94OC for 30 seconds; 50OC for 15seconds; 60OC for 4 minutes. Three primers A2, PRSec2A and RTSec1s were used for University of Ghana http://ugspace.ug.edu.gh 76 sequencing RT gene while two primers PRTS and P3G were used for sequencing the PR gene (Villahermosa et al, 2000; Fujisaki et al, 2007). The sequences of the primers used are shown in Table 1. 3.3.13 Purification of cycle sequenced products Cycle sequencing products were purified to remove excess dye terminators, primers and dNTPs using the AgenCourt CleanSeq Dye Terminator Removal kit according to manufacturer’s protocol (User manual, AgenCourt CleanSeq Dye Terminator Removal kit, 2006). Briefly, magnetic particles and 85% ethanol were added to the cycle-sequenced products and mixed. The tube or plate containing the mixture was placed in a magnetic field to enable DNA bound to the magnetic particles to attach to the walls. The liquid portion was discarded and the particles were washed with 85% ethanol and dried. The purified sequenced DNA was eluted from the particles with nuclease-free water, transferred into the wells of a 96-well optical plate and loaded onto the Genetic Analyzer. 3.3.14 Sequence Analysis on the 3130 ABI Genetic Analyzer This procedure is an automated polyacrylamide gel electrophoresis (PAGE). The sequenced samples were run using a polymer (POP-7) that was automatically injected into the capillary array. The 96-well optical plate, with purified samples, was properly assembled by first sealing with the 96-well septa, then placing it in the plate base with the correct orientation and covering it with the plate retainer. The monitor, computer and analyzer were switched on (in order of their appearance in this text). When the instrument status light was stable green, the Data Collection v3.0 software was launched. The manufacturer’s instructions (Applied Biosystems 3130/3130xl Genetic Analyzers, Getting Started Guide, 2004) were followed to University of Ghana http://ugspace.ug.edu.gh 77 load and run the plate in the analyzer in order to generate and store sequence data appropriately. At the end of the run, the stored sequence data was recalled by launching into the Sequence Analysis Software v5.2 and adding the samples. The electropherogram, details of run, base sequences and other parameters of the run were viewed in this software. The sequence data was saved and further analyzed by other software discussed in the next section. 3.3.15 Sequence data analysis Sequences were edited using the Seqman software (DNASTAR, USA) and aligned in BioEdit (http://www.mbio.ncsu.edu/Bioedit/bioedit.html). The HIV BLAST programme (http://www.hiv.lanl.gov) was used for subtype reference alignment and MEGA 4.2 (http://www.megasoftware.net/) was used for generating phylogenetic trees. Mutation data and drug resistance interpretations were obtained by submitting the sequences to the Stanford University HIV Drug Resistance Database. One or more sequences in fasta format were pasted into a textbox in the database and analyzed; a maximum of 100 sequences could be analyzed at a time. Nucleotide sequences were aligned to the consensus subtype B HIV-1 pol amimo acid sequence using a nucleotide to amino acid sequence local alignment program (Huang and Zhang, 1996). Nucleotide triplets containing mixed bases were translated into each of the possible amino acids they encode. Mutations were defined as the differences from consensus B reference sequence and further characterized as RT mutations (NRTI or NNRTI), PI mutations (Major and minor) and other mutations. Other mutations were those associated with drug resistance but are primarily accessory and polymorphic. Patients’ sequences were categorized as being susceptible or having potentially low-level, low-level, intermediate or high-level resistance according to the Stanford algorithm (Liu and Shafer, 2006). University of Ghana http://ugspace.ug.edu.gh 78 For subtype determination, each sequence was compared to a list of reference sequences for each HIV-1 group M subtypes A, B, C, D, F, G, H, J, K, CRF01_AE and CRF02_AG. The subtype of the closest reference is assigned to the submitted sequence. This method is generally accurate (Gifford et al, 2006) however, with exception to CRF01_AE and CRF02_AG, it does not accurately characterize circulating recombinant forms and unique recombinant forms. An HTML output was generated with the mutation data and subtype information. 3.3.16 Data Analyses Descriptive statistics such as means and median were used to describe the study population. Tables and charts were used to summarize the demographic data and clinical history of participants. The Pearson’s Chi Square was used to find the association between the presence of major drug resistance mutations, duration on treatment, viral load and the difference in CD4 count over the treatment period. The Pearson’s Chi Square was also used to find the association between the presence of major drug resistance mutations, adherence to antiretrovirals and herbal medicine use. 3.4 Staff training and capacity building The techniques and protocols used in the study were passed on to research assistants in the Virology Department of NMIMR through training and practical sessions. University of Ghana http://ugspace.ug.edu.gh 79 4.0 RESULTS 4.1 Study population Blood samples were obtained from three hundred and thirty-eight (338) patients at four study sites. The distribution of these among the sites is shown in Table 2. 4.2 Patient Information The mean age, gender composition and mean CD4 counts at time of sampling are shown in Table 2. The trend of mean CD4 counts over the treatment period is shown in Figure 13. Majority (93%) of the patients had undetectable viral loads. Twenty-four patients, representing 7%, had detectable viral loads with a mean of 1664 copies/ml (range 158 - 8751 copies/ml). The spread of viral loads is presented in Figure 14. HIV-1 infections were most prevalent (91.7%) while 0.9% was HIV-2 and 7.4% were HIV-1 and HIV-2 dually seropositive (Figure 15). University of Ghana http://ugspace.ug.edu.gh 80 Table 2: Summary of the characteristics of the study population Hospital Number Males Females Mean Age ^^Mean CD4 (cells/µl) Korle Bu Teaching 98 39 59 42 389 St. Martin de Porres 101 26 75 43 470 Atua Government 96 22 74 42 467 Kumasi South 43 8 35 41 534 Total 338 95 243 42 454 The summary demographic data of the study populations is shown for the four study sites. The respective numbers for patients enrolled, their gender distribution, mean age and mean CD4 count in cells/µl are presented.^^Mean CD4 represents the mean of CD4 counts taken at the time of sampling for all patients enrolled from the site. The values for the entire study population are shown under Total. University of Ghana http://ugspace.ug.edu.gh 81 Figure 13: Mean CD4 counts (cells/μl) for all enrolled patients from start of therapy to time of sampling. Start CD4 represents the mean CD4 count of patients at start of therapy; Previous CD4 represents the mean CD4 count during the visit (6 months on average) prior to the sampling date and Current CD4 represents the mean CD4 count at the time of sampling. CD4 counts were measured using BD FACSCount Instrument and reagents (BD Biosciences, USA).The chat shows increasing trend from start to time of sampling. University of Ghana http://ugspace.ug.edu.gh 82 Figure 14: Summary of HIV-1 viral loads for all enrolled patients at time of sampling. The percentages of patients with viral loads within the categories indicated are shown. A quantitative real-time PCR assay with detection limit of 153copies/ml was used to determine the viral loads. Majority (93%) of the patients had viral loads below the detection limit. Patients with detectable viral loads were further divided into two categories: those with viral loads less than 1000 copies/ml (i.e. 153-999 copies/ml) were 5% and those with viral loads greater than or equal to1000copies/ml (i.e. 1000 - 8751copies/ml) were 2%. University of Ghana http://ugspace.ug.edu.gh 83 Figure 15: Summary results of the serological typing of samples by Inno-lia HIV I/II Score assay. The percentage of study population (N=338) that had antibodies to HIV-1 only; HIV-2 only and both HIV-1and HIV-2 are shown. Majority of the patients (92%) had HIV-1 antibodies, confirming HIV-1 as the predominant HIV type in Ghana. University of Ghana http://ugspace.ug.edu.gh 84 4.3 History of antiretroviral therapy in patients studied All patients had been on antiretroviral therapy for at least 6 months in a range 6 to 30 months with the mean duration of treatment being 30 months. The majority (87%) of the participants were still on the first-line regimens. The first-line regimen comprised a combination of zidovudine (or stavudine), lamivudine and nevirapine (or efavirenz) for the majority (92%) of the patients. Combinations involving tenofovir, abacavir, didanosine, efavirenz nelfinavir and nevirapine were also used by the some patients (Table 3). Fourteen percent (N= 47) of patients were on second-line regimen and their regimen consisted primarily didanosine, abacavir and ritonavir-boosted lopinavir or nelfinavir. The details of the drug combinations taken by patients on second-line are presented in Table 3. University of Ghana http://ugspace.ug.edu.gh 85 Regimen NRTI NNRTI Patients N (%) First-line N=338 Zidovudine, Lamivudine Nevirapine 88 (26) Zidovudine, Lamivudine Efavirenz 83 (24) Stavudine, Lamivudine Nevirapine 68 (20) Stavudine, Lamivudine Efavirenz 73 (22) Other combinations 26 (8) NRTI PI No of Patients (%) Second-line N=47 Didanosine, Abacavir Lopinavir/r 30 (64) Abacavir, Didanosine Nelfinavir 5 (11) Lamivudine, Stavudine Nelfinavir 2 (4) Lamivudine, Didanosine Nelfinavir 1 (2) Zidovudine, Lamivudine Nelfinavir 3 (6) Other combinations 6 (13) Proportions of patients on the various first-line or second-line drug combinations are shown. NRTI - nucleoside reverse transcriptase inhibitors; NNRTI - non-nucleoside reverse transcriptase inhibitors; PI - protease inhibitors; Lopinavir/r –ritonavir-boosted Lopinavir. All patients on second-line regimens had previously been on one of the first-line combinations. Table 3: ART histories of patients showing drugs taken as first-line and second- line regimens University of Ghana http://ugspace.ug.edu.gh 86 4.4 Adherence to ART and herbal medicine use In order to assess adherence to the drugs, patients were asked if they took their drugs daily as scheduled. Nine percent (9%) of patients said they had not taken their drugs as scheduled for at least one month. Herbal medicine use was also investigated and 2% admitted they had used some form of herbal medicine for at least one month while on the ARVs. 4.5 Physicians assessment of patients Assessment of physicians’ rating of patients based on CD4 counts and clinical status showed that most patients (86%) were doing well. However, 11% were not doing so well and 3% were failing (Figure 16). University of Ghana http://ugspace.ug.edu.gh 87 Figure 16: Classification of all study patients (N=338) based on the physicians’ assesement of their clinical status. The figures shown are percentages of the study population that were either ‘Doing well’, ‘Not doing so well’ or ‘failing’ according to the physicians. This classification was guided by the CD4 counts and clinical symptoms of patients as stipulated in the guidelines for antiretroviral therapy in Ghana. University of Ghana http://ugspace.ug.edu.gh 88 4.6 Nested PCR Out of the 338 RNA samples analyzed for protease and reverse transcriptase genes, 152 and 96 respectively were successfully amplified. Of the 338 DNA samples analyzed, 182 PR genes and178RT genes were successfully amplified. The details of these results are found in Table 4. Representative gel photographs are shown in Figure 17 and 18. University of Ghana http://ugspace.ug.edu.gh 89 Table 4: Summary of PCR results showing numbers of samples with amplified genes of interest by study sites and type of sample Study Sites PR Gene RT Gene Plasma PBMC Plasma PBMC Korle-Bu Teaching (N=98) 34 73 37 67 St. Martin de Porres (N=101) 52 42 21 55 Atua Government (N=96) 54 44 23 33 Kumasi South (N=43) 12 23 15 23 Total (N=338) 152 182 96 178 Percent (%) 49.9 53.8 28.4 52.6 Nested PCR was used to attempt amplification of the protease (PR) and reverse transcriptase (RT) genes from plasma and peripheral blood mononuclear cells (PBMC) of all the samples collected from each study site. The numbers of positive amplicons obtained for each gene are shown for each site. The overall numbers and percentages of positive amplicons are shown under Total. University of Ghana http://ugspace.ug.edu.gh 90 Figure 17: Representative gel photograph showing results of amplification of the RT gene. Agarose gel (1.5%) was run using 1X TAE buffer. Lane 1 contained Trackit™ 100bp DNA ladder (Invitrogen, USA). The band sizes of the molecular weight marker increased from 100bp, at the top of the gel, towards the sample wells in steps of 100. Lanes 2 to 15 contained samples from participants, Lane 16 contained a negative control and Lane 17 contained a positive control. The expected size of the positive product was 887bp. Samples in lanes 3 and 9 were considered as failed amplification while samples in the other lanes (2, 4-8 and 10-14) were successful amplifications. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 887 bp The expected amplicon size 600 bp 100 bp 1500 bp University of Ghana http://ugspace.ug.edu.gh 91 Figure 18: Representative gel photograph showing the result of amplification of the protease gene. Agarose gel (2%) was run in 1X TAE buffer. Lane 1 contained Trackit™ 100bp DNA ladder (Invitrogen, USA). The band sizes of the molecular weight marker increased from 100bp, at the top of the gel, towards the sample wells in steps of 100. Lanes 2 to 15 contained samples from participants, lane 16 contained a negative control and lane 17 contained a positive control. The expected size of the positive product was 463bp. Samples in lanes 3, 5 and 9 were considered as ‘failed amplification’ whiles the other lanes (2, 4, 6-8 and 10-15) were successful amplifications. 1 2 3 4 .5 6 7 8 9 10 11 12 13 14 15 16 17 463bp The expected amplicon size 500 bp 100 bp 1500 bp 1000 bp University of Ghana http://ugspace.ug.edu.gh 92 4.7 Sequencing Out of the samples successfully amplified, 67% (n=65) and 55% (n=99) were successfully sequenced for the RT gene from plasma and PBMC respectively compared to 35% (n=54) and 42% (n=76) for the PR gene from plasma and PBMC respectively (Table 5). Although more samples were sequenced from PBMC than from plasma, not all samples that were successfully sequenced from plasma were successfully sequenced from PBMC. These sequences were all edited and submitted online to the Stanford HIV Drug Resistance Database (HIVDB) to generate drug resistance mutation information. A summary of the number of sequences with drug resistance mutations for the three drug classes is presented in Table 5. University of Ghana http://ugspace.ug.edu.gh 93 Table 5: Summary data showing the number of samples successfully sequenced and those with drug resistance mutations Sample type Plasma PBMC Gene PR RT PR RT Sequences obtained (N) 54 65 76 99 Number (%) with *DRM 15 (28) 30 (46)-NRTI 7 (9) 25 (25)- NRTI 32 (49)- NNRTI 26 (26)- NNRTI All positive amplicons were taken through direct sequencing. The total number of patients from whom protease (PR) and reverse transcriptase (RT) sequences were successfully obtained from either plasma or peripheral blood mononuclear cells is shown. The proportions (number and percentage) of these sequences that had drug resistance mutations (DRM) are also shown. RT sequences were analyzed for resistance to both nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) while PR sequences were analyzed for resistance to protease inhibitors. University of Ghana http://ugspace.ug.edu.gh 94 4.8 Prevalence of HIV drug resistance mutations Drug resistance mutations were mostly observed in the RT gene. Forty-six percent (46%) of the RT sequences obtained from plasma had major drug resistance mutations to the nucleoside reverse transcriptase inhibitors (NRTI). Forty-nine percent had major drug resistance mutations to the non-nucleoside reverse transcriptase inhibitors (NNRTI). Twenty-six percent (25%) of the RT sequences obtained from PBMC had drug resistance mutations to NRTI and 26% had NNRTI resistance. The major drug resistance mutations observed in the PR genes were from 28% of the plasma and 9% of the PBMC samples. The RT gene sequences obtained from patients on second-line regimen (N= 19) had more NRTI and NNRTI mutations (79% and 68% respectively) compared to 33% for NRTI and 41% for NNRTI in RT sequences obtained from patients on first-line regimen (Figure 19). University of Ghana http://ugspace.ug.edu.gh 95 Figure 19: The proportions of RT sequences obtained from patients on first-line regimen that had nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) resistance mutations (blue bars) compared to the proportions of RT sequences obtained from patients on second-line regimen with NRTI and NNRTI resistance mutations (wine bars). Higher proportions of 79% and 68% were observed for in second-line sequences for NRTI and NNRTI respectively compared to 33% and 41% of first- line sequences for NRTI and NNRTI mutations. University of Ghana http://ugspace.ug.edu.gh 96 4.8.1 Types of drug resistance mutations found among persons on first-line regimen The most frequently observed NRTI mutations among persons on first-line were M184V/I (78.6%), T215Y (28.6%) and K219E/Q/R (28.6%). Other less frequently observed drug resistance mutations were M41L, K65R, D67N and K70R. Three samples had T69D or T69S or T69N in RT (Table 6). Nine out of the thirteen patients whose RT sequences were analyzed had two or more NRTI mutations. Six patients had thymidine analogue mutations (TAMs) in addition to M184V (Table 6). Except for patient PDR 295 and PDR 71 who were on therapy for 14 and 21 months respectively before the study, all other patients with TAMs were on therapy for more than 50 months. The Q151M mutation was not found in the sequences analyzed. The NNRTI mutation commonly observed in persons on first-line were K103N (53%) and V90I (33%). V179E, V106A/I, Y181C, Y188I/L and K238T were also observed less frequently. Other mutations; A98G, E138A, M230L, F227L and H221Y were each found in one person (6%). The details of the combinations of these mutations observed in the patients are shown in Table 7. Except for sample PDR 223 that had N88S and L10V in the PR gene, all other sequences obtained from patients on the first-line regimen had no major drug resistance mutations in the PR gene. University of Ghana http://ugspace.ug.edu.gh 97 Table 6: Types of NRTI resistance mutations found in patients on first-line regimen and their clinical implications No. Patient ID Months on ART NRTIs taken NRTI mutations *NRTI resistance 1 PDR 71 21 AZT, 3TC M41LM, M184V, T215Y 3TC, ABC, AZT, D4T, DDI, FTC 2 PDR 88 21 D4T, 3TC M184MV 3TC, FTC 3 PDR 91 55 AZT, 3TC M41L, T69D, M184V, T215Y 3TC, ABC, AZT, D4T, DDI, FTC,TDF 4 PDR 102 18 D4T, 3TC, AZT T69AT, K70EK DDI 5 PDR 117 12 AZT, 3TC M184V 3TC, FTC 6 PDR 185 55 D4T, 3TC K65R, T69d, K219R 3TC, ABC, AZT, D4T, DDI, FTC, TDF 7 PDR 223 58 AZT, 3TC D67N, K70R, M184V, T215Y, K219Q 3TC, ABC, AZT, D4T, DDI, FTC 8 PDR270 36 D4T, 3TC M184V, K219EK 3TC, FTC 9 PDR 281 72 AZT, 3TC, M184V 3TC, FTC 10 PR 285 11 D4T, 3TC T69S, M184I 3TC, FTC 11 PDR 295 14 AZT, 3TC D67N, K70R, M184GV 3TC, FTC, AZT 12 PDR 304 48 AZT, 3TC M184V 3TC, FTC 13 PDR 310 60 AZT, 3TC D67N, K70R, M184V, T215Y, K219Q 3TC, ABC, D4T, AZT, FTC 14 PDR 351 44 AZT, 3TC M184V 3TC, FTC Patients on first-line regimen with RT gene sequences that had resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI). The duration on treatment, the NRTIs they had taken, the list of mutations found and the drug resistance implications of the mutations are shown for each patient. *NRTI resistance shows the names (acronyms) of drugs that the patient is resistant to as a result of the mutations found. Lamivudine-3TC; Stavudine- D4T; Zidovudine- AZT; Abacavir-ABC; Didanosine-DDI; Emtricitabine-FTC; Tenofovir-TDF. Red font shows high level resistance and blue font depicts intermediate level resistance. Drugs with low or potentially low resistance are not shown. University of Ghana http://ugspace.ug.edu.gh 98 Table 7: Types of NNRTI resistance mutations found in patients on first-line regimen and their clinical implications No Patient ID Months on ART NNRTIs taken NNRTI mutations *NNRTI resistance 1 PDR 71 21 EFV V90I, A98G, K103N, E138A, V179EV EFV, NVP 2 PDR 91 21 EFV K103N, M230L EFV, ETR NVP, RPV 3 PDR 117 12 NVP V106A, F227L EFV, NVP 4 PDR 121 28 NVP V90I, Y181C NVP 5 PDR 185 55 NVP K103N, Y181C, H221Y EFV, ETR, NVP, RPV 6 PDR 233 30 NVP K103KN NVP, EFV 7 PDR 281 72 NVP K103N, K238T EFV, NVP 8 PDR 285 11 NVP V179E, Y188I EFV, NVP 9 PDR 295 14 NVP Y188L EFV, NVP RPV 10 PDR 328 23 EFV V90I, K103N NVP, EFV 11 PDR 345 6 EFV V90I, K103N NVP, EFV 12 PDR 351 44 NVP K103N, K238T EFV, NVP Patients on first-line regimen who had RT gene sequences with resistance mutations to non- nucleoside reverse transcriptase inhibitors (NNRTI). The duration on treatment, the NNRTIs they had taken, the list of mutations found and the drug resistance implications of the mutations are shown for each patient. *NNRTI resistance shows the names (acronyms) of drugs that the patient is resistant to as a result of the mutations found. Efavirenz-EFV; Nevirapine-NVP; Etravirine- ETR; Rilpivirine-RPV. Red font shows high level resistance and blue font depicts intermediate level resistance. Drugs with potentially low or low level resistance are not shown. University of Ghana http://ugspace.ug.edu.gh 99 4.8.2 Types of drug resistance mutations found among persons on second -line regimen The patients on second-line are those who had failed the first line drugs, based on their CD4 counts and clinical symptoms, and were switched by their physicians to a new combination of drugs including protease inhibitors. The most frequently observed NRTI mutations were M184V (100%), T215Y/F/I (85%), M41L (54%), D67G/N (31%) and L210W (31%). Other mutations were observed at codon 215 in RT and these included T215ADSY and T215NSTY. One patient had two amino acid insertions at codon 69 in RT. E44D, T69D, K70R, L74V/I, V118I and K219Q were also observed in lesser proportions. The details of the combinations of resistance mutations observed in patients on second-line are shown in Table 8. The Q151M mutation was not found in any of the sequences analyzed. Various NNRTI mutations were found among patients on second-line regimen; the most frequent being K103N/S which occurred in 52% of the samples with resistance mutations, K101P/E/Q and A98G each of which occurred in 31% of samples with resistance mutations. Other NNRTI mutations that occurred less frequently were E138A, V179L, P225H, K238T, V90I, G190A, V108I, Y181C, V106M, H221Y, F227L and M230L. Table 9 shows the details of these mutations found in the patients. Resistance to protease inhibitors was observed in patients on second-line regimen (Table 10). The most frequently detected mutation was M46I, which occurred in 50% of sequences with PI resistance. Other DR mutations detected less frequently were N88S, I54V, I82F, L90M, E35G, A74V, L89V, 184V, V11I, L23I, L33I, G58E and L10I/V (Table 10). University of Ghana http://ugspace.ug.edu.gh 100 Table 8: Types of NRTI resistance mutations found in patients on second-line therapy, their clinical implication and the current NRTI taken by patients No. Patient ID NRTI mutations *NRTI resistance NRTI taken 1 PDR 1 M41L, E44D, L74V, V118I, M184V, L210W, T215Y 3TC, ABC, DDI, FTC, AZT, D4T, TDF DDI, ABC 2 PDR 2 V75M, M184V, T215Y 3TC, ABC, DDI, FTC, AZT, D4T TDF,3TC 3 PDR 4 L74V, M184V, L210W, T215Y 3TC, ABC, DDI, FTC, AZT, D4T, TDF DDI, ABC 4 PDR 5 T69i, M184V, L210W, T215NSTY 3TC, ABC, DDI, FTC, AZT, D4T DDI, ABC 5 PDR 6 M41L, D67G, K70R, V75M, M184I, T215F, K219Q 3TC, ABC, DDI, FTC, AZT, D4T, TDF DDI, ABC 6 PDR 9 M41L, L74I, M184V, T215F, K219W 3TC, ABC, DDI, FTC, AZT, D4T, TDF DDI, ABC 7 PDR 10 E44D, M184V, L210W, T215Y 3TC, FTC, ABC, AZT, D4T, DDI DDI, ABC 8 PDR 11 D67G, K70R, M184MV, T215IT 3TC, AZT, FTC, ABC, D4T DDI, ABC 9 PDR 19 M41L, E44D, D67N, M184V, T215F 3TC, ABC, DDI, FTC, AZT, D4T DDI, ABC 10 PDR 20 M184V, T215ADSY 3TC, FTC, ABC DDI, ABC 11 PDR 41 M41L, T69D, M184V, T215Y 3TC, ABC, DDI, FTC, AZT, D4T, TDF DDI, ABC 12 PDR 90 M41L, M184V, T215Y 3TC, ABC, DDI, FTC, AZT, D4T TDF, D4T 13 PDR 106 M41L, D67N, T69D, M184V, T215Y 3TC, ABC, DDI, FTC, AZT, D4T, TDF DDI, ABC Patients on second-line regimen who had RT sequences with resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI). The list of mutations found, the drug resistance implications of the mutations and the NRTI being taken at time of sampling are shown for each patient. *NRTI resistance shows the names (acronyms) of drugs that the patient is resistant to as a result of the mutations found. Lamivudine-3TC; Stavudine- D4T; Zidovudine- AZT; Abacavir-ABC; Didanosine-DDI; Emtricitabine-FTC; Tenofovir-TDF. Red font shows high level resistance and blue font depicts intermediate level resistance. Drugs with low or potentially low resistance are not shown. University of Ghana http://ugspace.ug.edu.gh 101 4.8.3 Occurrence of thymidine analog mutations (TAMs) Thymidine analog mutations were detected less in patients on first-line compared to those on second line. In those patients on first-line regimen, 6 out of the 15 (40%) of sequences with NRTI resistance mutations had at least 2 TAMs compared with 11 of 13 i.e. 85% of sequences from the second-line participants. Mutations occurring at position 215 and M41L were most frequently found in both groups of patients. Mutations such as L210W, D67N and K70R were detected mostly in patients on second-line regimen while K219Q was mainly found in those on first-line drugs. One patient on second-line had a double amino acid insertion at codon 69. Two patients on the first-line regimen had four TAMs each (D67N, K70R, T215Y and K219Q). Figure 20 shows a comparison of the prevalence of each TAMs between the two groups of patients. 4.8.4 Drug resistance mutations found in plasma and PBMC pairs The drug resistance mutations found in plasma were generally similar to those found in the PBMC of same patients (Table 11). In a few cases, mutations were detected in plasma but not in the PBMC and vice versa. A phylogenetic analysis of paired plasma and RT sequences (Fig. 22) showed that the pairs were closely related to each other than to sequences from other patients. University of Ghana http://ugspace.ug.edu.gh 102 Figure 20: Occurence of thymidine analogue mutations (TAMs) in patients on first-line regimen and those on second-line regimen.The types and frequency of detection of TAMs in patients on first-line regimen and those in patients on second-line regimens are shown. TAMs, known to cause cross-resistance among NRTIs, were detected more frquently in patients on second-line (wine bars) compared to patients on first-line (blue bars). Two of the TAMs (T69i and L210W) were only found in patients on second-line and not in patients on first-line. University of Ghana http://ugspace.ug.edu.gh 103 Table 9: Types of NNRTI resistance mutations observed in patients on second-line therapy and their clinical implications No. Patient ID NNRTI mutations *NNRTI resistance 1 PDR 1 K101P, K103N RPV, EFV, NVP, ETR 2 PDR 2 K101P, K103S, E138A RPV, EFV, NVP, ETR 3 PDR 4 A98G, K103N, V179LV, P225H, K238T EFV, NVP, ETR 4 PDR 5 K103N, V108I EFV, NVP 5 PDR 6 V90I, K101E, E138A, G190A EFV, NVP, RPV 6 PDR 9 K103N, V108I EFV, NVP 7 PDR 10 V90I, A98AG,Y181C, H221Y NVP, ETR, EFV, RPV 8 PDR 19 V106M, H221Y, F227Y EFV, NVP 9 PDR 20 K101KQ, K103N, Y181C, H221HY, P225HP RPV, EFV, NVP, ETR 10 PDR 41 K103N, M230L RPV, EFV, NVP, ETR 11 PDR 90 K103N, M230L EFV, NVP Patients on second-line regimen who had RT gene sequences with resistance mutations to non-nucleoside reverse transcriptase inhibitors (NNRTI). The list of mutations found and the drug resistance implications of the mutations are shown for each patient. *NNRTI resistance shows the names (acronyms) of drugs that the patient is resistant to as a result of the mutations found. Efavirenz-EFV; Nevirapine-NVP; Etravirine-ETR; Rilpivirine-RPV. Red font shows high level resistance and blue font depicts intermediate level resistance. Drugs with low or potentially low resistance are not shown. University of Ghana http://ugspace.ug.edu.gh 104 Table 10: PI resistance mutations in patients on second-line therapy, their clinical implications and the current PI taken by the patients No. Patient ID PI mutations *PI resistance PI taken 1 PDR 1 M46I, N88S, L10V ATV/r, NFV NFV 2 PDR 2 M46I, I54V, I82F, L90M, L10V ATV/r, IDV/r, NFV, SQV/r, FPV/r LPV/r 3 PDR 11 E35G, M46I, A71V, I84V, L89V NFV, ATV/r, FPV/r, IDV/r, SQV/r LPV/r 4 PDR 19 M46I, L74V, I84V FPV/r, IDV/r, NFV, ATV/r, LPV/r,SQV/r LPV/r 5 PDR 106 L90M, L10V, A71T, L89V NFV, ATV/r, FPV/r, SQV/r NFV 6 PDR 199 L90M, L23I, L33I, G58E NFV, SQV/r NFV Patients on second-line regimen who had protease gene sequences with resistance mutations to protease inhibitors. The list of mutations found and the drug resistance implications of the mutations are shown for each patient. *PI resistance shows the names (acronyms) of drugs that the patient is resistant to as a result of the mutations found. Nelfinavir-NFV; Fosamprenavir-FPV; Saquinavir-SQV; Lopinavir-LPV; Atazanavir-ATV; Indinavir-IDV; Ritonavir-r. Except for Nelfinavir, all other protease inhibitors were boosted with ritonavir denoted by /r. Red font shows high level resistance and blue font depicts intermediate level resistance. Drugs with low or potentially low level resistance are not shown. University of Ghana http://ugspace.ug.edu.gh 105 Table 11: Comparison of NRTI and NNRTI drug resistance mutations observed in sequences from plasma and PBMC pairs. Patient ID NRTI Mutations NNRTI mutations Plasma PBMC Plasma PBMC PDR 41 M41L, T69D, M184V, T215Y M41L, T69D, M184V, T215Y K103N, M230L K103N, M230L PDR 48 NONE M184MV *NONE NONE PDR 71 M41L, M184V, T215Y M41L, M184V, T215Y V90I, A98G, K103N, E138A, V179EV V90I, K103N, E138A, V179E PDR 90 M41L, M184V, T215Y V75IV K103N, V106I,V108I K101P, K103N, E138A PDR 106 M41L, D67N, T69D, M184V, T215Y M41L, D67DN, T69AT, M184V, T215Y A98G A98AG PDR 223 D67N, K70R, M184V, T215Y, K219Q D67N, K70R, M184V, T215Y, K219Q NONE V90I PDR 304 M184V NONE NONE V90I PDR 328 NONE M184IM V90I, K103N V90I, K103KN, V108IV, M230ML Patients who had protease (PR) and reverse transcriptase (RT) sequences from both plasma and peripheral blood mononuclear cells (PBMC) are shown. The mutations found in the PR and RT genes from plasma and PBMC are compared for each patient. *None implies absence of drug resistance mutations detected towards the particular drug class in the plasma or PBMC of that patient. University of Ghana http://ugspace.ug.edu.gh 106 4.9 Relationship between change in CD4 counts, viral load, duration on first line ART and the presence of drug resistance mutations The 65 sequences obtained were categorized based on the change in CD4 counts, viral load at sampling and duration on first-line therapy (Table12). The Pearson’s Chi Square test was used to test the relationship between these variables and the presence of drug resistance mutations. There was no statistically significant association between the variables tested and the presence of drug resistance mutations (Table 12). 4.10 Relationship between adherence to ART, herbal medicine use and the presence of drug resistance Self-reported data on adherence to therapy and herbal medicine use during ART was available for 53 of the 65 patients whose RT genes were successfully sequenced from plasma. Out of the 11 patients who admitted herbal medicine use during ART, 3 had NRTI mutations while 5 had NNRTI mutations. Seventeen of the 42 patients who did not use any herbal medicine during ART each had both NRTI and NNRTI mutations (Table 13). For adherence to ART, the 2 patients that admitted non-adherence both had NNRTI mutations while only 1 had NRTI mutations. Among the 51 patients who adhered, 19 and 20 had NRTI and NNRTI mutations respectively. Analysis of the relationship between herbal medicine use, adherence to ART versus presence of drug resistance mutations by the Pearson’s Chi Square test however revealed no statistically significant association between them (Table 13). University of Ghana http://ugspace.ug.edu.gh 107 4.11 Polymorphisms at drug resistance –associated positions in PR sequences Majority (72%) of the sequences analyzed did not have major drug resistance mutations in the PR gene. However, polymorphisms at drug-resistance associated positions were frequently detected in the PR sequences (Fig. 23). M36I was found in 97% of sequences while 93% had K20I and 22% had L10IV. Two sequences (PDR 10 and PDR 251) each had one amino acid insertion; Isoleucine at codon 36 and Asparagine at codon 37 respectively. 4.12 Prevalence of HIV-1 subtypes Out of the 65 RT sequences analyzed for HIV-1 subtypes, 61 (92.3%) were CRF02.AG recombinant, three (4.6%) were subtype G and one (1.5%) each was subtype B and K. These results were obtained from the Stanford HIV Drug Resistance Database and were confirmed by phylogenetic analysis (Figure 21) University of Ghana http://ugspace.ug.edu.gh 108 Table 12: Relationship between the change in CD4 counts, viral load at sampling, the duration on first-line antiretroviral therapy and the presence of drug resistance mutations Variable (N=65) Patients with mutations (%) NRTI NNRTI difference in CD4 count Neg (n=25) 48 56 Pos (n=40) 45 45 p Value 0.813 0.388 Viral load Undetectable (n=56) 46 48 Detectable (n=9) 56 56 p Value 0.542 0.683 Duration on first-line ART < 2yrs (n=24) 33 46 2-5 yrs (n=32) 59 59 >5yrs (n=9) 33 22 p Value 0.109 0.132 Associations between parameters were analyzed using the Pearson’s Chi Square test. The test of statistical significance was 2-sided and differences were considered significant at P< 0.05. Difference in CD4 count was ‘Negative’ if previous CD4 count was greater than current CD4 and ‘Positive’ if the opposite was true. Based on the p-values obtained, there was no statistically significant association between the development of drug resistance mutations and difference in CD4 count or viral load or duration on ART. University of Ghana http://ugspace.ug.edu.gh 109 Variable (N=53) Mutations (n) NRTI NNRTI Herbal Use Yes (n=11) 3 5 No (n=42) 17 17 p Value 0.421 0.765 Adherence to ART No (n=2) 1 2 Yes (n=51) 19 20 p Value 0.715 0.087 Associations between parameters were analyzed using the Pearson’s Chi Square test. The test of statistical significance was 2-sided and differences were considered significant at P< 0.05. Adherence was defined as not skipping any day without taking antiretrovirals or skipping less than one month of taking drugs cumulatively since the start of therapy. These were self- reported by patients. Based on the p-values obtained, there was no statistically significant association between the development of drug resistance mutations and self-reported adherence to antiretroviral therapy or herbal medicine use during antiretroviral therapy. Table 13: Relationship between adherence to ART, herbal medicine use and the presence of drug resistance mutations University of Ghana http://ugspace.ug.edu.gh 110 38RT 274RT 102RT 117RT 304RT 225RT 285RT 351RT 71RT GH264 P PRT 106RT 310RT 70RT 254RT 318RT 342RT Ref.02 AG.NG.x.IBNG.L39106 270RT 317RT 27RT 331RT 121RT 328RT 218RT 36RT 44RT Ref.02 AG.CM.99.pBD6 15.AY271690 Ref.36 cpx.CM.00.00CMNYU1162.EF087995 284RT Ref.06 cpx.GH.03.03GH173 06.AB286851 Ref.25 cpx.CM.06.06CM BA 040.EU693240 Ref.37 cpx.CM.00.00CMNYU926.EF116594 Ref.G.NG.92.92NG083.U88826 Ref.43 02G.SA.03.J11223.EU697904 92RT Ref.43 02G.SA.03.J11456.EU697909 Ref.38 BF1.UY.04.UY04 3987.FJ213781 Ref.39 BF.BR.03.03BRRJ103.EU735534 Ref.B.FR.83.HXB2 LAI IIIB BRU.K03455 Ref.B.US.98.1058 11.AY331295 90RT Ref.17 BF.PE.02.PE02 PCR0155.EU581828 Ref.23 BG.CU.03.CB118.AY900571 Ref.24 BG.CU.03.CB378.AY900574 Ref.K.CM.96.96CM MP535.AJ249239 Ref.45 cpx.CD.97.97CD MBFE185.FN392874 Ref.45 cpx.GA.97.97GA TB45.FN392877 Ref.49 cpx.GM.03.N26677.HQ385479 Ref.04 cpx.CY.94.94CY032 3.AF049337 Ref.09 cpx.CI.00.00IC 10092.AJ866553 183RT Ref.09 cpx.US.99.99DE4057.AY093607 Ref.A2.CD.97.97CDKTB48.AF286238 Ref.26 AU.CD.02.02CD KS069.FM877780 Ref.A1.AU.03.PS1044 Day0.DQ676872 Ref.01 AE.TH.90.CM240.U54771 Ref.22 01A1.CM.02.02CM 3097MN.GQ229529 Ref.N.CM.02.DJO0131.AY532635 Ref.N.CM.97.YBF106.AJ271370 Ref.CPZ.US.85.US Marilyn.AF103818 Ref.CPZ.CM.05.SIVcpzMT145.DQ373066 Ref.P.CM.06.U14788.HQ179987 Ref.O.BE.87.ANT70.L20587 Ref.O.CM.98.98CMU2901.AY169812 Ref.CPZ.CD.90.ANT.U42720 100 97 100 71 94 95 85 99 0.02 Figure 21: Phylogram (neighbour joining tree) of RT sequences (708 nucleotides) and selected HIV-1 subtype references. Phylogenetic analyses were conducted in MEGA 4 (Tamura et al, 2007).Bootstrap values 70% or greater are shown. Purple triangles represented study sequences. Majority of study sequences clustered with the circulating recombinant form (CRF02_AG). This subtype emerged due to the recombination of subtype A and G and it is the predominant HIV-1 subtype in West Africa. The study sequences clustered with reference CRF02_AG sequences Ref. 02 AG NG .x. IBNG.L39106 and Ref. 02 AG CM 99.pBDS 15 ay271690 from Nigeria and Cameroon respectively. CRF02_AG University of Ghana http://ugspace.ug.edu.gh 111 305RT 305dRT 10dRT 10RT 306RT 306dRT 106RT 106dRT 132RT 132dRT 57RT 57dRT 331RT 331dRT 328RT 328dRT 4dRT 4RT 324RT 324dRT 12dRT 12RT 223RT 223dRT 20dRT 20RT 71RT 71dRT 22dRT 22RT 11dRT 11RT 19dRT 19RT 100 99 98 99 100 100 99 98 97 99 99 99 99 95 82 93 93 96 51 47 38 45 62 8 18 23 17 10 13 3 2 0.005 Figure 22: Phylogenetic tree of paired RT sequences (708 nucleotides) obtained from plasma and PBMC of same patient showing the relationship between the sequences. Phylogenetic analyses were conducted in MEGA 4 (Tamura et al, 2007).Blue circles represent plasma samples and purple squares represent PBMC sequences. Sequences obtained from paired plasma and PBMC from the same patients clustered together. University of Ghana http://ugspace.ug.edu.gh 112 Figure 23: Dot plot of protease sequences from study aligned with the protease sequence of HIV-1 reference HXB2. All 99 amino acids (one-letter names) are shown for the reference sequence. The dots represent positions with similar amino acids in study sequences as the reference sequence. Alphabets are amino acid names and show positions where the amino acids in the study sequences are different from that at the same position of the reference sequence. The reference sequence is HIV-1 subtype B and majority of the study sequences are HIV-1 subtype CRF02_AG University of Ghana http://ugspace.ug.edu.gh 113 4.13 Capacity building at Virology Department, Noguchi Memorial Institute for Medical Research As part of this study, an optimized protocol for nested PCR and sequencing of protease and reverse transcriptase genes of HIV-1 was developed. This protocol was modified from two different protocols (Villahermosa et al, 2000; Fujisaki et al, 2007). The protocol was used to establish standard operating procedures in 2011 for HIV-1 genotyping in the Virology Department, Noguchi Memorial Institute for Medical Research. Five research assistants were trained on this protocol to perform HIV-1 drug resistance testing for patients. Three research assistants were also trained on DNA sequencing techniques which are now applied in studies on Influenza and Polio viruses. University of Ghana http://ugspace.ug.edu.gh 114 CHAPTER FIVE 5.0 DISCUSSION AND CONCLUSION 5.1 DISCUSSION 5.1.1 General Introduction This study sought to investigate drug resistance mutations present in HIV-infected patients on antiretroviral therapy in Ghana and relate the mutations to their drug histories, virologic and immunological profiles. Patients who had been on antiretroviral therapy for a mean duration of 30 months (range 6-86months) were studied. 5.1.2 HIV type, immunologic and virologic markers The majority of patients had HIV-1 infections (Figure 15) confirming findings of previous studies that HIV-1 is predominant in Ghana (Brandful et al, 1997; Ampofo et al, 1999; Fischetti et al, 2004; Brandful et al, 2012). However, the proportion of HIV-2 and HIV-1/2 dual infections (0.9% and 7.4% respectively) observed in this study (between November 2009 and July 2010) was higher than the recent national prevalence of 0.7% and 1.3%, respectively in 2011 (NACP, 2011). Access to highly active antiretroviral therapy (HAART) has expanded in resource-limited settings (RLS) such as Ghana. The laboratory infrastructure to monitor and identify patients failing treatment and requiring a switch in treatment regimen is however inadequate (Badri et al, 2008). Countries in such settings do not usually have routine plasma viral load monitoring, the gold standard used in developed countries for diagnosing virologic failure. Countries in sub-Saharan Africa such as Ghana therefore utilize the World Health Organization recommendations of CD4 cell count measurements and clinical symptoms to monitor ART in the absence of viral load (WHO, 2006). Investigations are therefore required into the usefulness of these markers as good indicators for ART success. University of Ghana http://ugspace.ug.edu.gh 115 In this study in Ghana, an increasing trend was observed in the mean values of CD4 counts recorded over the treatment period (Figure13). This trend suggested that the drugs used over the period had been effective. Also, since CD4 counts were used as a tool for monitoring treatment and switching therapy, this trend of increasing CD4 counts over the treatment period showed that the participants were generally doing well and there was no need to change their drugs. This finding was confirmed by the physician’s assessment of patients’ clinical status (Figure 16), which showed that 86% of the participants were doing well. Some individuals (data not shown) had decreased CD4 counts accounting for the 14% of the patients (Figure 16) rated by the physicians as not doing so well or failing on the drugs. However, analysis of the difference in CD4 counts at the time of sampling did not show significant association with the presence of drug resistance mutations in the RT gene. Although those with negative CD4 counts difference had relatively higher frequency of resistance mutations of 48% for NRTI and 56% for NNRTI compared to 46% for NRTI and 45% for NNRTI among those with positive CD4 count difference, these differences were however not statistically significant (Table 12) as also confirmed by the error bars (Figure 13). Thus the difference in CD4 count over a period could not indicate the presence or otherwise of drug resistance mutations. This observation was in agreement with an earlier report by Badri et al (2008) that measurement of CD4 count was an inadequate alternative to viral load measurement for the detection of virologic failure which is both a cause and consequence of the development of drug resistance mutations (Badri et al, 2008). Viral load was measured in all patients using real time quantitative RT-PCR assay. Only 7% (n=24) had detectable viral load. This suggested that only 7% of the study population may be experiencing virologic failure and may have drug resistance mutations. Fourteen percent (14%) of the study population were classified as failing or not doing so well on the drugs and University of Ghana http://ugspace.ug.edu.gh 116 this is double the number that had detectable viral loads. Thus, some of the patients classified as failing by the physicians had viral loads below the detection limit. The RT gene of nine of the patients with detectable viral load, representing 38%, was successfully sequenced and analyzed. Out of these, five had major drug resistance mutations. These five represent only 16% of all sequences with drug resistance mutations in the RT gene. The majority (84%) of sequences with drug resistance mutations in the RT gene were derived from patients with viral loads below detection limit. When the differences were statistically tested using Pearson’s Chi Square, no significant association was found between the presence of drug resistance mutations and detectable viral load (Table 12). This finding emphasized the observation that although several studies seek drug resistance mutations in patients experiencing virologic failure (Samati et al, 2003; Saravanan et al, 2012; Sigaloff et al, 2012), it is worthwhile to also investigate drug resistance mutations in patients on ART with persistently low or undetectable viral loads (Mackie et al, 2004; Martinez-Picado et al, 2007; Metzner et al, 2007). 5.1.3 PCR amplification of reverse transcriptase and protease genes Nested PCR was used to separately amplify an 887bp fragment of the reverse transcriptase coding genes and 463bp fragment of the protease coding genes of the pol region of HIV-1. The amplification success of the RT gene from plasma was the least followed by that of RT gene from PBMC then PR gene from plasma and finally the PR gene from PBMC (Table 4). The low success in amplification, a major limitation of this study, could be attributed to the mostly undetectable and low viral loads observed in these patients. However, some samples with undetectable viral loads were amplified while others with detectable viral loads were not amplified (data not shown) making it difficult to attribute the failure of amplification solely to viral load differences. The lengths of the fragments (887bp for RT and 463bp for PR) could University of Ghana http://ugspace.ug.edu.gh 117 have contributed to the higher success observed in the PR as compared with the RT genes. Proviral DNA from PBMC is known to be more easily amplified by PCR compared to RNA from plasma particularly in patients with low or undetectable viral loads (Kabamba-Mukadi et al, 2009) and so the differences observed in amplifying from PBMC compared to plasma were as expected. Template volumes were increased but that did not improve rate of successful amplifications. Other parameters of the protocol need further modification to achieve better amplification rates. 5.1.4 Sequencing of reverse transcriptase and protease genes Sequencing of the successfully amplified genes was done using reagents from Applied Biosystems on a Genetic Analyzer ABI 3130. The sequences were edited, assembled and aligned. For some patients, the RT or PR genes amplified were not successfully sequenced. This scenario was mostly observed for the protease gene. In some of these cases, some sequence data was obtained but the peaks in the electrophorogram looked so compressed like mixtures of bases at each position and therefore could not be edited and analyzed. Repeated sequencing using smaller amount of template DNA could not resolve these mixtures. Clonal sequencing could have resolved the mixtures involved but this was not within the scope of the current study. 5.1.5 Prevalence and characteristics of drug-resistance mutations Drug resistance mutations were observed both in patients on first-line regimen and those on second-line. The types of mutations and their implication for therapy are discussed in the following sections. University of Ghana http://ugspace.ug.edu.gh 118 5.1.5.1 Drug resistance mutations among patients on first-line regimens Among patients on first-line therapy, M184V was the most commonly detected mutation. This mutation was detected in 10 out of 15 participants, in addition to two other mutations at the same codon M184I and M184GV. Thus 80% of the sequences obtained from patients on first-line ART had a drug resistance mutation at codon 184 in the RT gene. This finding confirmed that of previous studies that M184V is the dominant NRTI mutation found among persons on first-line therapy (Shafer et al, 2000; Wallis et al, 2010; Shafer and Schapiro, 2008; Martinez-Cajas et al, 2009; Sigaloff et al, 2012). The mutations M184V and M184I cause high-level resistance to lamivudine and emtricitabine and low-level resistance to didanosine and abacavir. Thus the high frequency of M184IV observed in this study is not unusual since all these patients had been on lamivudine as part of their regimen. Previous findings by Ji and Loeb (1994) and Keulen et al (1999) showed that the M184I usually emerges before M184V due to the reverse transcriptase’s preferential mutation of G to A over A to G resulting in ATG to ATA (Methionine to Isoleucine) more quickly than ATG to GTA (Methionine to Valine) . The enzymatic efficiency of M184I is however lower than that of M184V therefore all viruses with mutations at this codon eventually develop the M184V (Frost et al, 2000). The observation of high prevalence of mutations at codon 184 in the RT gene is also in agreement with earlier findings that patients who develop virologic failure on their first treatment regimen are usually found to have HIV strains with resistance to only one drug in the regimen (Shafer, 2002; Gallego et al, 2001; Descamps et al, 2000; Murphy et al, 1999). According to these reports, the drug to which resistance most commonly developed is lamivudine or an NNRTI. Patients with M184V could however continue treatment with lamivudine or emtricitabine because these mutations increase susceptibility to zidovudine, University of Ghana http://ugspace.ug.edu.gh 119 tenofovir and stavudine and are associated with clinically significant decrease in HIV-1 replication (Miller et al, 2002; Diallo et al, 2003; Shafer and Schapiro, 2008). Results from this study also confirm that M184IV is the first NRTI mutation to emerge in patients treated with lamivudine-containing regimen (Descamps et al, 2000; Shafer, 2002) since it was the only mutation in patients that had a single drug resistance mutation (Table 6). Other mutations commonly detected were T215Y and K219E/Q/R. These mutations occurred with M184IV, M41L and K70R in some cases and with K65R and D69D/N/S in others (Table 6). The M41L, D67N, K70R, T215Y/F and K219Q/E are thymidine analogue mutations (TAMs), which usually develop during treatment with thymidine analogues (zidovudine and stavudine). TAMs are known to enhance the entry of pyrophosphates (PPi) and adenosine triphosphate (ATP) into a site adjacent to the incorporated analogue. The PPi or ATP then attacks the phosphodiester bond between the analogue and the growing chain resulting in the removal of the analogue and allowing polymerization to continue (Shafer et al, 2000; Shafer 2002; Clavel and Hance, 2004 and Schapiro, 2008). Due to this mechanism, the TAMs induce resistance to all the NRTIs to some extent. The mutation, T215Y, develops during treatment with zidovudine and stavudine and reduces susceptibility to abacavir, didanosine, and tenofovir particularly in combination with M41L and L210W. These, T215Y and T215F mutations result from two base-pair changes. Other mutations at codon 215 are transitions between the wild type and the mutant. These mutations (T215ADSY, T215NSTY and T215I) found in patients on this study do not reduce susceptibility of the drugs but are indicators of the presence of drug pressure (Shafer, 2002). The K219Q/E mutations decrease zidovudine and probably stavudine susceptibility when present with K70R or T215Y/F, but have little if any effect on the remaining NRTIs. K219R occurs commonly in heavily NRTI-treated patients. University of Ghana http://ugspace.ug.edu.gh 120 The mutations; K65R and D69N were also observed. Position 64-72 in RT forms a loop between the beta 2 and beta 3 strands in the finger region. This loop makes contact with the incoming dNTP during polymerization (Huang et al, 1998; Sarafianos et al, 1999). Mutations in this area affect the contact between the loop and the incoming dNTPs thus inhibiting polymerization. Two sequences had T69D and T69S. The mutations at codon 69 are known to cause resistance to each of the NRTIs when they occur with the TAMs (Winters and Merigan, 2001). Abacavir therapy selects for K65R and this mutation reduces susceptibility to abacavir, didanosine and tenofovir (Miller et al 2004). According to Hawkins et al (2009), K65R is also selected during treatment with stavudine-containing regimens in patients infected with HIV-1 subtypes other than subtype B (Hawkins et al 2009; Wallis et al, 2010). This could explain the occurrence of K65R in a patient without Abacavir experience in this study. An amino acid deletion at codon 69 in RT was also found. Amino acid deletions (d) between codon 66 and 71 are rare and usually only occur in combination with either multiple thymidine analogue mutations (TAMs) or the Q151M complex and, in these contexts, they are often associated with high-level multi-NRTI resistance (Rhee et al, 2006). In this particular patient (PDR 185), the amino acid deletion occurred with K65R and K219R. Generally, multi-nRTI resistance mutations were less frequently observed in patients on first- line regimen. The Q151M mutation was not observed in this study. This mutation, which is known to confer multi- NRTI resistance, is rare and occurs in only 5% of patients treated with DDI and AZT or d4T (Van Vaerenbergh et al, 2000; Shafer et al, 1995). It is therefore not strange that this mutation was not found in this study. In almost all of the patients on first-line regimen, lamivudine, stavudine and zidovudine were the NRTIs taken and the mutations observed were all indicative. Thymidine analogue mutations (M41L, T215Y, D67N, K70R University of Ghana http://ugspace.ug.edu.gh 121 and K219E/Q) were found among patients on first-line therapy and this is of great concern since these mutations could indicate cross resistance to the NRTIs (Shafer, 2002). The duration on therapy seemed to have contributed to the number and types of drug resistance mutations found in the patients on first-line regimen. Majority of the patients on first-line therapy had been on the same drugs for a median of 36 months and had accumulated these mutations over the treatment period. Four out of the six patients who had RT sequences with >2 NRTI mutations with TAMs (Table 6) had been on therapy for more than 50 months. Thus the longer patients are maintained on same drugs; the more likely they are to develop more mutations including multi-drug resistance ones (Kantor et al, 2004). Although there was no statistically significant association found between duration on therapy and the presence of the mutations (Table 12), it was clear that these mutations accumulated with time in the presence of drug pressure. According to Kantor et al, 2004, the maintenance of patients on the same drugs in the presence of one or two drug resistance mutations leads to the development of more mutations particularly cross-resistant mutations that will render future drugs ineffective. It is important to determine at what time point in therapy multi-NRTI resistance mutations usually develop but the cross-sectional nature of this study could not provide this information. Even in patients that had more than two mutations at the time of analysis, it was difficult to tell, using a one-time sequence analysis, which of the mutations was first to develop, except for M184V/I, which is usually the first mutation to appear in patient who had taken lamivudine (Descamps et al, 2000; Shafer, 2002). A longitudinal study would provide data that will better explain the evolution of resistance mutations. The mutations that are selected for after the failure of treatment with NNRTIs are all located in the pocket targeted by these compounds, and they reduce the affinity of the drug (Boyer et University of Ghana http://ugspace.ug.edu.gh 122 al., 1993; Richman et al., 1994; Bacheler et al., 2000; Ren et al., 2001; Hsiou et al., 2001). The mutations develop in a drug-specific manner due to the specific interaction of the drugs with the hydrophobic pocket but cause cross resistance to other drugs in the class. For example, K103N is an EFV- induced mutation but causes high resistance to both EFV and NVP. The Y181C mutation emerges during NVP use but induces high level resistance to EFV. The most commonly detected NNRTI resistance mutation among patients on first-line was K103N. This was expected since all the patients studied had been on either EFV or NVP as part of their regimen. The sequential use of NVP and EFV (in either order) is not recommended because of cross-resistance between these drugs (Antinori et al, 2002). However, this was done in some of the patients in this study perhaps due to drug availability. Although more patients used NVP compared to EFV (Table 3), the Y181C mutation was less frequently detected compared to K103N. The presence of the K103N mutation meant that there was high resistance to both NVP and EFV. Other NNRTI mutations detected are shown in Table 7. The V90I mutation, a substitution of valine by isoleucine, is a common polymorphism that is selected for by etravirine and rilpivirine and is associated with reduced etravirine susceptibility in combination with other NNRTI-resistance mutations (Liu and Shafer, 2006). The occurrence of this mutation in the study population at a level as high as 40% is contrary to expectation since neither etravirine nor rilpirvirine was used by any patient. This could mean that nevirapine and or efavirenz use may also select for this mutation but this needs further research. It has been reported that A98G reduces NVP and EFV susceptibility by about 5-fold and 3-fold, respectively. The V106A mutation causes high-level resistance to NVP and low-level resistance to EFV. Whilst V179D/E alone can reduce NVP and EFV susceptibility approximately2-fold, the combination of K103R and V179D reduces susceptibility to NVP and EFV approximately10-fold. The Nevirapine related mutation (Y181C) causes high-level resistance to NVP, and decrease susceptibility to EFV, ETR and University of Ghana http://ugspace.ug.edu.gh 123 RPV by approximately 2-fold and 5-fold respectively (Liu and Shafer, 2006). Other mutations found were Y188L/H/C and F227L that Y188L causes high-level resistance to NVP, EFV, and RPV. The F227L mutation usually occurs in combination with V106A and is associated with high level resistance to nevirapine and intermediate resistance to efavirenz while M230L causes intermediate/high-level resistance to each of the NNRTIs. The mutations K238T and K238N are NNRTI-selected mutations that usually occur in combination with K103N. In combination with K103N, they cause high-level resistance to Nevirapine and efavirenz. While E138A may contribute to reduced etravirine and rilpilvirine susceptibility in combination with other NNRTI-resistance mutations, H221Y is seen in patients receiving NNRTI and contributes to decreased NNRTI susceptibility in combination with other NNRTI-resistance mutations. Therefore, all the NNRTI mutations found among patients in this study had contributed in one way or the other to decreased susceptibility of NVP and EFV in particular and may lead to decreased susceptibility to etravirine and rilpivirine that were not taken by patients in this study. All the patients on first-line regimen were still receiving either NVP or EFV. Generally, these patients were described as doing well by the Physicians but the resistance mutations found in them could reduce the effectiveness of the NNRTI in their regimen and contribute to poor clinical outcomes. Protease inhibitor (PI) mutations were found in only one participant on first-line regimen, which is not surprising, since the first-line regimen does not usually contain PIs. Although there was no clinical history of PI use, this patient (PDR 223) had N88S and L10V mutations. The N88S mutation causes high-level resistance to NFV and ATV/r and low-level resistance to IDV/r; it increases susceptibility to FPV/r (Liu and Shafer, 2006). L10I/V/F/R/Y are associated with resistance to most PIs when present with other mutations (Liu and Shafer, University of Ghana http://ugspace.ug.edu.gh 124 2006). L10I/V also occurs in 5-10% of untreated persons. It is therefore likely that the L10V mutation was present before the patient begun treatment. The source of N88S could not be traced to any drugs used since the participant had no history of PI use. However, the presence of these mutations could affect future administration of second-line regimen which contains PIs. The pattern of resistance observed in patients on first-line regimen indicates that drug resistance testing would be most useful in the choice of appropriate drugs for the second-line regimen to achieve optimum treatment outcomes. 5.1.5.2 Drug resistance mutations among patients on second-line regimen In this study, various NRTI resistance mutations were found among patients on second-line regimen. The M184V was most frequently detected. This mutation was found in all the sequences that had drug resistance mutations and was closely followed by T215Y/F/I and M41L. The mutations D67GN, L210W, E44D, T69D, K70R, L74V/I and K219Q were also found in this study (Table 8). The presence of M184V in all the samples confirms previous use of lamivudine in the first-line therapy. Thymidine analogue mutations (M41L, D69N, K70R, L210W T215F/Y, and K219Q) were more frequently detected in these samples compared to samples from patients on first-line regimens. Thus, HIV strains in these patients were more resistant to many of the NRTIs. When compared with mutations detected among patients on first-line, the mutations observed among the first-line group were drug-specific while those observed in the second-line were class-specific. Thymidine analogue mutations were frequently observed in the second-line group in addition to the 69 insertion complex of mutations (Table 8). University of Ghana http://ugspace.ug.edu.gh 125 Some of the drug resistance mutations found affected the very drugs that the patients were taking at the time of study. Didanosine and abacavir were the most commonly used combination of NRTI for the second-line group and all of the viral sequences obtained had indications of either high level or intermediate level resistance to either or both (Table 8). This may render the NRTIs in the second-line regimen ineffective and further limit future options of drug choices. The accumulation of TAMs and other complex mutations, such as 69i causes decreased activity of NRTIs, with consequences for the effectiveness of the currently available second-line regimens in Ghana. Previous findings have shown that once cross-resistance has developed, standard second-line regimens will primarily offer the benefits of the boosted PI, with limited or no additional effect of the NRTI backbone (Sigaloff et al 2012). Patients will thus practically receive PI mono-therapy, which lowers the barrier for selection of PI resistance. In the patients on second-line regimen, NNRTIs were not part of the regimen but NNRTI mutations similar to those detected among patients on first-line regimen were observed (Table 9). This observation was concordant with a previous study that majority (73%) of patients in whom NNRTIs had been discontinued still harboured NNRTI mutations (Saravanan et al, 2012). Persistence of NNRTI mutations long after discontinuation of the NNRTI may be related to the low overall impact on viral fitness of mutations such as K103N, Y181C and G190 (Iglesias-Ussel et al, 2002; Joly et al, 2004). However, the presence of these mutations needs to be evaluated in case the need arises to re-introduce NNRTIs in future regimens. The protease inhibitor administered to patients on the study was NFV or LPV/r. The most frequently detected PI resistance mutations were M46I and L90M occurring in 50% and 37.5% of the sequences. The I84V mutation, which is an ATV/r-related mutation, was also University of Ghana http://ugspace.ug.edu.gh 126 observed in 20% of the patients although there was no ATV/r use. Other mutations observed less frequently and in various combinations included N88S, I54V, I82F, L74V, A71T, L89V, L23I, L33I, G58E and E35G (Table 10). The presence of PI mutations in these patients was expected since they had been on PIs as part of their second- line regimen. The levels of PI resistance mutations observed were lower than those of NRTI and NNRTI which could mean that the PIs were still effective. This difference could also be attributed to the longer duration of patients on NRTI and NNRTI compared to PI. The resistance profiles of patients on second-line mirrors the effect of switching drugs without drug resistance data. These patients have accumulated various NRTI resistance mutations in addition to PI mutations, rendering their regimen sub-optimal. Shafer (2002) alludes to the fact that patients whose physicians have access to drug resistance data respond better to salvage therapy than those whose physicians depend on other markers than drug resistance data and make ‘blind’ switches (Shafer 2002). It is therefore strongly recommended that patients failing second-line regimen in Ghana should undergo genotypic resistance testing to guide the composition of their subsequent regimens. 5.1.5.3 Drug resistance mutations in paired plasma and PBMC Generally, the NRTI drug resistance mutations observed in plasma and their corresponding PBMC were similar (Table 11). One mutation M184MV or M184IM each was detected in PBMC and not in plasma in two patients (PDR 48 and PDR 328). In these cases, it was possible that these mutations were selected for earlier and archived while the patients were on lamivudine but disappeared from plasma after the drug was discontinued. These patients also showed the simultaneous presence of the mutant with the wild type indicating a gradual reversal to the wild type. Another patient (PDR 304) also had M184V in plasma and no mutation in PBMC. In this case, M184V, being the first mutation to emerge in patients using University of Ghana http://ugspace.ug.edu.gh 127 lamivudine-containing regimen (Shafer, 2002) may be recent and not yet archived in PBMC. In one patient (PDR 90), the three mutations (M41L, M184V and T215Y) found in plasma were different from that found in PBMC (V75IV). This was the only case of a completely different mutation profile in plasma compared to PBMC for NRTI. The PBMC sequence however showed hypermutation due to the presence of APOBEC 3G/F and this could explain why the resistance mutations were not observed in the PBMC. Apolipoprotein B editing complex (APOBEC) group of proteins are host restriction factors that inhibit HIV replication by inducing dG-to-dA hypermutations in the proviral DNA (Sheehy et al., 2002; Liddament et al., 2004; Bishop et al., 2006). These proteins are expressed in lymphocytes, the major target cells for HIV-1 infection (Liddament et al., 2004; Wiegand et al., 2004). They reduce the rate of HIV replication and by extension the development of mutations. The NNRTI mutations were also similar in plasma and PBMC (Table 11). Where there were more mutations detected in either plasma or PBMC, the cumulative implications of these mutations for drug resistance were similar for both compartments. Patient PDR 328 for example, had 2 more mutations (M230L and V108I) in PBMC in addition to V90I and K103N observed in both compartments but these mutations cumulatively had reduced NVP and EFV susceptibility so the clinical implications were the same. PI mutations were observed mainly in plasma component of the patients on second-line therapy. The three patients that had mutations in both plasma and PBMC were PDR 4, PDR 11 and PDR 19. One of them (PDR 4) had the same mutation L10I in both plasma and PBMC. The patient PDR 11 had M46LM and A71AV in PBMC whereas its plasma that had I84V, E35G and L89V in addition. The additional mutations in plasma might have emerged recently and were not yet archived in PBMC since those mutations (M46LM and A71AV) University of Ghana http://ugspace.ug.edu.gh 128 found in the PBMC were co-existing with the wild type. Thus in general, the profile of mutations observed in plasma were similar to those observed in PBMC. 5.1.6 Effect of change in CD4 counts, viral load at sampling and duration on first-line ART on drug resistance mutations Duration on ART, change in CD4 counts and viral loads are factors known to affect the presence of drug resistance mutations in HIV patients. In this study however, the differences observed were not statistically significant. This may be due to the small number of sequences (65) analyzed. 5.1.7 Effect of adherence to antiretrovirals or herbal medicine use on the presence of drug resistance mutations Data on adherence to ART and herbal medicine taken during ART showed some differences between patients that took herbal medicine and those who did not and between those who adhered and those who did not. However, these differences were not statistically significant (Table 13). These differences are worth further investigations particularly in a longitudinal study. 5.1.8 Polymorphisms at DR sites in the PR sequences Polymorphisms at resistance-associated positions were observed in the PR genes analyzed in this study. The mutations K20I and M36I were found in 96% and 97% of the sequences respectively. The L10I or L10V mutations were also observed in 22% of the sequences confirming the fact that polymorphisms associated with PI resistance in subtype B do occur naturally in non-B subtypes (Turner et al, 2004; Kantor et al, 2006). Majority of the sequences in this study were CRF02_AG and known to be associated with K20I, M36I and University of Ghana http://ugspace.ug.edu.gh 129 L10IV which have implications for nelfinavir and tipranavir susceptibility (Turner et al, 2004; Delgado et al, 2008). Kinomoto et al (2004) also reported that HIV-1 protease sequences obtained from ART- naive patients from Ghana were differentially less susceptible to protease inhibitors but did not directly link their findings to any of the mutations observed. Amino acid insertions at codons 35 and 37 in the protease gene, similar to those reported by Delgado et al (2008) were also detected in patients in this study. The implication of these mutations on PI susceptibility however needs further investigation. 5.1.9 HIV-1 Subtype information The majority of the reverse transcriptase sequences were subtyped as CRF02_AG by the Stanford HIV Database. This finding agrees with those of previous studies that CRF02_AG is the predominant HIV-1 subtype in Ghana (Delgado et al, 2008; Brandful et al, 2012). The other subtypes found in this study were B, G and K. Subtype B is reported as the predominant HIV-1 subtype in Europe and North America (Osmanov et al, 2000; Kantor et al, 2005) and finding it in Ghana could be due to importation. Subtypes G and K have been previously reported in Ghana and other West African countries (Brandful et al, 1998; Kantor et al, 2005). Although unique recombinant forms are also known to occur in Ghana (Delgado et al, 2008), none were described in this study probably because detailed analysis was not done to identify recombinants. University of Ghana http://ugspace.ug.edu.gh 130 5.2 Conclusions This study has clearly showed that HIV-1 patients on ART in Ghana may not be deriving optimal benefit because of the limited markers being used to monitor treatment and effect changes in regimens. The CD4 counts and viral loads of the patients studied confirmed the physicians’ assertion that majority of the patients were doing well. However, 46% and 49% of these patients had major drug resistance mutations to NRTIs and NNRTIs respectively. Also, TAMs, which render NRTIs ineffective, were found in both patients on first-line (33%) and those on second-line (79%) regimens. There were PI resistance mutations present in 28% of the PR sequences obtained from patients on second-line regimen. Similar patterns of drug resistance mutations were observed across study sites and were comparable to patterns observed elsewhere with similar drug regimens. Thus, the types of antiretroviral drugs taken by patients were the main driving force for the development of resistance mutations. The profiles of drug resistance mutations found in patients on first-line regimens suggested that the duration of a patient on same drug combination has an influence on the type and number of drug resistance mutations developed. There was however no statistically significant association between duration on therapy and the presence of drug resistance mutations probably because this was a cross-sectional study. There was also no statistically significant association between adherence to therapy or herbal medicine use while on therapy and the presence of drug resistance mutations. University of Ghana http://ugspace.ug.edu.gh 131 The HIV-1 sequences obtained were mostly classified as CRF02_AG recombinants and this confirmed the dominance of this HIV-1 subtype in Ghana as shown by previous studies. This study also found similar resistance profiles in plasma compared to peripheral blood mononuclear cells of paired sequences in both the RT and PR genes. Thus PBMC could be used as an alternative to plasma for drug resistance testing. The drug resistance mutation data generated in this study indicates the clear need for the use of genotypic resistance data in patient management particularly to inform the choice of drugs to change regimens. This will reduce the accumulation of multi- nucleoside and thymidine analog mutations in patients and enhance treatment success. For patients who will fail the current second-line regimen, the data showed that there will be very little benefit with available drugs if switched without individual drug resistance profiling The study has provided vital drug resistance data to guide policy on ART monitoring in Ghana. It has also contributed to improved protocols for HIV-1 genotyping at the Virology Department of the Noguchi Memorial Institute for Medical Research and helped to build capacity for drug resistance testing of patients who fail ART in Ghana. University of Ghana http://ugspace.ug.edu.gh 132 5.3 Recommendations 1. Regular viral loads should be used to monitor patients on ART in Ghana and drug resistance testing should be done before switching to a new regimen. 2. A longitudinal study is recommended to establish a cohort of patients on ART in Ghana to better understand the evolution of drug resistance mutations during treatment. 3. A phenotypic study is recommended to better understand the drug resistance implications of the polymorphisms observed in the protease gene of HIV-1 subtype CRF02_AG from Ghana. 4. 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Sample ID__________________ Age___________ Sex______________ 3. Date of Collection_______________ Current CD4 Count___________________ 4. Previous CD4 count_____________ Date analyzed_______________________ 5. Date started ART_______________ CD4 count at start of ART________________ 6. ARV currently on______________________ Duration_________________ 7. Treatment history: ARV_____________________________ Dates (from- to) _____________________ ARV_____________________________ Dates (from- to) ______________________ Please make a cross (X) near the appropriate answer for questions 8-10 8. Have you missed any drug days since you started? Yes_______ No________ 8b. If Yes, for how long_______________________________________________ 9. Have you taken any herbal medicine while on ART? Yes_______ No________ 9b. If Yes, for how long_______________________________________________ 10. Physician’s comment on performance of patient: Doing well___________ Not doing so well__________ Failing____________ Other (please specify______________________________________________________ Thank you for the sample and your time!!! University of Ghana http://ugspace.ug.edu.gh 166 Appendix II PATIENT CONSENT FORM Research Title Characterization of HIV Drug-Resistant Strains in HIV-Infected persons on antiretrovirals (ARV) in selected HIV Care Centres in Ghana Principal Investigator Evelyn Yayra Bonney (Mrs.) Address Noguchi Memorial Institute for Medical Research, University of Ghana, Legon Accra, Ghana Introduction This Consent Form contains information about the research named above. In order to be sure that you are informed about being in this research, we are asking you to read (or have read to you) this Consent Form. You will also be asked to sign it (or make your mark in front of a witness). We will give you a copy of this form. This consent form might contain some words that are unfamiliar to you. Please ask us to explain anything you may not understand Reason for the Research You are being asked to take part in research to find out whether the medicine that you are given at the clinic (ARV) can control the growth of the HIV you have in your blood now. Research Purpose/General Information about Research This is a research to find out whether the HIV in persons on ARV is being successfully managed with the drugs that are currently in use in Ghana. When the ARV can longer control the growth of the HIV a person is carrying, we say the virus is a drug-resistant strain. The development of drug-resistant strains makes ARV no longer useful to the person using it and sometimes affects the effectiveness of the other drugs that can be used by the patient if they belong to the same class as the current ARV. Some people seem to be doing well clinically but still have drug-resistant strains and these strains will eventually make all the treatment efforts fruitless. CD4 counts cannot tell whether a person has drug-resistant strain or not. This research will use a more advanced method to find out the types of strains people on ARV have so that the success of the current ARV program in the country can be evaluated and modified if necessary. Your Part in the Research University of Ghana http://ugspace.ug.edu.gh 167 If you agree to be in the research, you will be asked to give 7ml of your blood sample, taken from your veins, only once, although the project is expected to last over a three year period. About 380 adults (both males and females) from 4 HIV care centres in Ghana, who have taken the drugs for at least 6 months, will take part in this study. Possible Risks and Discomforts Participants may have some discomfort and pain when blood sample is being taken. Possible Benefits The data generated from this research would be useful in monitoring therapy and redesigning treatment regimens. This will directly benefit participants by informing change in treatment regimens if necessary. The project will also benefit all persons living with HIV/AIDS by providing information for the better management of the infection. Alternatives to Participation You will continue to benefit from the ART programme even if you decline to participate in this research project Confidentiality We will protect information about you and your part in this research to the best of our ability. No report generated from this research will name, or be linked to, the persons involved. Where necessary however, the researcher and your doctor may need to discuss your specific results so as to enable a better management of your condition. Compensation You will not be paid for participating in this research, since you do not have to take part in this research. If You Have a Problem or Have Other Questions Please call Evelyn Yayra Bonney (0244 785677) if you have further questions about the research. Your rights as a participant This research project has been reviewed and approved by the IRB of the Noguchi Memorial Institute for Medical Research. If you have any questions about your rights as a research participant you may contact Rev. Dr. Ayete-Nyampong, Chairperson, NMIMR-IRB, mobile 0208152360 University of Ghana http://ugspace.ug.edu.gh 168 Appendix III PARTICIPANT’S AGREEMENT The above document describing the benefits, risks and procedures for the research title “Characterization of HIV Drug Resistant Strains in HIV-Infected persons on antiretrovirals in selected HIV Care Centres in Ghana” has been read and explained to me. I have been given an opportunity to have any questions about the research answered to my satisfaction. I agree to participate as a volunteer. _______________________ _______________________________________________ Date Signature or mark of volunteer If volunteers cannot read the form themselves, a witness must sign here: I was present while the benefits, risks and procedures were read to the volunteer. All questions were answered and the volunteer has agreed to take part in the research. _______________________ ____________________________________________ Date Signature of Witness I certify that the nature and purpose, the potential benefits, and possible risks associated with participating in this research have been explained to the above individual. _________________________ _______________________________________________ Date Signature of Person Who Obtained Consent University of Ghana http://ugspace.ug.edu.gh 169 Appendix IV MATERIALS A. Reagents for laboratory analyses Lymphocyte separation medium- (Histopaque®-1077) [SIGMA, USA] Phosphate buffered saline (PBS) [SIGMA, USA] Foetal bovine serum (FBS) [SIGMA, USA] Dimethyl sulphoxide (DMSO) [SIGMA, USA] INNO-LIA HIV-1/2 Confirmatory Assay (Innogenetics, Belgium) Absolute ethanol (molecular biology grade) [SIGMA, USA] Nuclease-free water (Ambion, USA) Nucleic acid purification kit (Roche Diagnostics, Germany) QIAamp DNA Blood kit (QIAGEN, USA) QIAamp viral RNA kit (QIAGEN, USA) Taqman One-Step RT-PCR Reagents (ABI, USA) One Step RT-PCR Kit (QIAGEN, USA) AmpliTaq Gold Master Mix Reagents (ABI, USA) Agarose (SIGMA, USA) Ethidium bromide (SIGMA, USA) Tris-Acetate-EDTA (TAE) [Ambion, USA) DNA molecular weight 100bp ladder (Invitrogen, USA) QIAquick PCR purification kit (QIAGEN, USA) Big Dye Terminator Cycle Sequencing Kit vs. 3.1 (ABI, USA) AgenCourt CleanSeq Dye Terminator Removal kit (Beckman Coulter, USA) Sequencing Buffer with EDTA 5X (ABI, USA) University of Ghana http://ugspace.ug.edu.gh 170 Performance Optimized Polymer-POP 7 (ABI, USA) Primers (Eurogentec, Belgium B. Laboratory equipment Biosafety Cabinet Class IIA (AirTech Services, India) Biological Safety Cabinet Class II (LABGARD, USA) Centrifuge (H-900) [Kokusan, Japan] Platform rocker, STR6 (Bibby, UK) Autoclave SS-325 (Tomy, Japan) Vortex Genie-2 (Scientific Industries, USA) Microcentrifuge 5415D (Eppendorf, USA) Heat block (Haep labor Consult, Germany) Pipetman Classic: p1000, p200, p20, p10 (Gilson S.A.S, France) Aerosol-resistant pipette tips: 1000µl, 200µl, 20µl, 10µl (Gilson S.A.S, France) MBP ART® self-sealing barrier tips: 1000µl, 200µl, 20µl, 10µl (SIGMA, USA) AirClean 600 PCR Workstation (AirClean Systems, USA) Real Time PCR System 7300 (ABI, USA) GeneAmp PCR System 2700 and 2720 (ABI, USA) Microwave oven (LG Electronics Inc., Ghana) Electrophoresis system (Mupid-2 Plus) [Japan] Gel logic 100 Imaging System (Eastman Kodak Company, USA) High Performance Ultraviolet Transilluminator (UVP, UK) Genetic Analyzer 3130 (ABI, USA) C. Consumables for laboratory analyses RNase-free 15ml centrifuge tubes (Ambion, USA) University of Ghana http://ugspace.ug.edu.gh 171 Nalgene 1.8ml cryovials (Nalge Nunc, USA) Nalgene cryoboxes (Nalge Nunc, USA) Latex examination gloves GN32; powder-free, Fine Touch (Hand Safe, UK) RNase-free 1.5ml microfuge tubes (Ambion, USA) Sterile RNase-free 0.2ml thin walled PCR tubes (Ambion, USA) KimTech Science Precision wipes (Kimberly-Clark® Professional, USA) D. Software for sequence analysis Seqman (DNAStar, Madison, WI) Bioedit (http://www.mbio.ncsu.edu/Bioedit/bioedit.html) MEGA 4.1 (http://www.megasoftware.net/) Stanford University HIVdb Program (http://hivdb6.stanford.edu) Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html) BLAST (http://www.ncbi.nlm.nih.gov/blast/blast overview.shtml) University of Ghana http://ugspace.ug.edu.gh 172 Appendix V ETHICAL CLEARANCE CERTIFICATES The ethical clearance certificates received from the Institutional Review Board of Noguchi Memorial Institute for Medical research are found on Pages 179-182. The study first received ethical clearance in March 2010 (Page 182) and the clearance was renewed annually for its 4- year duration. University of Ghana http://ugspace.ug.edu.gh 173 University of Ghana http://ugspace.ug.edu.gh 174 University of Ghana http://ugspace.ug.edu.gh 175 University of Ghana http://ugspace.ug.edu.gh 176 University of Ghana http://ugspace.ug.edu.gh 177 Appendix VI AMINO ACID NAMES, ONE-LETTER CODE AND ACRONYMS Name One letter code Acronym Alanine A Ala Cysteine C Cys Aspartic acid D Asp Glutamic acid E Glu Phenylalanine F Phe Glycine G Gly Histidine H His Isoleucine I Ile Lysine K Lys Leucine L Leu Methionine M Met Asparagine N Asn Proline P Pro Glutamine Q Gln Arginine R Arg Serine S Ser Threonine T Thr Valine V Val Tryptophan W Trp Tyrosine Y Tyr University of Ghana http://ugspace.ug.edu.gh