Vaginal microbial diversity of the genital tract of South African adolescent females

1 Chapter 1: Literature Review . 3 1.1 Human-Immunodeficiency Virus in South Africa . 3 1.2 The female genital tract (FGT) immune response . 4 1.3 FGT Microbiota . 7 1.4 Bacterial vaginosis (BV) . 9 1.5 Sexually Transmitted Infections (STIs) . 11 1.6 Hormonal Contraceptives . 12 1.7 Technical analysis of FGT bacteria . 15 1.8 Aims of this Study . 16 1.9 Objectives of this Study . 17 1.10 Hypothesis . 17 Chapter 2: . 18 2.1 Study Design . 18 2.2 Recruitment of participants . 18 2.3 Exclusion criteria . 18 2.4 Participants and sample collection. 19 2.5 Human-immunodeficiency virus (HIV) testing . 19 2.6 Bacterial vaginosis (BV) testing . 19 2.7 Sexually Transmitted Infections (STIs) testing . 20 2.8 Next Generation Sequencing (NGS) of 16S rRNA . 20 2.9 Cohort characteristics . 21 Chapter 3: Methods and Materials . 23 3.1. Bacterial reference strains . 23 3.1.1 Bacterial Culturing . 23 3.1.1.1 Lactobacillus spp. growth conditions . 23 3.1.1.2 Lactobacillus iners, Prevotella bivia and Gardnerella vaginalis growth conditions. 24 3.1.2 DNA Extraction . 24 3.1.3 Primer Design . 25 3.2 Polymerase Chain Reaction . 26 3.2.1 Polymerase Chain Reaction (PCR) of ATCC reference strains (Primer confirmation) . 26 3.2.2 Gel electrophoresis . 27 3.2.3 Serial dilution calculations for the known standard controls: . 27 3.3 qPCR optimization. 31 3.3.1 qPCR Optimization Outcomes.32 3.3.2 Lactobacillus crispatus.34 3.3.3 Gardnerella vaginalis . 39 3.3.4 Prevotella bivia . 41 3.4 Real-Time PCR (qPCR) Protocol.47 3.5 Analysis . 49 3.6 Statistical considerations . 51 3.6. 1 Statistical software used for data analysis . 51 3.6. 2 Statistical tests used for data analysis in this study . 51 3.6. 3 Conceptual Framework . 54 3.7 Sequencing and Analysis. 54 Chapter 4: Results . 56 4.1 P. bivia Sequencing . 56 4.1.1 NCBI Blast Analysis . 57 4.1.2 Sequence Alignment . 61 4.2 Real-Time PCR (qPCR) Results . 71 4.2.1 Descriptive statistics . 72 4.3 Comparison of absolute bacterial quantities to BV status, inflammation levels, age, hormonal contraceptive and STI status, bacterial versus viral STIs and HPV. 74 4.3.1 Association between the quantities of the bacteria of interest and BV status . 74 4.3.2 Association between bacteria of interest and inflammatory immunological factor levels . 83 4.3.3 Association between the quantities (copies/ng) of bacteria of interest and age . 92 4.3.4 Association between the quantities (copies/ng) of vaginal bacteria and hormonal contraceptives . 100 4.3.5 Association between the quantities (copies/ng) of the bacteria of interest and the absence or presence of any one STI in the WISH cohort . 109 4.3.6 Association between the quantities (copies/ng) of the bacteria of interest and the presence of bacterial or viral STIs in the WISH cohort . 117 4.3.7 Association between the quantities (copies/ng) of bacteria of interest and the absence or presence of low and high risk HPV subtypes in the WISH cohort . 123 4.5 Overview . 132 Chapter 5: Discussion . 134 Chapter 6: Conclusion . 141 References . 142 Appendix A . 163 Appendix B . 167 Appendix C . 168 Appendix D . 173 Appendix E . 188