Person:

Musonda, Rosemary

To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; −0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

Laboratory cross-sectional assays are useful for the estimation of HIV incidence, but are known to misclassify individuals with long-standing infection as recently infected. The false recent rate (FRR) varies widely across geographic areas; therefore, accurate estimates of HIV incidence require a locally defined FRR. We determined FRR for Botswana, where HIV-1 subtype C infection is predominant, using the BED capture enzyme immunoassay (BED), a Bio-Rad Avidity Index (BAI) assay (a modification of the Bio-Rad HIV1/2+O EIA), and two multiassay algorithms (MAA) that included clinical data. To estimate FRR, stored blood samples from 512 antiretroviral (ARV)-naive HIV-1 subtype C-infected individuals from a prospective cohort in Botswana were tested at 18-24 months postenrollment. The following FRR mean (95% CI) values were obtained: BED 6.05% (4.15-8.48), BAI 5.57% (3.70-8.0), BED-BAI 2.25% (1.13-4.0), and a combination of BED-BAI with CD4 (>200) and viral load (>400) threshold 1.43% (0.58-2.93). The interassay agreement between BED and BAI was 92.8% (95% CI, 90.1-94.5) for recent/long-term classification. Misclassification was associated with viral suppression for BED [adjusted OR (aOR) 10.31; p=0.008], BAI [aOR 9.72; p=0.019], and MAA1 [aOR 16.6; p=0.006]. Employing MAA can reduce FRR to <2%. A local FRR can improve cross-sectional HIV incidence estimates.

BACKGROUND: Clinical laboratories in Botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers' kits and textbooks. OBJECTIVES: The aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in Botswana. METHOD: A total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of Botswana, and immunological and haematological laboratory parameters were measured. RESULTS: The mean age was 28.8 (95% Confidence Interval [CI] 27.7-29.8) years, with a median of 27 years and a range 18-66 years. The mean haemoglobin level was significantly lower for women (12.4 g/dL; 95% CI 12.1% - 12.7%) than men (15.1 g/dL; 95% CI 14.9% - 15.3%). The women's haemoglobin reference values (9.0 g/dL - 15.0 g/dL) levels were lower than observed in predominantly White populations (12.0 g/dL - 16.0 g/dL), but comparable with regional consensus reference intervals (9.5 g/dL - 15.8 g/dL) recently defined for East and Southern Africa. CONCLUSION: The established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally.

Background: Hepatitis B virus (HBV) is a major global health problem especially in sub-Saharan Africa and in East Asia. Ten hepatitis B virus genotypes have been described that differ by geographic distribution, disease progression, and response to treatment. Escape mutations within the surface open reading frame (ORF) affect HBV antigenicity leading to failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. However, the molecular characteristics of HBV in Botswana, a highly endemic country, are unknown. We describe the molecular characteristics of HBV and prevalence of escape mutants among HIV/HBV coinfected individuals Botswana. Methods: DNA was extracted from archived plasma samples from 81 HIV/HBV co-infected participants from various clinical studies at the Botswana Harvard AIDS Institute Partnership. A 415 base pair (bp) fragment of the polymerase gene was amplified by semi-nested PCR. In a subset of samples, a 2100 bp fragment was amplified. The PCR product was genotyped using Big Dye sequencing chemistry and the sequences were analysed for genotypes and mutations. Results: Of the 81 samples included, 70 (86 %) samples were successfully genotyped. Genotype A was found in 56 (80 %) participants, D in 13 (18.6 %), and 1 (1.4 %) was genotype E. Escape mutations previously linked with failure of diagnosis or escaping active vaccination and passive immunoglobulin therapy were detected in 12 (17.1 %) participants at positions 100, 119, 122, 123, 124, 126, 129, 130, 133, 134 and 140 of the S ORF. Genotypes and escape mutations were not significantly associated with aspartate aminotransferase (AST), alanine aminotransferase (ALT) and AST platelet ratio index (APRI). Conclusion: Genotypes A, D and E were found in this cohort of HIV coinfected patients in Botswana, consistent with the findings from the sub-Saharan Africa region. Some escape mutations which have previously been associated with diagnosis failure, escaping vaccine and immunoglobulin therapy were also observed and are important in guiding future policies related to vaccine implementation, therapeutic guidelines, and diagnostic guidelines. They are also important for identifying patients who are at an increased risk of disease progression and to choose optimal therapy. Future research should focus on determining the clinical significance of the different HBV genotypes and mutations found in this population.

Introduction: A dried blood spot (DBS) on filter paper has been used for different tests globally and has gained popularities in resource limited settings especially during HIV/AIDS epidemic. We assessed the efficiency of molecular characterization of HIV-1 subtypes using DBS collected under field conditions in northern Tanzania. Materials and Methods In 2011 and 2012, 60 DBS samples were collected under field conditions from exposed and newly diagnosed HIV-1 infected children from Kilimanjaro (n=20), Arusha (n=20), Tanga (n=10) and Manyara (n=10). Results and discussion Of 60 DBS analyzed at both Protease (PR) and Reverse Transcriptase (RT) regions, 45 (75%) were analyzed, including 17 (85%) from Kilimanjaro, 15 (75%) from Arusha, 8 (80%) from Tanga, and 5 (50%) from Manyara region. All 45 DBS characterized had viral load above 1000 copies/mL with mean log10 viral loads of 3.87 copies/mL (SD 0.995). The phylogenetic results indicated presence of subtype and circulating recombinant form (CRF). In which, 24 were subtype A1 (53.33%), 16 were subtype C (35.55%), 3 were subtype D (6.67%) and 2 were CRF10_CD (4.35%). All major mutations were detected in the RT region, none from protease (PR) region. The mutations detected were Y181C (n=8), K103 (n=4) and G190A (n=1), conferring resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), and M184V (n=1), conferring resistance to lamivudine and emtricitabine. Conclusions: Our results indicate that DBS collected from field conditions in resource scarcity areas can be used to determine the phylogeny of the virus and drug resistance mutations in areas with diverse HIV-1 group M subtypes.

Objectives: In resource-limited settings, it is a challenge to get quality clinical specimens due to poor infrastructure for their collection, transportation, processing and storage. Using dried blood spots (DBS) might be an alternative to plasma for HIV-1 drug resistance testing in this setting. The objectives of this study were to determine mutations associated with antiretroviral resistance among children <18 months old born to HIV-1-infected mothers enrolled in prevention of mother-to-child transmission services in northern Tanzania. Patients and methods Kilimanjaro Christian Medical Center (KCMC) Clinical Laboratory is the zonal centre for early infant diagnosis using DBS in northern Tanzania. DBS were collected from January 2011 to December 2012. Mothers were kept on triple therapy and single-dose nevirapine before pregnancy and during labour, respectively. Infants were given single-dose nevirapine and most of them were breastfed. Genotypic resistance was determined in those with a viral load of >400 copies/mL. Results: Genotypic resistance mutations were detected in 13 of 46 children (28%). HIV-1 genotypes were A1 (n = 27), C (n = 10), A/D (n = 4), D (n = 3) and CRF10_CD (n = 2). The median age was 12 weeks (IQR 6–28). The mean log10 viral load was 3.87 copies/mL (SD 0.995). All major mutations were detected in the reverse transcriptase gene and none in the protease gene region. The most frequent mutations were Y181C (n = 8) and K103N (n = 4), conferring resistance to non-nucleoside reverse transcriptase inhibitors. Conclusions: One-third of infants newly diagnosed with HIV in northern Tanzania harboured major drug resistance mutations to currently used antiretroviral regimens. These mutations were detected from DBS collected from the field and stored at room temperature. Surveillance of drug resistance among this population in resource-limited settings is warranted.

The first aim of the study is to assess the distribution of HIV-1 RNA levels in subtype C infection. Among 4,348 drug-naïve HIV-positive individuals participating in clinical studies in Botswana, the median baseline plasma HIV-1 RNA levels differed between the general population cohorts (4.1–4.2 log10) and cART-initiating cohorts (5.1–5.3 log10) by about one log10. The proportion of individuals with high (≥50,000 (4.7 log10) copies/ml) HIV-1 RNA levels ranged from 24%–28% in the general HIV-positive population cohorts to 65%–83% in cART-initiating cohorts. The second aim is to estimate the proportion of individuals who maintain high HIV-1 RNA levels for an extended time and the duration of this period. For this analysis, we estimate the proportion of individuals who could be identified by repeated 6- vs. 12-month-interval HIV testing, as well as the potential reduction of HIV transmission time that can be achieved by testing and ARV treating. Longitudinal analysis of 42 seroconverters revealed that 33% (95% CI: 20%–50%) of individuals maintain high HIV-1 RNA levels for at least 180 days post seroconversion (p/s) and the median duration of high viral load period was 350 (269; 428) days p/s. We found that it would be possible to identify all HIV-infected individuals with viral load ≥50,000 (4.7 log10) copies/ml using repeated six-month-interval HIV testing. Assuming individuals with high viral load initiate cART after being identified, the period of high transmissibility due to high viral load can potentially be reduced by 77% (95% CI: 71%–82%). Therefore, if HIV-infected individuals maintaining high levels of plasma HIV-1 RNA for extended period of time contribute disproportionally to HIV transmission, a modified “test-and-treat” strategy targeting such individuals by repeated HIV testing (followed by initiation of cART) might be a useful public health strategy for mitigating the HIV epidemic in some communities.

Background: Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness.

Background. Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection has emerged as an important cause of morbidity and mortality. We determined the response to Truvada-based first-line combination antiretroviral therapy (cART) in HIV/HBV-coinfected verus HIV-monoinfected patients in Botswana. Methods. Hepatitis B virus surface antigen (HBsAg), HBV e antigen (HBeAg), and HBV deoxyribonucleic acid (DNA) load were determined from baseline and follow-up visits in a longitudinal cART cohort of Truvada-based regimen. We assessed predictors of HBV serostatus and viral suppression (undetectable HBV DNA) using logistic regression techniques. Results. Of 300 participants, 28 were HBsAg positive, giving an HIV/HBV prevalence of 9.3% (95% confidence interval [CI], 6.3–13.2), and 5 of these, 17.9% (95% CI, 6.1–36.9), were HBeAg positive. There was a reduced CD4+ T-cell gain in HIV/HBV-coinfected compared with HIV-monoinfected patients. Hepatitis B virus surface antigen and HBeAg loss was 38% and 60%, respectively, at 24 months post-cART initiation. The HBV DNA suppression rates increased with time on cART from 54% to 75% in 6 and 24 months, respectively. Conclusions. Human immunodeficiency virus/HBV coinfection negatively affected immunologic recovery compared with HIV-1C monoinfection. Hepatitis B virus screening before cART initiation could help improve HBV/HIV treatment outcomes and help determine treatment options when there is a need to switch regimens.