Background: Individuals living with human immunodeficiency virus (HIV) who experience virological failure (VF) after combination antiretroviral therapy (cART) initiation may have had low-frequency drug resistance mutations (DRMs) at cART initiation. There are no data on low-frequency DRMs among cART-naïve HIV-positive individuals in Botswana. Methods: We evaluated the prevalence of low-frequency DRMs among cART-naïve individuals previously sequenced using Sanger sequencing. The generated pol amplicons were sequenced by next-generation sequencing. Results: We observed low-frequency DRMs (detected at 20%. K65R was the most common low-frequency DRM detected in 8 individuals. Eighty-two of the 103 individuals had follow-up viral load data while on cART. Twenty-seven of the 82 individuals harbored low-frequency DRMs. Only 12 of 82 individuals experienced VF. The following low-frequency DRMs were observed in four individuals experiencing VF: K65R, K103N, V108I, and Y188C. No statistically significant difference was observed in the prevalence of low-frequency DRMs between individuals experiencing VF (4/12) and those not experiencing VF (23/70) (P = .97). However, individuals with non-nucleoside reverse transcriptase inhibitors-associated low-frequency DRMs were 2.68 times more likely to experience VF (odds ratio, 2.68; 95% confidential interval, 0.4–13.9) compared with those without (P = .22). Conclusion: Next-generation sequencing was able to detect low-frequency DRMs in this cohort in Botswana, but these DRMs did not contribute significantly to VF.

Background:HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs). Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods:A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results:Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants. There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion:The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Results from the first audit of an intensive care unit in Botswana

Cerebrospinal fluid (CSF) viral escape has been poorly described among people with HIV-associated cryptococcal meningitis. We determined the prevalence of CSF viral escape and HIV-1 viral load (VL) trajectories in individuals treated for HIV-associated cryptococcal meningitis. A retrospective longitudinal study was performed using paired CSF and plasma collected prior to and during the antifungal treatment of 83 participants recruited at the Botswana site of the phase-3 AMBITION-cm trial (2018–2021). HIV-1 RNA levels were quantified then CSF viral escape (CSF HIV-1 RNA ≥ 0.5 log₁₀ higher than plasma) and HIV-1 VL trajectories were assessed. CSF viral escape occurred in 20/62 (32.3%; 95% confidence interval [CI]: 21.9–44.6%), 13/52 (25.0%; 95% CI: 15.2–38.2%) and 1/33 (3.0%; 95% CI: 0.16–15.3%) participants at days 1, 7 and 14 respectively. CSF viral escape was significantly lower on day 14 compared to days 1 and 7, p = 0.003 and p = 0.02, respectively. HIV-1 VL decreased significantly from day 1 to day 14 post antifungal therapy in the CSF but not in the plasma (β = −0.47; 95% CI: −0.69 to −0.25; p < 0.001). CSF viral escape is high among individuals presenting with HIV-associated cryptococcal meningitis; however, antifungal therapy may reverse this, highlighting the importance of rapid initiation of antifungal therapy in these patients.

Background:HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs). Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods:A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results:Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants. There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion:The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Treated human immunodeficiency virus (HIV) is associated with persistent systemic inflammation, even after many years of sustained viral suppression following initiation of antiretroviral therapy (ART). Albuminuria is common among people living with HIV (PLWH), but the impact of persistent systemic inflammation on outcome of albuminuria is not well understood. Thawed serum samples from PLWH who participated in an albuminuria prevalence study in Gaborone, Botswana, between January 2020 and March 2022, were selected randomly for a cross-sectional study of the link between inflammation and albuminuria. Systemic inflammation (interleukin [IL] 1β, IL 6, and soluble cluster of differentiation-163 [sCD163]) was assessed using enzyme linked immunosorbent assay, and albuminuria was reported as urinary albumin–creatinine ratio (ACR) (mg/g), as obtained from the parent study. The association between systemic inflammation and albuminuria was first explored by ACR quartiles, graphically using simple linear models, and then using general additive models for the adjusted analysis. The study population comprised 715 ART treated PLWH, with a mean age of 49.9 (SD 10.7) years, median HIV disease duration of 13.5 (IQR 8.7–16.7) years, and 398/715 (55.7%) were male. The relationship between log transformed ACR and sCD163 was linear, with regression coefficient β = 0.10, P-value = .02 but was nonlinear for log transformed IL-1β and IL-6, β = 0.10, P -value = .82 and β = ‐0.04, P-value = .36, respectively. In the final adjusted general additive models, sCD163 was not associated with ACR, P-value = .137. IL-1β, IL-6, and sCD163 were not associated with ACR among ART treated PLWH. Novel strategies to identify inflammatory pathways that may promote albuminuria among PLWH should consider other innovative and sensitive markers of both systemic and organ specific inflammation.