Long-term non-progression and risk factors for disease progression among children living with HIV in Botswana and Uganda: A retrospective cohort study.

OBJECTIVES: We utilize a large retrospective study cohort derived from electronic medical records to estimate the prevalence of long-term non-progression (LTNP) and determine the factors associated with progression among children infected with HIV in Botswana and Uganda. METHODS: Electronic medical records from large tertiary HIV clinical centers in Botswana and Uganda were queried to identify LTNP children 0-18 years enrolled between June 2003 and May 2014 and extract demographic and nutritional parameters. Multivariate subdistribution hazard analyses were used to examine demographic factors and nutritional status in progression in the pre-antiretroviral therapy era. RESULTS: Between the two countries, 14,246 antiretroviral therapy-naïve children infected with HIV were enrolled into clinical care. The overall proportion of LTNP was 6.3% (9.5% in Botswana vs 5.9% in Uganda). The median progression-free survival for the cohort was 6.3 years, although this was lower in Botswana than in Uganda (6.6 vs 8.8 years; P <0.001). At baseline, the adjusted subdistribution hazard ratio (aHRsd) of progression was increased among underweight children (aHRsd 1.42; 95% confidence interval [CI]: 1.32-1.53), enrolled after 2010 (aHRsd 1.32; 95% CI 1.22-1.42), and those from Botswana (aHRsd 2; 95% CI 1.91-2.10). CONCLUSIONS: In our study, the prevalence of pediatric LTNP was lower than that observed among adult populations, but progression-free survival was higher than expected. Underweight, year of enrollment into care, and country of origin are independent predictors of progression among children.

Comparison of an in-house 'home-brew' and commercial ViroSeq integrase genotyping assays on HIV-1 subtype C samples.

BACKGROUND: Roll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals. METHODS: We used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time. RESULTS: Among 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C. CONCLUSIONS: The IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

Four-class drug-resistant HIV-1 subtype C in a treatment experienced individual on dolutegravir-based antiretroviral therapy in Botswana.

: There are limited data on the effectiveness of dolutegravir (DTG)-based combination antiretroviral therapy (ART) in real-life settings in southern Africa where HIV-1 subtype C predominates. We report a patient infected with HIV-1 subtype C on DTG-based ART previously exposed to raltegravir who developed multidrug resistance mutations to four antiretroviral classes. There is need for drug resistance monitoring and clinical vigilance to ensure effectiveness of HIV treatment programs even in the era of DTG-based ART.

Prevalence of Rilpivirine and Etravirine Resistance Mutations in HIV-1 Subtype C-Infected Patients Failing Nevirapine or Efavirenz-Based Combination Antiretroviral Therapy in Botswana.

Rilpivirine (RPV) and Etravirine (ETR) are approved second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment. There is a cross-resistance HIV mutation profile between first- and second-generation NNRTI drugs. We determined the prevalence of HIV-1 drug resistance mutations (DRMs) to RPV and ETR in Botswana. A total of 168 HIV-1 polymerase gene sequences from participants failing nevirapine (NVP)- or efavirenz (EFV)-containing regimens were analyzed for DRMs using the Stanford University HIV drug resistance database. Forty-one sequences were from an adult antiretroviral therapy (ART) study, the Tshepo study, and 127 from a prevention of mother-to-child transmission (PMTCT) study, the Mashi study, all conducted in Botswana. Prevalence of RPV and ETR highest DRM in the adult ART study (n = 41) were K101E (26.2%), E138A (23.8%), and Y181C (26.2%). The PMTCT cohort's (n = 127) high prevalence mutations were Y181C (15.7%), E138A (15%), and K101E (11%). A total of 42.9% and 3.2% of patients in the adult ART study and PMTCT study, respectively, had three or more NNRTI mutations at virologic failure. We identified HIV-1 mutations conferring resistance to RPV and ETR even though they have not been used in Botswana. Of concern was the high proportion of sequences from the adult ART study that displayed multiple DRMs; as the number of NNRTI mutations increases, the level of cross-resistance increases. It is plausible that patients displaying such profiles maybe at increased risk of failing second-generation NNRTI drugs, hence, calls for genotyping in patients with prior NVP or efavirenz exposure before prescription of RPV- or ETR-containing cART.

Correction: Features of Recently Transmitted HIV-1 Clade C Viruses that Impact Antibody Recognition: Implications for Active and Passive Immunization.

[This corrects the article DOI: 10.1371/journal.ppat.1005742.].

Features of Recently Transmitted HIV-1 Clade C Viruses that Impact Antibody Recognition: Implications for Active and Passive Immunization.

The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a global priority, however their potential effectiveness is challenged by very high HIV-1 envelope diversity. Two large prophylactic trials in high incidence, clade C epidemic regions in southern Africa are imminent; passive administration of the monoclonal antibody VRC01, and active immunization with a clade C modified RV144-like vaccines. We have created a large representative panel of C clade viruses to enable assessment of antibody responses to vaccines and natural infection in Southern Africa, and we investigated the genotypic and neutralization properties of recently transmitted clade C viruses to determine how viral diversity impacted antibody recognition. We further explore the implications of these findings for the potential effectiveness of these trials. A panel of 200 HIV-1 Envelope pseudoviruses was constructed from clade C viruses collected within the first 100 days following infection. Viruses collected pre-seroconversion were significantly more resistant to serum neutralization compared to post-seroconversion viruses (p = 0.001). Over 13 years of the study as the epidemic matured, HIV-1 diversified (p = 0.0009) and became more neutralization resistant to monoclonal antibodies VRC01, PG9 and 4E10. When tested at therapeutic levels (10ug/ml), VRC01 only neutralized 80% of viruses in the panel, although it did exhibit potent neutralization activity against sensitive viruses (IC50 titres of 0.42 µg/ml). The Gp120 amino acid similarity between the clade C panel and candidate C-clade vaccine protein boosts (Ce1086 and TV1) was 77%, which is 8% more distant than between CRF01_AE viruses and the RV144 CRF01_AE immunogen. Furthermore, two vaccine signature sites, K169 in V2 and I307 in V3, associated with reduced infection risk in RV144, occurred less frequently in clade C panel viruses than in CRF01_AE viruses from Thailand. Increased resistance of pre-seroconversion viruses and evidence of antigenic drift highlights the value of using panels of very recently transmitted viruses and suggests that interventions may need to be modified over time to track the changing epidemic. Furthermore, high divergence such as that observed in the older clade C epidemic in southern Africa may impact vaccine efficacy, although the correlates of infection risk are yet to be defined in the clade C setting. Findings from this study of acute/early clade C viruses will aid vaccine development, and enable identification of new broad and potent antibodies to combat the HIV-1 C-clade epidemic in southern Africa.

Increased CXCR4 use of HIV-1 subtype C identified by population sequencing in patients failing antiretroviral treatment compared with treatment-naive patients in Botswana.

HIV-1 uses the coreceptors CCR5 and/or CXCR4 for cell entry. Monotropic CCR5-using variants are found early in the infection while CXCR4-using variants may appear after progression to AIDS. CXCR4 use may consist of both monotropic and dualtropic viruses. The viral phenotype is important in evaluating the response to CCR5 inhibitors, a new class of antiviral drugs. The coreceptor use of HIV-1 was investigated using population sequencing in 24 patients from Botswana, carrying HIV-1 subtype C and failing antiretroviral treatment, while 26 treatment-naive patients acted as controls. Single genome sequencing was used to discern minor HIV-1 populations in the treatment-experienced group. The Geno2Pheno method was employed to predict the coreceptor use phenotype from HIV-1 env gp120 V3 DNA sequences. The glycan-charge model adjusted for subtype C was also used for phenotype prediction. The viral phenotype of population sequences was predicted using Geno2Pheno in 24/24 treatment-experienced patients, of whom eight (33%) were predicted to harbor CXCR4-using strains as compared to 2/26 in the treatment-naive group (p=0.03). Single genome sequencing generated 4-23 clones/patient in the treatment-experienced group. Altogether, 90/295 (31%) putative CXCR4-using clones were identified. In 10/24 (42%) treated patients at least one clone was predicted to be CXCR4-using, further increasing the amount of identified treatment-experienced patients with CXCR4 use. Although subtype C is usually associated with comparatively little CXCR4 use, the frequency of CXCR4 use in treatment-experienced patients with subtype C can be higher, which may have implications for the administration of CCR5 inhibitors in this patient group.