Reduced CCR5 expression among Uganda HIV controllers.

BACKGROUND: Several mechanisms including reduced CCR5 expression, protective HLA, viral restriction factors, broadly neutralizing antibodies, and more efficient T-cell responses, have been reported to account for HIV control among HIV controllers. However, no one mechanism universally accounts for HIV control among all controllers. In this study we determined whether reduced CCR5 expression accounts for HIV control among Ugandan HIV controllers. We determined CCR5 expression among Ugandan HIV controllers compared with treated HIV non-controllers through ex-vivo characterization of CD4 + T cells isolated from archived PBMCs collected from the two distinct groups. RESULTS: The percentage of CCR5 + CD4 + T cells was similar between HIV controllers and treated HIV non-controllers (ECs vs. NCs, P = 0.6010; VCs vs. NCs, P = 0.0702) but T cells from controllers had significantly reduced CCR5 expression on their cell surface (ECs vs. NCs, P = 0.0210; VCs vs. NCs, P = 0.0312). Furthermore, we identified rs1799987 SNP among a subset of HIV controllers, a mutation previously reported to reduce CCR5 expression. In stark contrast, we identified the rs41469351 SNP to be common among HIV non-controllers. This SNP has previously been shown to be associated with increased perinatal HIV transmission, vaginal shedding of HIV-infected cells and increased risk of death. CONCLUSION: CCR5 has a non-redundant role in HIV control among Ugandan HIV controllers. HIV controllers maintain high CD4 + T cells despite being ART naïve partly because their CD4 + T cells have significantly reduced CCR5 densities.

Long-term HIV treatment outcomes and associated factors in sub-Saharan Africa: multicountry longitudinal cohort analysis.

OBJECTIVE: In a multicountry prospective cohort of persons with HIV from six countries between 2007 and 2015, we evaluated long-term outcomes of first-line non-nucleoside reverse-transcriptase inhibitor-based antiretroviral therapy (ART), and risk factors for loss-to-follow-up, mortality, virological failure, and incomplete CD4 + T-cell recovery. METHODS: We calculated cumulative incidence of lost-to-follow-up, death, virological failure (VL ≥ 1000 cps/ml) and incomplete CD4 + T-cell recovery (<500 cells/µl) at successive years, using Kaplan-Meier and Cox regression. RESULTS: Of 2735 participants, 58.0% were female, median age was 37 (interquartile range [IQR] 32-43) years, and median pre-ART CD4 + T-cell count was 135 (IQR 63-205)/µl. Total follow-up time was 7208 person-years (median 24.3 months, IQR 18.7-58.3). Deaths by any cause and loss to follow-up occurred mostly during the first year of ART (84%, 201/240 and 56%, 199/353, respectively). During their first 6 years of ART, 71% (95% confidence interval [CI] 69.0-73.7) were retained on first-line, and among those 90-93% sustained viral suppression (<1000 cps/ml); CD4 + T-cell recovery was incomplete in 60% (220/363) of participants. The risk factors associated with poor outcomes during long-term ART were: for loss-to-follow-up, recent VL ≥1000 cps/ml, recent CD4 + T-cell count ≤50 cells/µl, age <30 years, being underweight; for mortality, recent CD4 + T-cell count ≤50 cells/µl; and, for virological failure, age <40 years, recent CD4 + T-cell count ≤200 cells/µl, poor adherence, male sex, and low-level viremia. CONCLUSION: To achieve long-term ART success towards the UNAIDS targets, early ART initiation is crucial, coupled with careful monitoring and retention support, particularly in the first year of ART. Male and youth-centred care delivery models are needed to improve outcomes for those vulnerable groups.

Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda.

BACKGROUND: Our understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia. RESULTS: In this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4+ T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4+ counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001). CONCLUSIONS: The smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs.

High-level resistance to bictegravir and cabotegravir in subtype A- and D-infected HIV-1 patients failing raltegravir with multiple resistance mutations.

OBJECTIVES: The second-generation integrase strand transfer inhibitor (INSTI) bictegravir is becoming accessible in low- and middle-income countries (LMICs), and another INSTI, cabotegravir, has recently been approved as a long-acting injectable. Data on bictegravir and cabotegravir susceptibility in raltegravir-experienced HIV-1 subtype A- and D-infected patients carrying drug resistance mutations (DRMs) remain very scarce in LMICs. PATIENTS AND METHODS: HIV-1 integrase (IN)-recombinant viruses from eight patients failing raltegravir-based third-line therapy in Uganda were genotypically and phenotypically tested for susceptibility to bictegravir and cabotegravir. Ability of these viruses to integrate into human genomes was assessed in MT-4 cells. RESULTS: HIV-1 IN-recombinant viruses harbouring single primary mutations (N155H or Y143R/S) or in combination with secondary INSTI mutations (T97A, M50I, L74IM, E157Q, G163R or V151I) were susceptible to both bictegravir and cabotegravir. However, combinations of primary INSTI-resistance mutations such as E138A/G140A/G163R/Q148R or E138K/G140A/S147G/Q148K led to decreased susceptibility to both cabotegravir (fold change in EC50 values from 429 to 1000×) and bictegravir (60 to 100×), exhibiting a high degree of cross-resistance. However, these same IN-recombinant viruses showed impaired integration capacity (14% to 48%) relative to the WT HIV-1 NL4-3 strain in the absence of drug. CONCLUSIONS: Though not currently widely accessible in most LMICs, bictegravir and cabotegravir offer a valid alternative to HIV-infected individuals harbouring subtype A and D HIV-1 variants with reduced susceptibility to first-generation INSTIs but previous exposure to raltegravir may reduce efficacy, more so with cabotegravir.

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1.

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.

Accumulation of integrase strand transfer inhibitor resistance mutations confers high-level resistance to dolutegravir in non-B subtype HIV-1 strains from patients failing raltegravir in Uganda.

BACKGROUND: Increasing first-line treatment failures in low- and middle-income countries (LMICs) have led to increased use of integrase strand transfer inhibitors (INSTIs) such as dolutegravir. However, HIV-1 susceptibility to INSTIs in LMICs, especially with previous raltegravir exposure, is poorly understood due to infrequent reporting of INSTI failures and testing for INSTI drug resistance mutations (DRMs). METHODS: A total of 51 non-subtype B HIV-1 infected patients failing third-line (raltegravir-based) therapy in Uganda were initially selected for the study. DRMs were detected using Sanger and deep sequencing. HIV integrase genes of 13 patients were cloned and replication capacities (RCs) and phenotypic susceptibilities to dolutegravir, raltegravir and elvitegravir were determined with TZM-bl cells. Spearman's correlation coefficient was used to determine cross-resistance between INSTIs. RESULTS: INSTI DRMs were detected in 47% of patients. HIV integrase-recombinant virus carrying one primary INSTI DRM (N155H or Y143R/S) was susceptible to dolutegravir but highly resistant to raltegravir and elvitegravir (>50-fold change). Two patients, one with E138A/G140A/Q148R/G163R and one with E138K/G140A/S147G/Q148K, displayed the highest reported resistance to raltegravir, elvitegravir and even dolutegravir. The former multi-DRM virus had WT RC whereas the latter had lower RCs than WT. CONCLUSIONS: In HIV-1 subtype A- and D-infected patients failing raltegravir and harbouring INSTI DRMs, there is high-level resistance to elvitegravir and raltegravir. More routine monitoring of INSTI treatment may be advised in LMICs, considering that multiple INSTI DRMs may have accumulated during prolonged exposure to raltegravir during virological failure, leading to high-level INSTI resistance, including dolutegravir resistance.

Comparison of HIV drug resistance profiles across HIV-1 subtypes A and D for patients receiving a tenofovir-based and zidovudine-based first line regimens in Uganda.

BACKGROUND: Resistance to antiretroviral drugs is a major challenge among Human Immunodeficiency Virus (HIV) positive patients receiving antiretroviral therapy (ART). Mutations that arise as a result of this are diverse across the various drugs, drug classes, drug regimens and subtypes. In Uganda, there is a paucity of information on how these mutations differ among the different drug regimens and the predominant HIV-1 subtypes. The purpose of this study was to determine mutation profile differences between first-line drug regimens: TDF/3TC/EFV and AZT/3TC/EFV and HIV-1 subtypes: A and D in Uganda. The study also investigated the potential usage of rilpivirine, doravirine and etravirine in patients who failed treatment on efavirenz. METHODS: A retrospective study was conducted on 182 archived plasma samples obtained from patients who were experiencing virological failure between 2006 and 2017 at five Joint Clinical Research Center (JCRC) sites in Uganda. Sanger sequencing of the Reverse Transcriptase (RT) gene from codons 1-300 was done. Mutation scores were generated using the Stanford University HIV Drug Resistance Database. A Chi-square test was used to determine the association between drug resistance mutations (DRMs) and drug regimens or HIV-1 subtypes. RESULTS: The prevalence of DRMs was 84.6% among patients failing a first-line efavirenz (EFV)-based regimen. The most prevalent Nucleoside Reverse Transcriptase Inhibitor (NRTI) mutations were M184V/I (67.3%), K219/Q/E (22.6%) and K65R (21.1%). While K103N (50.8%) and G190A/S/E/G (29.1%) were the most prevalent Non-Nucleoside Reverse Transcriptase Inhibitor (NNTRI) mutations. As expected, discriminatory DRMs such as K65R, L74I, and Y115F were noted in Tenofovir (TDF) containing regimens while the Thymidine Analogue Mutations (TAMs) L210W and T215 mutations were in Zidovudine (AZT)-based regimens. No significant difference (p = 0.336) was found for overall DRMs between HIV-1 subtypes A and D. Among the patients who had resistance to EFV, 37 (23.6%) were susceptible to newer NNRTIs such as Rilpivirine and Etravirine. CONCLUSION: Accumulation of DRMs between AZT/3TC/EFV and TDF/3TC/EFV is comparable but individual mutations that confer resistance to particular drugs should be considered at virological failure. Having either HIV-1 subtype A or D is not associated with the acquisition of DRMs, therefore HIV diversity should not determine the choice of treatment. Rilpivirine, etravirine and doravirine had minimal benefits for patients who failed on efavirenz.

First-line HIV treatment failures in non-B subtypes and recombinants: a cross-sectional analysis of multiple populations in Uganda.

BACKGROUND: Our understanding of HIV-1 and antiretroviral treatment (ART) is strongly biased towards subtype B, the predominant subtype in North America and western Europe. Efforts to characterize the response to first-line treatments in other HIV-1 subtypes have been hindered by the availability of large study cohorts in resource-limited settings. To maximize our statistical power, we combined HIV-1 sequence and clinical data from every available study population associated with the Joint Clinical Research Centre (JCRC) in Uganda. These records were combined with contemporaneous ART-naive records from Uganda in the Stanford HIVdb database. METHODS: Treatment failures were defined by the presence of HIV genotype records with sample collection dates after the ART start dates in the JCRC database. Drug resistances were predicted by the Stanford HIVdb algorithm, and HIV subtype classification and recombination detection was performed with SCUEAL. We used Bayesian network analysis to evaluate associations between drug exposures and subtypes, and binomial regression for associations with recombination. RESULTS: This is the largest database of first-line treatment failures ([Formula: see text]) in Uganda to date, with a predicted statistical power of 80% to detect subtype associations at an odds ratio of [Formula: see text]. In the subset where drug regimen data were available, we observed that use of 3TC was associated with a higher rate of first line treatment failure, whereas regimens containing AZT and TDF were associated with reduced rates of failure. In the complete database, we found limited evidence of associations between HIV-1 subtypes and treatment failure, with the exception of a significantly lower frequency of failures among A/D recombinants that comprised about 7% of the population. First-line treatment failure was significantly associated with reduced numbers of recombination breakpoints across subtypes. CONCLUSIONS: Expanding access to first-line ART should confer the anticipated public health benefits in Uganda, despite known differences in the pathogenesis of HIV-1 subtypes. Furthermore, the impact of ART may actually be enhanced by frequent inter-subtype recombination in this region.

Higher sequence diversity in the vaginal tract than in blood at early HIV-1 infection.

In the majority of cases, human immunodeficiency virus type 1 (HIV-1) infection is transmitted through sexual intercourse. A single founder virus in the blood of the newly infected donor emerges from a genetic bottleneck, while in rarer instances multiple viruses are responsible for systemic infection. We sought to characterize the sequence diversity at early infection, between two distinct anatomical sites; the female reproductive tract vs. systemic compartment. We recruited 72 women from Uganda and Zimbabwe within seven months of HIV-1 infection. Using next generation deep sequencing, we analyzed the total genetic diversity within the C2-V3-C3 envelope region of HIV-1 isolated from the female genital tract at early infection and compared this to the diversity of HIV-1 in plasma. We then compared intra-patient viral diversity in matched cervical and blood samples with three or seven months post infection. Genetic analysis of the C2-V3-C3 region of HIV-1 env revealed that early HIV-1 isolates within blood displayed a more homogeneous genotype (mean 1.67 clones, range 1-5 clones) than clones in the female genital tract (mean 5.7 clones, range 3-10 clones) (p<0.0001). The higher env diversity observed within the genital tract compared to plasma was independent of HIV-1 subtype (A, C and D). Our analysis of early mucosal infections in women revealed high HIV-1 diversity in the vaginal tract but few transmitted clones in the blood. These novel in vivo finding suggest a possible mucosal sieve effect, leading to the establishment of a homogenous systemic infection.

Absence of HIV-1 Drug Resistance Mutations Supports the Use of Dolutegravir in Uganda.

To screen for drug resistance and possible treatment with Dolutegravir (DTG) in treatment-naive patients and those experiencing virologic failure during first-, second-, and third-line combined antiretroviral therapy (cART) in Uganda. Samples from 417 patients in Uganda were analyzed for predicted drug resistance upon failing a first- (N = 158), second- (N = 121), or third-line [all 51 involving Raltegravir (RAL)] treatment regimen. HIV-1 pol gene was amplified and sequenced from plasma samples. Drug susceptibility was interpreted using the Stanford HIV database algorithm and SCUEAL was used for HIV-1 subtyping. Frequency of resistance to nucleoside reverse transcriptase inhibitors (NRTIs) (95%) and non-NRTI (NNRTI, 96%) was high in first-line treatment failures. Despite lack of NNRTI-based treatment for years, NNRTI resistance remained stable in 55% of patients failing second-line or third-line treatment, and was also at 10% in treatment-naive Ugandans. DTG resistance (n = 366) was not observed in treatment-naive individuals or individuals failing first- and second-line cART, and only found in two patients failing third-line cART, while 47% of the latter had RAL- and Elvitegravir-resistant HIV-1. Secondary mutations associated with DTG resistance were found in 2%-10% of patients failing third-line cART. Of 14 drugs currently available for cART in Uganda, resistance was readily observed to all antiretroviral drugs (except for DTG) in Ugandan patients failing first-, second-, or even third-line treatment regimens. The high NNRTI resistance in first-line treatment in Uganda even among treatment-naive patients calls for the use of DTG to reach the UNAIDS 90:90:90 goals.

Sensitive detection of HIV-1 resistance to Zidovudine and impact on treatment outcomes in low- to middle-income countries.

BACKGROUND: Thymidine analogs, namely AZT (Zidovudine or Retrovir™) and d4T (Stavudine or Zerit™) are antiretroviral drugs still employed in over 75% of first line combination antiretroviral therapy (cART) in Kampala, Uganda despite aversion to prescribing these drugs for cART in high income countries due in part to adverse events. For this study, we explored how the continued use of these thymidine analogs in cART could impact emergence of drug resistance and impact on future treatment success in Uganda, a low-income country. METHODS: We examined the drug resistance genotypes by Sanger sequencing of 262 HIV-infected patients failing a first line combined antiretroviral treatment containing either AZT or d4T, which represents approximately 5% of the patients at the Joint Clinical Research Center receiving a AZT or d4T containing treatment. Next generation sequencing (DEEPGEN™HIV) and multiplex oligonucleotide ligation assays (AfriPOLA) were then performed on a subset of patient samples to detect low frequency drug resistant mutations. CD4 cell counts, viral RNA loads, and treatment changes were analyzed in a cohort of treatment success and failures. RESULTS: Over 80% of patients failing first line AZT/d4T-containing cART had predicted drug resistance to 3TC (Lamivudine) and non-nucleoside RT inhibitors (NNRTIs) in the treatment regimen but only 45% had resistance AZT/d4T associated resistance mutations (TAMs). TAMs were however detected at low frequency within the patients HIV quasispecies (1-20%) in 21 of 34 individuals who were failing first-line AZT-containing cART and lacked TAMs by Sanger. Due to lack of TAMs by Sanger, AZT was typically maintained in second-line therapies and these patients had a low frequency of subsequent virologic success. CONCLUSIONS: Our findings suggest that continued use of AZT and d4T in first-line treatment in low-to-middle income countries may lead to misdiagnosis of HIV-1 drug resistance and possibly enhance a succession of second- and third-line treatment failures.

Nucleoside reverse-transcriptase inhibitor cross-resistance and outcomes from second-line antiretroviral therapy in the public health approach: an observational analysis within the randomised, open-label, EARNEST trial.

BACKGROUND: Cross-resistance after first-line antiretroviral therapy (ART) failure is expected to impair activity of nucleoside reverse-transcriptase inhibitors (NRTIs) in second-line therapy for patients with HIV, but evidence for the effect of cross-resistance on virological outcomes is limited. We aimed to assess the association between the activity, predicted by resistance testing, of the NRTIs used in second-line therapy and treatment outcomes for patients infected with HIV. METHODS: We did an observational analysis of additional data from a published open-label, randomised trial of second-line ART (EARNEST) in sub-Saharan Africa. 1277 adults or adolescents infected with HIV in whom first-line ART had failed (assessed by WHO criteria with virological confirmation) were randomly assigned to a boosted protease inhibitor (standardised to ritonavir-boosted lopinavir) with two to three NRTIs (clinician-selected, without resistance testing); or with raltegravir; or alone as protease inhibitor monotherapy (discontinued after week 96). We tested genotypic resistance on stored baseline samples in patients in the protease inhibitor and NRTI group and calculated the predicted activity of prescribed second-line NRTIs. We measured viral load in stored samples for all patients obtained every 12-16 weeks. This trial is registered with Controlled-Trials.com (number ISRCTN 37737787) and ClinicalTrials.gov (number NCT00988039). FINDINGS: Baseline genotypes were available in 391 (92%) of 426 patients in the protease inhibitor and NRTI group. 176 (89%) of 198 patients prescribed a protease inhibitor with no predicted-active NRTIs had viral suppression (viral load <400 copies per mL) at week 144, compared with 312 (81%) of 383 patients in the protease inhibitor and raltegravir group at week 144 (p=0·02) and 233 (61%) of 280 patients in the protease inhibitor monotherapy group at week 96 (p<0·0001). Compared with results with no active NRTIs, 95 (85%) of 112 patients with one predicted-active NRTI had viral suppression (p=0·3) and 20 (77%) of 26 patients with two or three active NRTIs had viral suppression (p=0·08). Over all follow-up, greater predicted NRTI activity was associated with worse viral load suppression (global p=0·0004). INTERPRETATION: Genotypic resistance testing might not accurately predict NRTI activity in protease inhibitor-based second-line ART. Our results do not support the introduction of routine resistance testing in ART programmes in low-income settings for the purpose of selecting second-line NRTIs. FUNDING: European and Developing Countries Clinical Trials Partnership, UK Medical Research Council, Institito de Salud Carlos III, Irish Aid, Swedish International Development Cooperation Agency, Instituto Superiore di Sanita, WHO, Merck.

HIV Drug Resistance Mutations in Non-B Subtypes After Prolonged Virological Failure on NNRTI-Based First-Line Regimens in Sub-Saharan Africa.

OBJECTIVE: To determine drug resistance mutation (DRM) patterns in a large cohort of patients failing nonnucleoside reverse transcriptase inhibitor (NNRTI)-based first-line antiretroviral therapy regimens in programs without routine viral load (VL) monitoring and to examine intersubtype differences in DRMs. DESIGN: Sequences from 787 adults/adolescents who failed an NNRTI-based first-line regimen in 13 clinics in Uganda, Kenya, Zimbabwe, and Malawi were analyzed. Multivariable logistic regression was used to determine the association between specific DRMs and Stanford intermediate-/high-level resistance and factors including REGA subtype, first-line antiretroviral therapy drugs, CD4, and VL at failure. RESULTS: The median first-line treatment duration was 4 years (interquartile range 30-43 months); 42% of participants had VL ≥100,000 copies/mL and 63% participants had CD4 <100 cells/mm. Viral subtype distribution was A1 (40%; Uganda and Kenya), C (31%; Zimbabwe and Malawi), and D (25%; Uganda and Kenya), and recombinant/unclassified (5%). In general, DRMs were more common in subtype-C than in subtype-A and/or subtype-D (nucleoside reverse transcriptase inhibitor mutations K65R and Q151M; NNRTI mutations E138A, V106M, Y181C, K101E, and H221Y). The presence of tenofovir resistance was similar between subtypes [P (adjusted) = 0.32], but resistance to zidovudine, abacavir, etravirine, or rilpivirine was more common in subtype-C than in subtype-D/subtype-A [P (adjusted) < 0.02]. CONCLUSIONS: Non-B subtypes differ in DRMs at first-line failure, which impacts on residual nucleoside reverse transcriptase inhibitor and NNRTI susceptibility. In particular, higher rates of etravirine and rilpivirine resistance in subtype-C may limit their potential utility in salvage regimens.

Second-line HIV Treatment in Ugandan Children: Favorable Outcomes and No Protease Inhibitor Resistance.

Background: Data on pediatric second-line antiretroviral treatment (ART) outcomes are scarce, but essential to evaluate second-line and design third-line regimens. Methods: Children ≤12 years switching to second-line ART containing a protease inhibitor (PI) in Uganda were followed for 24 months. Viral load (VL) was determined at switch to second-line and every 6 months thereafter; genotypic resistance testing was done if VL ≥ 1000 cps/ml. Results: 60 children were included in the analysis; all had ≥1 drug resistance mutations at switch. Twelve children (20.0%) experienced treatment failure; no PI mutations were detected. Sub-optimal adherence and underweight were associated with treatment failure. Conclusions: No PI mutations occurred in children failing second-line ART, which is reassuring as pediatric third-line is not routinely available in these settings. Poor adherence rather than HIV drug resistance is likely to be the main mechanism for treatment failure and should receive close attention in children on second-line ART.

Infecting HIV-1 Subtype Predicts Disease Progression in Women of Sub-Saharan Africa.

INTRODUCTION: Long-term natural history cohorts of HIV-1 in the absence of treatment provide the best measure of virulence by different viral subtypes. METHODS: Newly HIV infected Ugandan and Zimbabwean women (N=303) were recruited and monitored for clinical, social, behavioral, immunological and viral parameters for 3 to 9.5years. RESULTS: Ugandan and Zimbabwean women infected with HIV-1 subtype C had 2.5-fold slower rates of CD4 T-cell declines and higher frequencies of long-term non-progression than those infected with subtype A or D (GEE model, P<0.001), a difference not associated with any other clinical parameters. Relative replicative fitness and entry efficiency of HIV-1 variants directly correlated with virulence in the patients, subtype D>A>C (P<0.001, ANOVA). DISCUSSION: HIV-1 subtype C was less virulent than either A or D in humans; the latter being the most virulent. Longer periods of asymptomatic HIV-1 subtype C could explain the continued expansion and dominance of subtype C in the global epidemic.

Accumulation of HIV-1 drug resistance after continued virological failure on first-line ART in adults and children in sub-Saharan Africa.

OBJECTIVES: Limited availability of viral load (VL) monitoring in HIV treatment programmes in sub-Saharan Africa can delay switching to second-line ART, leading to the accumulation of drug resistance mutations (DRMs). The objective of this study was to evaluate the accumulation of resistance to reverse transcriptase inhibitors after continued virological failure on first-line ART, among adults and children in sub-Saharan Africa. METHODS: HIV-1-positive adults and children on an NNRTI-based first-line ART were included. Retrospective VL and, if VL ≥1000 copies/mL, pol genotypic testing was performed. Among participants with continued virological failure (≥2 VL ≥1000 copies/mL), drug resistance was evaluated. RESULTS: At first virological failure, DRM(s) were detected in 87% of participants: K103N (38.7%), G190A (21.8%), Y181C (20.2%), V106M (8.4%), K101E (8.4%), any E138 (7.6%) and V108I (7.6%) associated with NNRTIs, and M184V (69.7%), any thymidine analogue mutation (9.2%), K65R (5.9%) and K70R (5.0%) associated with NRTIs. New DRMs accumulated with an average rate of 1.45 (SD 2.07) DRM per year; 0.62 (SD 1.11) NNRTI DRMs and 0.84 (SD 1.38) NRTI DRMs per year, respectively. The predicted susceptibility declined significantly after continued virological failure for all reverse transcriptase inhibitors (all P < 0.001). Acquired drug resistance patterns were similar in adults and children. CONCLUSIONS: Patterns of drug resistance after virological failure on first-line ART are similar in adults and children in sub-Saharan Africa. Improved VL monitoring to prevent accumulation of mutations, and new drug classes to construct fully active regimens, are required.

Once vs twice-daily abacavir and lamivudine in African children.

BACKGROUND: Antiretroviral therapy (ART) adherence is critical for successful HIV treatment outcomes. Once-daily dosing could improve adherence. Plasma concentrations of once-daily vs twice-daily abacavir + lamivudine are bioequivalent in children, but no randomized trial has compared virological outcomes. METHODS: Children taking abacavir + lamivudine-containing first-line regimens twice daily for more than 36 weeks in the ARROW trial (NCT02028676, ISRCTN24791884) were randomized to continue twice-daily vs move to once-daily abacavir + lamivudine (open-label). Co-primary outcomes were viral load suppression at week 48 (12% noninferiority margin, measured retrospectively) and lamivudine or abacavir-related grade 3/4 adverse events. RESULTS: Six hundred and sixty-nine children (median 5 years, range 1-16) were randomized to twice daily (n = 333) vs once daily (n = 336) after median 1.8 years on twice-daily abacavir + lamivudine-containing first-line ART. Children were followed for median 114 weeks. At week 48, 242/331 (73%) twice daily vs 236/330 (72%) once daily had viral load less than 80 copies/ml [difference -1.6% (95% confidence interval -8.4,+5.2%) P = 0.65]; 79% twice daily vs 78% once daily had viral load less than 400 copies/ml (P = 0.76) (week 96 results similar). One grade 3/4 adverse event was judged uncertainly related to abacavir + lamivudine (hepatitis; once daily). At week 48, 9% twice daily vs 10% once daily reported missing one or more ART pills in the last 4 weeks (P = 0.74) and 8 vs 8% at week 96 (P = 0.90). Carers strongly preferred once-daily dosing. There was no difference between randomized groups in postbaseline drug-resistance mutations or drug-susceptibility; WHO 3/4 events; ART-modifying, grade 3/4 or serious adverse events; CD4% or weight-for-age/height-for-age (all P > 0.15). CONCLUSION: Once-daily abacavir + lamivudine was noninferior to twice daily in viral load suppression, with similar resistance, adherence, clinical, immunological and safety outcomes. Abacavir + lamivudine provides the first once-daily nucleoside backbone across childhood that can be used to simplify ART.

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure.

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.

HIV Drug Resistance Among Children Initiating First-Line Antiretroviral Treatment in Uganda.

BACKGROUND: There are limited data on primary human immunodeficiency virus drug resistance (HIVDR) in pediatric populations. This study aimed to assess the prevalence of primary HIVDR and associated risk factors among children initiating first-line antiretroviral therapy (ART) in Uganda. METHODS: At three Ugandan clinics, children (age <12 years) requiring ART were recruited between January 2010 and August 2011. Before starting ART, blood was collected for viral load and pol gene sequencing. Drug resistance mutations were determined using the 2010 International AIDS Society-USA mutation list. Risk factors for HIVDR were assessed with multivariate regression analysis. RESULTS: Three hundred nineteen HIV-infected children with a median age of 4.9 years were enrolled. Sequencing was successful in 279 children (87.5%). HIVDR was present in 10% of all children and 15.2% of children <3 years. Nucleoside reverse transcriptase inhibitors (NRTIs), non-NRTI (NNRTI), and dual-class resistance was present in 5.7%, 7.5%, and 3.2%, respectively. HIVDR occurred in 35.7% of prevention of mother-to-child transmission (PMTCT)-exposed children, 15.6% in children with unknown PMTCT history, and 7.7% among antiretroviral-naive children. History of PMTCT exposure [adjusted odds ratio (AOR): 2.6, 95% CI: 1.3-5.1] or unknown PMTCT status (AOR: 3.8, 95% CI: 1.1-13.5), low CD4 (AOR: 2.2, 95% CI: 1.3-3.6), current breastfeeding (AOR: 7.4, 95% CI: 2.6-21), and current maternal ART use (AOR: 6.4, 95% CI: 3.4-11.9) emerged as risk factors for primary HIVDR in multivariate analysis. CONCLUSION: Pretreatment HIVDR is high, especially in children with PMTCT exposure. Protease inhibitor (PI)-based regimens are advocated by the World Health Organization, but availability in children is limited. Children with (unknown) PMTCT exposure, low CD4 count, current breastfeeding, or maternal ART need to be prioritized to receive PI-based regimens.

Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

BACKGROUND: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates. METHODS: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing. RESULTS: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells. CONCLUSIONS: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.