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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 (+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 (+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 +T-cell count ≤50 cells/μl; and, for virological failure, age +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.

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.

IntroductionBy Sept 2018, only 59.3% of all HIV positive children on cART under 5 years were achieving virological suppression compared to 88.4% in the general population in Uganda. CCR5 promoter genotype specifically single nucleotide polymorphisms have been linked to modulate patient virological status. However, the few studies that have studied the association in infants have utilized allele-specific PCR a genotypic method limited to detecting already known SNPs. By using Sanger sequencing, we explored the association taking into account novel mutations. Methods Following ethical approvals, a cross sectional study was conducted using archived buffy coat samples from a pediatric HIV drug resistance study between May 2019 and January 2022 at the Joint Clinical Research Centre in Kampala, Uganda. 100 HIV seropositive infant buffy coat samples were sequenced for CCR5 SNP genotype following nucleic acid extraction, polymerase chain reaction and sanger sequencing. Odds ratios were used to determine the association between CCR5 SNPs and infant HIV plasma loads. Results 10 SNPs were identified with frequencies as follows; 58227G/A (21.8%), 58636A/G (28.7%), 58934T/G (74.7%), 59029G/A (48.3%), 59353T/C (55.2%), 59356C/T (23.0%), 59402G/A (94.2%) 59653C/T (66.7%), 59802A/C (34.5%), and 60024A/G (34.5%). Notably, 58227G/A, 58636A/G, 59802A/C, and 60024A/G are novel SNPs. However, none of these SNPs was significantly associated with infant virological failure but SNPs 59029G/A and 59353T/C showed higher odds of occurring in non-suppressors whereas 59356C/T and 59402G/A appeared to correlate with reduced plasma HIV loads. Infant virological suppression remained at 59% and in agreement with the national data. Conclusion Our study augments previous studies that CCR5 promoter SNPs play a role in modulating patient virological status however, strong conclusions could be drawn from either utilizing in-vitro studies or large epidemiological studies.

Objective To determine the prevalence of multi-drug resistant variants among patients failing on a nucleoside reverse transcriptase inhibitor (NRTI) based regimen with a detectable viral load ≥ 1000 copies/ml among patients harboring HIV subtype A, C and D. Methods Samples were obtained from patients who were failing on an NRTI based regimen. Sanger based sequencing was performed as part of the standard of care. Mutation analysis was performed using the Stanford HIV drug Resistance database. A subset of these patient samples was further analyzed using the Next Generation Sequencing (NGS) technology and analysis of the drug resistance mutations was performed at the 20% and 1% cut off Results Analysis of the Non-nucleoside reverse transcriptase inhibitor (NNRTI) coding region revealed that the K101 and the Y181 mutations were more predominant among subtype C than subtype A and D. Although Thymidine analog mutations (TAMs) were prevalent in all subtypes, our analyses showed that these mutations occurred in significantly less proportions among subtype C infections when compared with the subtype A and D counterparts. Furthermore, the Q151M mutation complex which involves mutations in multiple domains was significantly more prominent among patients harboring subtype C variants. Analysis using NGS revealed that minority drug resistant mutations that confer multi-drug resistance (MDR) were present even in patients who exhibited a susceptible genotype based on the Sanger sequencing technique. Conclusion Although HIV-1 MDR variants occur in all subtypes, their predominance is subtype specific with TAMs being significantly more predominant among subtype A and D while the Q151M complex being significantly more predominant among patients harboring subtype C viruses. Even in patients with a susceptible genotype based on Sanger technology, minority variants are present and their evolution to full blown MDR occurs over time such that by the time they are detectable, cross resistance to other drugs has occurred in some cases.

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, and the mechanisms involved in this selection process have not been elucidated., 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.

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.

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.

The global HIV-1 pandemic comprises many genetically divergent subtypes. Most of our understanding of drug resistance in HIV-1 derives from subtype B, which predominates in North America and western Europe. However, about 90% of the pandemic represents non-subtype B infections. Here, we use deep sequencing to analyze HIV-1 from infected individuals in Uganda who were either treatment-naïve or who experienced virologic failure on ART without the expected patterns of drug resistance. Our objective was to detect potentially novel associations between mutations in HIV-1 integrase and treatment outcomes in Uganda, where most infections are subtypes A or D. We retrieved a total of 380 archived plasma samples from patients at the Joint Clinical Research Centre (Kampala), of which 328 were integrase inhibitor-naïve and 52 were raltegravir (RAL)-based treatment failures. Next, we developed a bioinformatic pipeline for alignment and variant calling of the deep sequence data obtained from these samples from a MiSeq platform (Illumina). To detect associations between within-patient polymorphisms and treatment outcomes, we used a support vector machine (SVM) for feature selection with multiple imputation to account for partial reads and low quality base calls. Candidate point mutations of interest were experimentally introduced into the HIV-1 subtype B NL4-3 backbone to determine susceptibility to RAL in U87.CD4.CXCR4 cells. Finally, we carried out replication capacity experiments with wild-type and mutant viruses in TZM-bl cells in the presence and absence of RAL. Our analyses not only identified the known major mutation N155H and accessory mutations G163R and V151I, but also novel mutations I203M and I208L as most highly associated with RAL failure. The I203M and I208L mutations resulted in significantly decreased susceptibility to RAL (44.0-fold and 54.9-fold, respectively) compared to wild-type virus (EC50=0.32 nM), and may represent novel pathways of HIV-1 resistance to modern treatments.Author summaryThere are many different types of HIV-1 around the world. Most of the research on how HIV-1 can become resistant to drug treatment has focused on the type (B) that is the most common in high-income countries. However, about 90% of infections around the world are caused by a type other than B. We used next-generation sequencing to analyze samples of HIV-1 from patients in Uganda (mostly infected by types A and D) for whom drug treatment failed to work, and whose infections did not fit the classic pattern of adaptation based on B. Next, we used machine learning to detect mutations in these virus populations that could explain the treatment outcomes. Finally, we experimentally added two candidate mutations identified by our analysis to a laboratory strain of HIV-1 and confirmed that they conferred drug resistance to the virus. Our study reveals new pathways that other types of HIV-1 may use to evolve resistance to drugs that make up the current recommended treatment for newly diagnosed individuals.

BackgroundAlthough minority HIV-1 drug-resistant HIV-1 variants may be selected under antiretroviral pressure, leading to therapy failure, their clinical significance remains controversial. This is particularly relevant in the case of prevention of mother-to-child transmission (MTCT), where transmitted drug resistance can affect treatment outcomes.MethodsAn ultrasensitive HIV-1 genotyping assay based on deep sequencing (DEEPGENHIV) with a 1% mutation frequency sensitivity, was used to quantify MTCT drug-resistant variants in 38 prenatally HIV-infected children experiencing (Group I, n=27) or not (Group II, n=11) virologic failure 12 months after initiating first-line antiretroviral therapy (ART) as part of a paediatric cohort in Uganda.ResultsInfants were infected with subtype A(n=20), D(n=16) or C(n=2) HIV-1 strains, distributed equally between both patients’ groups. Similarly, no significant difference was observed in intra-patient HIV-1 diversity among viruses obtained from Group I or II individuals at baseline. DEEPGENHIV was able to detect all the mutations originally detected in samples obtained from four control patients in Group II, where drug resistance was identified at baseline using Sanger sequencing, e.g. K65R (78% mutation frequency), K103N (47%), or M184V (85%). More importantly, a series of low abundance (ConclusionDEEPGENHIV improves the detection of minority viral variants in infants following MTCT; however, most of the emergent HIV-1 drug resistance mutations were not present at low frequency at baseline in subjects failing ART, most likely being generated and selected following exposure to treatment. Further studies, using this or other ultrasensitive assays, are needed to better understand the transmission, dynamics and overall evolution of minority drug-resistant viruses in MTCT.

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.

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.

BackgroundResistance 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.MethodsA 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.ResultsThe 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.ConclusionAccumulation 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.

Additional file 1. Additional text, figures and tables are provided in support of results in the main text, including methodological details on HIV-1 sequence processing.

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, Author summary During chronic HIV-1 infection, high viral diversity can be found in the blood and semen of donors. However, a single HIV-1 clone establishes productive infection in the recipient following heterosexual transmission. To investigate the genetic bottleneck occurring at the earliest stages of heterosexual HIV-1 transmission, we characterized the HIV-1 envelope sequence diversity at very early and early stages of infection in the female reproductive tract and matched plasma samples from a cohort of Ugandan and Zimbabwean women. A more diverse viral population was observed in the endocervical swab samples compared to plasma. Endocervical samples harbored a larger number of viral clones, while in the majority of plasma samples only a single clone was present early in infection. Interestingly, these observations were independent of HIV-1 subtype, hormonal contraceptive use or the number of sex acts and partners. Furthermore, in the cases of higher HIV-1 diversity in the blood during early infection, faster CD4 T cell decline were observed during chronic disease suggesting faster disease progression. Our findings provide novel in vivo evidence for the existence of an intra-patient genetic bottleneck restricting the HIV-1 from the vaginal tract to the blood during early heterosexual HIV-1 transmission.

BACKGROUND: Implementation of ultrasensitive HIV drug resistance tests for routine clinical use is hampered by uncertainty about the clinical relevance of drug-resistant minority variants. We assessed different detection thresholds for pretreatment drug resistance to predict an increased risk of virological failure.METHODS: We did a case-control study nested within a prospective multicountry cohort. Our study included patients from 12 clinical sites in Kenya, Nigeria, South Africa, Uganda, and Zambia. We defined cases as patients with virological failure (ie, those who had either viral load ≥400 copies per mL at 12 months or had switched to second-line antiretroviral therapy [ART] as a result of virological failure before 12 months) and controls as those with viral suppression (viral load FINDINGS: Paired viral load results before ART and at month 12 of follow-up were available from 1896 participants. We identified 178 patients with virological failure and selected 338 matched controls. We excluded 117 patients from pretreatment drug resistance analysis; therefore, 152 cases of virological failure and 247 controls were included in the final analysis. With the IAS-USA mutation list, at a detection threshold of 20% or more in patients with pretreatment drug resistance, the adjusted odds ratio (OR) for virological failure was 9·2 (95% CI 4·2-20·1) compared with those without pretreatment drug resistance. Lowering the threshold resulted in adjusted ORs of virological failure of 6·8 (95% CI 3·3-13·9) at the 10% threshold, 7·6 (3·4-17·1) at the 5% threshold, and 4·5 (2·0-10·2) at the 1% threshold. Lowering the detection threshold from 20% improved the sensitivity (ie, ability to identify cases) from 12% (n=18) to 13% (n=19) at detection threshold 10%, to 15% (n=23) at detection threshold 5%, and to 17% (n=26) at detection threshold 1%, but caused a slight reduction in specificity (ie, ability to identify controls) from 98% (n=241) to 96% (n=238) at the 10% threshold, 96% (n=236) at the 5% threshold, and a larger reduction to 92% (n=227) at the 1% threshold. Diagnostic ORs were 5·4 (95% CI 2·1-13·9) at the 20% threshold, 3·8 (1·7-8·6) at the 10% threshold, 3·8 (1·8-8·1) at the 5% threshold, and 2·3 (1·2-4·2) at the 1% threshold. Use of the Stanford HIVDB genotypic sensitivity scores yielded similar ORs for virological failure, sensitivities, specificities, and diagnostic ORs.INTERPRETATION: Ultrasensitive resistance testing for pretreatment drug resistance improved identification of people at risk of virological failure; however, this came with a reduction in our ability to identify people with viral suppression, especially at very low thresholds. Further modelling is needed to estimate the optimal trade-off for the 5% and 20% thresholds, balancing improved case finding against unnecessary regimen switching.FUNDING: The Netherlands Ministry of Foreign Affairs, IrsiCaixa, and European Union.

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. Electronic supplementary material The online version of this article (doi: 10.1186/s40249-017-0377-0) contains supplementary material, which is available to authorized users.

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 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.

Little is known about the differences in clinical manifestations between women with various HIV-1 subtypes during acute (AI) and early (EI) HIV infection. In a longitudinal cohort study, clinical signs and symptoms among Uganda and Zimbabwe women with AI and EI were compared with HIV-negative controls; symptoms were assessed quarterly for 15 to 24 months. Early HIV infection was defined as the first visit during which a woman tested HIV antibody positive. Women who were HIV negative serologically but DNA polymerase chain reaction positive were considered AI. In all, 26 women were classified AI and 192 EI, with 654 HIV-negative controls. Primary HIV infection (AI and EI) was associated with unexplained fever ( P

Supplemental Digital Content is Available in the Text., 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

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.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).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 load400 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 (p0·0001). 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).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.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.

We sought to determine the pattern of resistance-associated mutations (RAMs) among HIV-1-infected children failing first-line antiretroviral therapy (ART) and ascertain their response to second-line regimens in 48 weeks of follow-up. The design involved a cohort study within an HIV care program. We studied records of 142 children on ART with virological failure to first-line ART and switched to second-line ART with prior genotypic resistance testing. The pattern of RAMs was determined in frequency runs and the factors associated with accumulation of >= 3 thymidine analogue mutations (TAMs) and K103N were determined using multivariate logistic models. Changes in weight, height, CD4, and viral load at weeks 24 and 48 after switch to second-line therapy were determined using descriptive statistics. The children were mean age 10.9+/-4.6 years and 55.6% were male. The commonest nucleoside reverse transcriptase inhibitor (NRTI) RAM was M184V in 129/142 (90.8%) children. TAMs, >= 3 TAMs, 69 insertion complex, K65R/N, and Q151M were observed in 43.0%, 10.6%, 18.3%, 2.8%, and 2.1% of the children, respectively. The commonest nonnucleoside reverse transcriptase inhibitor (NNRTI) RAM was K103N in 72/142 (50.7%) children. The starting ART regimen was associated with accumulation of both >= 3 TAMs (p = 0.046) and K103N (p < 0.0001), while a history of poor adherence was associated with K103N accumulation (p = 0.0388). After 24 weeks and 48 weeks of follow-up on lopinavir-ritonavir based second-line ART, 86/108 (79.6%) and 84.5% (87/103) of the children had viral loads < 400 copies/ml, respectively. The mean CD4 absolute count increased by 173 cells/mu l and 267cells/mu l at weeks 24 and 48, respectively. Increments were also observed in mean weight (1.6 kg and 4.3 kg) and height (1.8 cm and 5.8 cm) at weeks 24 and 48, respectively. Multiple RAMs were observed among HIV-1-infected children with virological failure on first-line ART with M184V and K103N most frequent. The children responded favorably to boosted PI-based second-line ART.

HIV-infected children are at high risk of acquiring drug-resistant viruses, which is of particular concern in settings where antiretroviral drug options are limited. We aimed to assess resistance patterns and predict viral drug susceptibility among children with first-line antiretroviral therapy (ART) failure in Uganda. A cross-sectional analysis of children switching ART regimens due to first-line failure was performed at three clinical sites in Uganda. HIV-RNA determination and genotypic resistance testing on all specimens with HIV-RNA >1,000 copies/ml were performed. Major drug resistance mutations were scored using the 2011 International Antiviral Society-USA list. The Stanford algorithm was used to predict drug susceptibility. At the time of switch, 44 genotypic resistance tests were available for 50 children. All children harbored virus with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance [95% confidence interval (CI) 92-100%] and NRTI resistance was present in 98% (95% CI 88-100%). Forty-six percent (95% CI 30-61%) of children harbored ≥2 thymidine analog mutations. M184V was identified as the only NRTI mutation in 27% (95% CI 15-43%). HIV susceptibility to NRTIs, with the exception of tenofovir, was reduced in ≥60% of children. Ugandan children experiencing first-line ART failure in our study harbored high rates of dual-class and accumulated HIV drug resistance. Methods to prevent treatment failure, including adequate pediatric formulations and alternative second-line treatment options, are urgently needed.

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors.

Background. After the scale-up of antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection in Africa, increasing numbers of patients have pretreatment drug resistance. Methods. In a large multicountry cohort of patients starting standard first-line ART in six African countries, pol genotyping was retrospectively performed if viral load (VL) >1000 cps/mL. Pretreatment drug resistance was defined as a decreased susceptibility to >1 prescribed drug. We assessed the effect of pretreatment drug resistance on all-cause mortality, new AIDS events and switch to second-line ART due to presumed treatment failure, using Cox models. Results. Among 2579 participants for whom a pretreatment genotype was available, 5.5% had pretreatment drug resistance. Pretreatment drug resistance was associated with an increased risk of regimen switch (adjusted hazard ratio [aHR] 3.80; 95% confidence interval [CI], 1.49-9.68; P =. 005) but was not associated with mortality (aHR 0.75, 95% CI,. 24-2.35; P =. 617) or new AIDS events (aHR 1.06, 95% CI,. 68-1.64; P =. 807). During three years of follow up, 106 (4.1%) participants switched to second-line, of whom 18 (17.0%) switched with VL < 1000 cps/mL, 7 (6.6%) with VL > 1000 cps/mL and no drug resistance mutations (DRMs), 46 (43.4%) with VL > 1000 cps/mL and >1 DRMs; no HIV RNA data was available for 32 (30.2%) participants. Conclusions. Given rising pretreatment HIV drug resistance levels in sub-Saharan Africa, these findings underscore the need for expanded access to second-line ART. VL monitoring can improve the accuracy of failure detection and efficiency of switching practices.

Exclusive breastfeeding (EBF) is being promoted by WHO/UNICEF as the recommended mode of feeding for all infants from birth to around 6 months of age. It is also recommended for the initial few months, for infants born to HIV-positive mothers in the developing world where conditions may not be ideal for replacement feeding. A cross-sectional study was done to find the prevalence and factors associated with EBF from delivery, amongst mothers of infants 0-6 months of age in a rural Ugandan community with a high HIV/AIDS burden. A prevalence of EBF of 35.1 per cent was found. There was a very high use of prelacteal feeds. Factors positively associated with EBF from birth, after logistic regression, were: delivery from a health unit (OR 2.07; 95 per cent confidence interval, 1.2-3.6); and mother having a normal vaginal delivery (OR 10.54; 95 per cent CI, 3.0-36.6). Factors showing negative association were: male gender of child (OR 0.44; 95 per cent CI, 0.3-0.7); and age of child over 3 months (OR 0.48; 95 per cent CI, 0.3-0.7). Mothers knowledge of her HIV serostatus was not associated with EBF (p = 0.78). Breastfeeding is almost universal in this area but it is rarely exclusive from birth.

Analysis of samples from Uganda using serologic HIV incidence assays reveal that individuals with subtype D infection often have weak humoral immune responses to HIV infection. It is unclear whether this reflects a poor initial response to infection or a waning antibody response later in infection.Samples (N = 2614) were obtained from 114 women aged 18-45 years in the Ugandan Genital Shedding and Disease Progression Study cohort (2001-2009; 82 subtype A, 32 subtype D; median 23 samples/women, range 3-41 samples, median follow-up of 6.6 years). Samples were analyzed using the BED capture immunoassay (cutoff, 0.8 OD-n) and the avidity assay (cutoff, 90% Avidity Index). Antibody maturation was assessed by having the BED capture enzyme immunoassay (BED-CEIA) or avidity value exceed the assay cutoff 1 or 2 years after infection. The waning antibody response was measured by having the BED-CEIA or avidity value fall20% below the maximum value.For the BED-CEIA, 8 women with subtype A infection and 3 women with subtype D infection never progressed previously the cutoff value (median, 5.9 years follow-up after infection). Six women with subtype D infection never achieved an avidity index90%. Subtype did not impact the proportion of women whose assay values regressed by20% of the maximal value (for BED-CEIA: 33% for A, 41% for D, P = 0.51; for avidity: 1% for A, 6% for D, P = 0.19).The higher frequency of misclassification of individuals with long-term subtype D infection as recently infected using serologic incidence assays reflects a weak initial antibody response to HIV infection that is sustained over time.

To determine the impact of HIV-1 subtype on treatment outcomes and the emergence of drug resistance in the resource limited setting of Kampala, Uganda.The Joint Clinical Research Centre (JCRC) in Kampala, Uganda has provided over 2000 drug-resistant genotypes (DRGs) over the past 10 years as standard of care for patients failing therapy and 1403 from treatment-naive and experienced patients over the past 10 years have been analyzed for this study.Viral loads, CD4 cell count, treatment histories and other relevant clinical data was compared with the infecting HIV-1 subtype and DRGs of Ugandan patients failing treatment.Patients failing HAART with DRGs (n = 937) were more frequently infected with subtype D than expected on the basis of the subtype distribution in the treatment-naive population (n = 655) in Kampala (P0.001). Higher proportions of treatment failures among subtype D-infected patients were driven by resistance to nucleoside reverse transcriptase inhibitors (NRTI) (P0.0002) more than to non-NRTIs (P0.04) or protease inhibitors.Higher rates of treatment failure among subtype D as compared with subtype A-infected Ugandans was analogous to the faster disease progression in subtype D-infected patients. The mechanism(s) by which drug resistance may emerge faster in subtype D HIV-1 may relate to higher replicative fitness and increased propensity for a CXCR4 tropism.

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.

Summary Background There are few data on the epidemiology of primary HIV-1 drug resistance after the roll-out of antiretroviral treatment (ART) in sub-Saharan Africa. We aimed to assess the prevalence of primary resistance in six African countries after ART roll-out and if wider use of ART in sub-Saharan Africa is associated with rising prevalence of drug resistance. Methods We did a cross-sectional study in antiretroviral-naive adults infected with HIV-1 who had not started first-line ART, recruited between 2007 and 2009 from 11 regions in Kenya, Nigeria, South Africa, Uganda, Zambia, and Zimbabwe. We did population-based sequencing of the pol gene on plasma specimens with greater than 1000 copies per mL of HIV RNA. We identified drug-resistance mutations with the WHO list for transmitted resistance. The prevalence of sequences containing at least one drug-resistance mutation was calculated accounting for the sampling weights of the sites. We assessed the risk factors of resistance with multilevel logistic regression with random coefficients. Findings 2436 (94.1%) of 2590 participants had a pretreatment genotypic resistance result. 1486 participants (57.4%) were women, 1575 (60.8%) had WHO clinical stage 3 or 4 disease, and the median CD4 count was 133 cells per μL (IQR 62–204). Overall sample-weighted drug-resistance prevalence was 5.6% (139 of 2436; 95% CI 4.6–6.7), ranging from 1.1% (two of 176; 0.0–2.7) in Pretoria, South Africa, to 12.3% (22 of 179; 7.5–17.1) in Kampala, Uganda. The pooled prevalence for all three Ugandan sites was 11.6% (66 of 570; 8.9–14.2), compared with 3.5% (73 of 1866; 2.5–4.5) for all other sites. Drug class-specific resistance prevalence was 2.5% (54 of 2436; 1.8–3.2) for nucleoside reverse-transcriptase inhibitors (NRTIs), 3.3% (83 of 2436; 2.5–4.2) for non-NRTIs (NNRTIs), 1.3% (31 of 2436; 0.8–1.8) for protease inhibitors, and 1.2% (25 of 2436; 0.7–1.7) for dual-class resistance to NRTIs and NNRTIs. The most common drug-resistance mutations were K103N (43 [1.8%] of 2436), thymidine analogue mutations (33 [1.6%] of 2436), M184V (25 [1.2%] of 2436), and Y181C/I (19 [0.7%] of 2436). The odds ratio for drug resistance associated with each additional year since the start of the ART roll-out in a region was 1.38 (95% CI 1.13–1.68; p=0.001). Interpretation The higher prevalence of primary drug resistance in Uganda than in other African countries is probably related to the earlier start of ART roll-out in Uganda. Resistance surveillance and prevention should be prioritised in settings where ART programmes are scaled up. Funding Ministry of Foreign Affairs of the Netherlands.

HIV-infected women need highly effective contraception to reduce unintended pregnancies and mother-to-child HIV transmission. Previous studies report conflicting results regarding the effect of hormonal contraception on HIV disease progression.HIV-infected women in Uganda and Zimbabwe were recruited immediately after seroconversion; CD4 testing and clinical examinations were conducted quarterly. The study end point was time to AIDS (two successive CD4 200 cells/mm or less or World Health Organization advanced Stage 3 or Stage 4 disease). We used marginal structural Cox survival models to estimate the effect of cumulative exposure to depot-medroxyprogesterone acetate and oral contraceptives on time to AIDS.Three hundred three HIV-infected women contributed 1408 person-years. One hundred eleven women (37%) developed AIDS. Cumulative probability of AIDS was 50% at 7 years and did not vary by country. AIDS incidence was 6.6, 9.3, and 8.8 per 100 person-years for depot-medroxyprogesterone acetate, oral contraceptive, and nonhormonal users. Neither depot-medroxyprogesterone acetate (adjusted hazard ratio, 0.90; 95% confidence interval, 0.76-1.08) nor oral contraceptives ( adjusted hazard ratio, 1.07; 95% confidence interval, 0.89-1.29) were associated with HIV disease progression. Alternative exposure definitions of hormonal contraception use during the year before AIDS or at the time of HIV infection produced similar results. Sexually transmitted infection symptoms were associated with faster progression, whereas young age at HIV infection (18-24 years) was associated with slower progression. Adding baseline CD4 level and set point viral load to models did not change the hormonal contraception results, but Subtype D infection became associated with disease progression.Hormonal contraceptive use was not associated with more rapid HIV disease progression, but older age, sexually transmitted infection symptoms, and Subtype D infection were.

Antiretroviral therapy programs in Africa are currently providing treatment for almost two million people. The long-term success of large scale antiretroviral therapy programs in sub-Saharan Africa remains uncertain because of the limited information currently available on rates of virologic failure and selection for drug-resistant variants in the different HIV subtypes. This article provides a comprehensive review of the published literature on the prevalence of primary and secondary HIV drug resistance with different subtypes and in various settings across sub-Saharan Africa.

Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust cloning/virus production systems to study drug sensitivity, replication kinetics, or to develop personalized vaccines. Extreme HIV-1 heterogeneity leaves few restriction enzyme sites for bacterial cloning strategies. In this study, we describe an alternative approach that involves direct introduction of any HIV-1 coding regions (e.g., any gene from a patient sample) into an HIV-1 DNA vector using yeast recombination. This technique uses positive and negative selectable markers in yeast and avoids the need for purification and screening of the DNA substrates and cloning products. Replication-competent virus is then produced from a modified mammalian 293T packaging cell line transfected with this yeast-derived HIV-1 vector. Although HIV-1 served as the prototype, this cloning strategy is now being developed for other diverse virus species such as hepatitis C virus and influenza virus.

Human immunodeficiency virus type 1 (HIV-1) subtype C is the dominant subtype globally, due largely to the incidence of subtype C infections in sub-Saharan Africa and east Asia. We compared the relative replicative fitness (ex vivo) of the major (M) group of HIV-1 subtypes A, B, C, D, and CRF01_AE and group O isolates. To estimate pathogenic fitness, pairwise competitions were performed between CCR5-tropic (R5) or CXCR4-tropic (X4) virus isolates in peripheral blood mononuclear cells (PBMC). A general fitness order was observed among 33 HIV-1 isolates; subtype B and D HIV-1 isolates were slightly more fit than the subtype A and dramatically more fit than the 12 subtype C isolates. All group M isolates were more fit (ex vivo) than the group O isolates. To estimate ex vivo transmission fitness, a subset of primary HIV-1 isolates were examined in primary human explants from penile, cervical, and rectal tissues. Only R5 isolates and no X4 HIV-1 isolates could replicate in these tissues, whereas the spread to PM1 cells was dependent on active replication and passive virus transfer. In tissue competition experiments, subtype C isolates could compete with and, in some cases, even win over subtype A and D isolates. However, when the migratory cells from infected tissues were mixed with a susceptible cell line, the subtype C isolates were outcompeted by other subtypes, as observed in experiments with PBMC. These findings suggest that subtype C HIV-1 isolates might have equal transmission fitness but reduced pathogenic fitness relative to other group M HIV-1 isolates.

Studies of HIV-1 replication kinetics and fitness require an accurate determination of the level of infectious HIV-1 present in virus stocks. The standard technique for measuring the level of replication-competent infectious virus in culture supernatants or patient samples is the tissue culture dose for 50% infectivity (TCID(50)), which provides an accurate assessment of the level of infectious HIV-1. However, it is a time-consuming technique which typically takes two or more weeks to complete and requires PHA-stimulated PBMC from HIV-1 seronegative donors or an appropriate cell line. Thus rapid, cell-free surrogate measures for TCID(50) are desirable. Here, we introduce the virtual TCID(50) technique: a new cell-free method estimating a surrogate of infectious titer by comparing the reverse transcriptase activity in virus stock to that of reference viruses with a known TCID(50) value. We have demonstrated that the virtual TCID(50) obtained through this technique is comparable to the actual infectious TCID(50). This method greatly simplifies the process of accurate HIV-1 titration and is particularly beneficial for studies which require titration of large number of HIV-1 isolates.

The objectives were to determine whether there are differences in coreceptor use in subjects infected with HIV-1 envelope subtypes A and D that could explain the differences in progression rates between these subtypes in a rural Ugandan cohort. HIV-1 was subtyped in env by V3 sequencing or heteroduplex mobility assay. Coreceptor use was determined by the ability of the isolates to replicate in U87 CD4+ cells expressing different coreceptors. The Fisher exact test was used to examine the relation between coreceptor use and subtype clinical stage and V3 charge. The Kruskall-Wallis nonparametric test was used to examine the association between median CD4 cell counts coreceptor use and subtype. Logistic regression was used to examine predicted coreceptor use at different CD4 groupings. Isolates from 66 participants were analyzed. Thirty-one were infected with subtype A and 35 were infected with subtype D. Although this work was based on a small sample size we found statistically significant differences. The probability of having an X4 virus was higher in subtype D infections than in subtype A infections among those with a non-AIDS clinical status (Fisher exact test P = 0.040). Logistic regression analysis in which we predicted X4 use by subtype and stratified by CD4 group confirmed these findings among those with a CD4 count > 200 cells/µL (likelihood ratio test P = 0.003). R5 viruses were associated with higher median CD4 cell counts than X4 or X4/R5 (Kruskall-Wallis test P = 0.0045). AV3 charge of +5 and greater was highly associated with X4 virus (Fisher exact test P = 0.006). These subtype differences in coreceptor use may partially explain the faster progression rates we have previously reported in individuals infected with subtype D compared with subtype A. Our observations may have implications for the future use of coreceptor inhibitors in this population. (authors)

Additional file 1. Characteristics of primary HIV-1 isolates.

Additional file 1. Characteristics of primary HIV-1 isolates.

Multilingual abstracts in the five official working languages of the United Nations. (PDF 667Â kb)

Multilingual abstracts in the five official working languages of the United Nations. (PDF 667Â kb)

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. Electronic supplementary material The online version of this article (doi:10.1186/s12981-015-0066-7) contains supplementary material, which is available to authorized users.

Background Viral load monitoring (VLM) to identify individuals failing antiretroviral therapy (ART) is not widely available in resource-limited settings. We compared the genotypic resistance patterns between clients with VLM versus immunological monitoring (IM). Methods Between 2004–2008, 559 ART naïve clients were enrolled in a prospective cohort, initiated on ART, and monitored with viral load (VL) and CD4+ cell counts every 6 months (VLM group). From February 2008 through June 2009, 998 clients on ART for 36–40 months (corresponding to the follow-up time of the VLM group) at the same clinic and monitored with CD4+ cell counts every 6 months were recruited into a cross sectional study (IM group). Samples from VLM clients at 12, 24 and 36 months and IM clients at 36–40 months with VL > 2000 copies/ml underwent genotypic drug resistance testing. Results Baseline characteristics were similar. Virologic failure (VL > 400 copies/ml) at 12, 24 and 36 months in the VLM group were 12%, 6% and 8% respectively, and in the IM group 10% at 36–40 months. Samples from 39 VLM and 70 IM clients were genotyped. 23/39 (59%) clients in the VLM group (at 12, 24 or 36 months) compared to 63/70 (90%) in the IM group, (P P (P < 0.0001). Conclusions Routine VL monitoring reduced the rate of accumulated genotypic resistance to commonly used ART in Uganda.