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: Exposure of infants to antiretroviral drugs for prevention of mother-to-child transmission can induce resistance to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). Data from nine national surveys of pretreatment drug resistance in children newly diagnosed with HIV show high levels of resistance to NRTIs included in first-line antiretroviral treatment (ART) regimens (dual abacavir-lamivudine/emtricitabine resistance). Additional research is needed to determine the impact of NRTI resistance on treatment response and optimize infant ART.

Introduction: To achieve viral suppression among more than 90% of people on antiretroviral therapy (ART), improved understanding is warranted of the modifiable causes of HIV viremic episodes. We assessed the relative contributions of drug-resistance, nonadherence and low-level viremia (LLV) (viral load 50-999 cps/ml) on viremic episodes in sub-Saharan Africa., Methods: In a multicountry adult cohort initiating nonnucleoside reverse transcriptase inhibitor-based first-line ART, viremic episodes (viral load ≥1000 cps/ml) were classified as first, viral nonsuppression at 12 months; second, virological rebound at 24 months (after initial viral suppression at 12 months); third, failure to achieve viral resuppression at 24 months (after viremic episode at 12 months). We used adjusted odds ratios from multivariable logistic regression to estimate attributable fractions for each risk factor., Results: Of 2737 cohort participants, 1935 had data on pretreatment drug resistance (PDR) and at least 1 viral load outcome. Viral nonsuppression episodes [173/1935 (8.9%)] were attributable to nonadherence in 30% (35% in men vs. 24% in women) and to PDR to nonnucleoside reverse transcriptase inhibitors in 10% (15% in women vs. 6% in men). Notably, at contemporary PDR prevalences of 10-25%, PDR would explain 13-30% of viral nonsuppression. Virological rebound episodes [96/1515 (6.3%)] were mostly attributable to LLV (29%) and nonadherence (14%), and only rarely to PDR (1.1%). Failures to achieve viral resuppression [66/81 (81.5%)] were mostly attributable to the presence of acquired drug resistance (34%) and only rarely to nonadherence (2.4%)., Conclusion: Effective adherence interventions could substantially reduce viral nonsuppression (especially in men) and virological rebound (especially during LLV), but would have limited effect on improving viral resuppression. Alternative ART regimens could circumvent PDR and acquired resistance.

Background: The presence of high-abundance drug-resistant HIV-1 jeopardizes success of antiretroviral therapy (ART). Despite numerous investigations, the clinical impact of low-abundance drug-resistant HIV-1 variants (LA-DRVs) at levels <15%-25% of the virus population in antiretroviral (ARV) drug-naive individuals remains controversial., Methods: We systematically reviewed 103 studies assessing prevalence, detection methods, technical and clinical detection cutoffs, and clinical significance of LA-DRVs in antiretroviral drug-naive adults., Results: In total, 14 919 ARV drug-naive individuals were included. Prevalence of LA-DRVs (ie, proportion of individuals harboring LA-DRVs) was 0%-100%. Technical detection cutoffs showed a 4 log range (0.001%-10%); 42/103 (40.8%) studies investigating the impact of LA-DRVs on ART; 25 studies included only individuals on first-line nonnucleoside reverse transcriptase inhibitor-based ART regimens. Eleven of those 25 studies (44.0%) reported a significantly association between preexisting LA-DRVs and risk of virological failure whereas 14/25 (56.0%) did not., Conclusions: Comparability of the 103 studies is hampered by high heterogeneity of the studies' designs and use of different methods to detect LA-DRVs. Thus, evaluating clinical impact of LA-DRVs on first-line ART remains challenging. We, the WHO HIVResNet working group, defined central areas of future investigations to guide further efforts to implement ultrasensitive resistance testing in routine settings., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

To improve virological suppression and address the emerging threat of HIV drug resistance, many low-income and middle-income countries are moving away from non-nucleoside reverse transcriptase inhibitors (NNRTI) and transitioning to dolutegravir as part of a more affordable and standardised antiretroviral therapy (ART). Although this transition could decrease the effect of rising NNRTI resistance and yield improved ART outcomes, it also presents new challenges. First, current safety concerns for dolutegravir use in women of childbearing potential require alternative solutions. Second, pre-existing resistance to the co-administered nucleoside reverse transcriptase inhibitors might reduce effectiveness and durability of dolutegravir, particularly if there is scarce access to viral load tests to monitor treatment outcomes. Third, there is inadequate information on the genetic correlates of resistance to dolutegravir, particularly in patients infected with HIV-1 non-B subtypes. Finally, clinical management of patients with confirmed virological failure on a dolutegravir-based regimen can pose challenges because of uncertainty around whether dolutegravir resistance has actually developed and switching is needed, or whether only interventions to improve adherence without switching are sufficient. These considerations should be addressed to consolidate expected gains from widespread introduction of dolutegravir in low-income and middle-income countries., (Copyright © 2019 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.)

Background: Many lines of evidence point to HIV-1 subtype-specific differences in the development of drug resistance mutations. While variation between subtype C and others has been extensively explored, there has been less emphasis on subtypes common to West Africa. We examined a previously described national survey of pretreatment drug resistance in HIV-1-infected Nigerian children aged <18 months, to explore the association between subtypes and patterns of resistance., Methods: Five hundred and forty-nine dried blood spots, from 15 early infant diagnostic facilities in Nigeria, were amplified and HIV-1 polymerase was sequenced. Four hundred and twenty-four were analysed for surveillance drug resistance mutations (SDRMs). Associations between subtype and SDRMs were evaluated by Fisher's exact test and logistic regression analysis, controlling for geographical region and exposure., Results: Using the sub-subtypes of HIV-1 G defined by Delatorre et al. (PLoS One 2014., 9: e98908) the most common subtypes were CRF02&#95;AG (174, 41.0%), GWA-I (128, 30.2%), GWA-II (24, 5.7%), GCA (11, 2.6%), A (21, 5.0%) and CRF06&#95;cpx (18, 4.2%). One hundred and ninety infants (44.8%) had ≥1 NNRTI mutation, 92 infants (21.7%) had ≥1 NRTI mutation and 6 infants (1.4%) had ≥1 PI mutation. By logistic regression, 67N was more common in GWA-II/GCA than CRF02&#95;AG (OR 12.0, P = 0.006), as was 70R (OR 23.1, P = 0.007), 184I/V (OR 2.92, P = 0.020), the presence of ≥1 thymidine analogue mutation (TAM) (OR 3.87, P = 0.014), ≥1 type 2 TAM (OR 7.61, P = 0.001) and ≥1 NRTI mutation (OR 3.26, P = 0.005)., Conclusions: This dataset reveals differences among SDRMs by subtype; in particular, between the GWA-II and GCA subclades, compared with CRF02&#95;AG and GWA-I.

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 <400 copies per mL at 12 months) on first-line non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy. We assessed pretreatment drug resistance with Illumina MiSeq next-generation sequencing, using the International Antiviral Society (IAS)-USA mutation list or the Stanford HIV Drug Resistance Database (HIVDB) genotypic sensitivity score. We calculated diagnostic accuracy measures and assessed the odds of virological failure using conditional logistic regression for 1%, 5%, and 10% pretreatment drug resistance detection thresholds, compared with the conventional 20% or more used in Sanger-based sequencing., 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In ART programs in sub-Saharan Africa, a growing proportion of HIV-infected persons initiating first-line antiretroviral therapy (ART) have a history of prior antiretroviral drug use (PAU). We assessed the effect of PAU on the risk of pre-treatment drug resistance (PDR) and virological failure (VF) in a multicountry cohort of HIV-infected adults initiated on a standard non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line ART. Multivariate logistic regression was used to assess the associations between PAU, PDR and VF (defined as viral load ≥400 cps/mL). Causal mediation analysis was used to assess the proportion of the effect of PAU on VF that could be eliminated by intervening on PDR. Of 2737 participants, 122 (4.5%) had a history of PAU. Participants with PAU had a 7.2-fold (95% CI 4.4-11.7) risk of carrying PDR and a 3.1-fold (95% CI 1.6-6.1) increased risk of VF, compared to antiretroviral-naïve participants. Controlling for PDR would eliminate nearly half the effect of PAU on the risk of VF. Patients with a history of PAU are at increased risk of ART failure, which is to a large extent attributable to PDR. These findings support the recent WHO recommendations for use of differentiated, non-NNRTI-based empiric first-line therapy in patients with PAU.

After 15 years of global scale-up of antiretroviral therapy (ART), rising prevalence of HIV drug resistance in many low-income and middle-income countries (LMICs) poses a growing threat to the HIV response, with the potential to drive an increase in mortality, HIV incidence, and costs. To achieve UNAIDS global targets, enhanced strategies are needed to improve quality of ART services and durability of available ART regimens, and to curb resistance. These strategies include roll out of drugs with greater efficacy and higher genetic barriers to resistance than those that are currently widely used, universal access to and improved effectiveness of viral load monitoring, patient-centred care delivery models, and reliable drug supply chains, in conjunction with frameworks for resistance monitoring and prevention. In this Review, we assess contemporary data on HIV drug resistance in LMICs and their implications for the HIV response, highlighting the potential impact and resistance risks of novel ART strategies and knowledge gaps., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: To investigate the prevalence and patterns of major and accessory resistance mutations associated with integrase strand transfer inhibitors (INSTIs), across diverse HIV-1 subtypes in sub-Saharan Africa., Methods: pol gene sequences were obtained using Illumina next-generation sequencing from 425 INSTI-naive HIV-infected adults from Kenya (21.2%), Nigeria (7.3%), South Africa (22.8%), Uganda (25.2%) and Zambia (23.5%). Drug resistance interpretation was based on the IAS 2017 mutation list and accessory mutations from Stanford HIVdb with resistance penalty scores of ≥10 to at least 1 INSTI. Resistance was further classified based on sensitivity thresholds of ≥20% (Sanger sequencing) and 1%-20% for low-frequency variants (next-generation sequencing)., Results: Of 425 genotypes, 48.7% were subtype C, 28.5% A, 10.1% D, 2.8% G and 9.9% were recombinants. Major INSTI resistance mutations were detected only at <20% threshold, at a prevalence of 2.4% (2.5% in subtype A, 2.4% C, 0% D, 8.3% G and 2.4% in recombinants) and included T66A/I (0.7%), E92G (0.5%), Y143C/S (0.7%), S147G (0.2%) and Q148R (0.5%). Accessory mutations occurred at a prevalence of 15.1% at the ≥20% threshold (23.1% in subtype A, 8.7% C, 11.6% D, 25% G and 23.8% in recombinants), and included L74I/M (10.4%), Q95K (0.5%), T97A (4%), E157Q (0.7%) and G163R/K (0.7%)., Conclusions: Major INSTI resistance mutations were rare and only occurred at low-level resistance detection thresholds. INSTI-based regimens are expected to be effective across the different major HIV-1 subtypes in the region.

Background: WHO recommends protease-inhibitor-based first-line regimen in infants because of risk of drug resistance from failed prophylaxis used in prevention of mother-to-child transmission (PMTCT). However, cost and logistics impede implementation in sub-Saharan Africa, and >75% of children still receive nonnucleoside reverse transcriptase inhibitor-based regimen (NNRTI) used in PMTCT., Methods: We assessed the national pretreatment drug resistance prevalence of HIV-infected children aged <18 months in Nigeria, using WHO-recommended HIV drug resistance surveillance protocol. We used remnant dried blood spots collected between June 2014 and July 2015 from 15 early infant diagnosis facilities spread across all the 6 geopolitical regions of Nigeria. Sampling was through a probability proportional-to-size approach. HIV drug resistance was determined by population-based sequencing., Results: Overall, in 48% of infants (205 of 430) drug resistance mutations (DRM) were detected, conferring resistance to predominantly NNRTIs (45%). NRTI and multiclass NRTI/NNRTI resistance were present at 22% and 20%, respectively, while resistance to protease inhibitors was at 2%. Among 204 infants with exposure to drugs for PMTCT, 57% had DRMs, conferring NNRTI resistance in 54% and multiclass NRTI/NNRTI resistance in 29%. DRMs were also detected in 34% of 132 PMTCT unexposed infants., Conclusion: A high frequency of PDR, mainly NNRTI-associated, was observed in a nationwide surveillance among newly diagnosed HIV-infected children in Nigeria. PDR prevalence was equally high in PMTCT-unexposed infants. Our results support the use of protease inhibitor-based first-line regimens in HIV-infected young children regardless of PMTCT history and underscore the need to accelerate implementation of the newly disseminated guideline in Nigeria.

Global scale-up of antiretroviral treatment has dramatically changed the prospects of HIV/AIDS disease, rendering life-long chronic care and treatment a reality for millions of HIV-infected patients. Affordable technologies to monitor antiretroviral treatment are needed to ensure long-term durability of limited available drug regimens. HIV drug resistance tests can complement existing strategies in optimizing clinical decision-making for patients with treatment failure, in addition to facilitating population-based surveillance of HIV drug resistance. This review assesses the current landscape of HIV drug resistance technologies and discusses the strengths and limitations of existing assays available for expanding testing in resource-limited settings. These include sequencing-based assays (Sanger sequencing assays and nextgeneration sequencing), point mutation assays, and genotype-free data-based prediction systems. Sanger assays are currently considered the gold standard genotyping technology, though only available at a limited number of resource-limited setting reference and regional laboratories, but high capital and test costs have limited their wide expansion. Point mutation assays present opportunities for simplified laboratory assays, but HIV genetic variability, extensive codon redundancy at or near the mutation target sites with limited multiplexing capability have restricted their utility. Next-generation sequencing, despite high costs, may have potential to reduce the testing cost significantly through multiplexing in high-throughput facilities, although the level of bioinformatics expertise required for data analysis is currently still complex and expensive and lacks standardization. Web-based genotype-free prediction systems may provide enhanced antiretroviral treatment decision-making without the need for laboratory testing, but require further clinical field evaluation and implementation scientific research in resource-limited settings.

Background: Pretreatment HIV drug resistance (PDR) can impair virological response to ART, jeopardizing effective treatment for children., Methods: Children aged ≤12 years initiated first-line ART in Uganda during 2010-11. Baseline and 6 monthly viral load (VL) and genotypic resistance testing if VL >1000 copies/mL was done. The 2015 IAS-USA mutation list and Stanford algorithm were used to score drug resistance mutations (DRMs) and susceptibility. Virological failure (VF) was defined as two consecutive VLs >1000 copies/mL or death after 6 months of ART. Factors associated with failure and acquired drug resistance (ADR) were assessed in a logistic regression analysis., Results: Among 317 children enrolled, median age was 4.9 years and 91.5% received NNRTI-based regimens. PDR was detected in 47/278 (16.9%) children, of whom 22 (7.9%) had resistance against their first-line regimen and were therefore on a partially active regimen. After 24 months of follow-up, 92/287 (32.1%) had experienced VF. Children with PDR had a higher risk of VF (OR 15.25, P < 0.001) and ADR (OR 3.58, P = 0.01)., Conclusions: Almost one-third of children experienced VF within 24 months of NNRTI-based first-line treatment. PDR was the strongest predictor of VF and ADR, and therefore presents a major threat in children. There is a need for ART regimens that maximize effectiveness of first-line therapy for long-term treatment success in the presence of PDR or incorporation of routine VL testing to detect VF and change treatment in time, in order to prevent clinical deterioration and accumulation of additional drug resistance. Children ≤3 years should be initiated on a PI-based regimen as per WHO guidelines., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

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., (© The Author [2016]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

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., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: As antiretroviral therapy (ART) programs in sub-Saharan Africa mature, increasing numbers of persons with human immunodeficiency virus (HIV) infection will experience treatment failure, and require second- or third-line ART. Data on second-line failure and development of protease inhibitor (PI) resistance in sub-Saharan Africa are scarce., Methods: HIV-1-infected adults were included if they received >180 days of PI-based second-line ART. We assessed risk factors for having a detectable viral load (VL, ≥400 cps/mL) using Cox models. If VL was ≥1000 cps/mL, genotyping was performed., Results: Of 227 included participants, 14.6%, 15.2% and 11.1% had VLs ≥400 cps/mL at 12, 24, and 36 months, respectively. Risk factors for a detectable VL were as follows: exposure to nonstandard nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based (hazard ratio, 7.10; 95% confidence interval, 3.40-14.83; P < .001) or PI-based (7.59; 3.02-19.07; P = .001) first-line regimen compared with zidovudine/lamivudine/NNRTI, PI resistance at switch (6.69; 2.49-17.98; P < .001), and suboptimal adherence (3.05; 1.71-5.42; P = .025). Among participants with VLs ≥1000 cps/mL, 22 of 32 (69%) harbored drug resistance mutation(s), and 7 of 32 (22%) harbored PI resistance., Conclusions: Although VL suppression rates were high, PI resistance was detected in 22% of participants with VLs ≥1000 cps/mL. To ensure long-term ART success, intensified support for adherence, VL and drug resistance testing, and third-line drugs will be necessary., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

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.

Background: K65R is a relatively rare drug resistance mutation (DRM) selected by the NRTIs tenofovir, didanosine, abacavir and stavudine and confers cross-resistance to all NRTIs except zidovudine. Selection by other NRTIs is uncommon., Objectives: In this study we investigated the frequency of emergence of the K65R mutation and factors associated with it in HIV-1-infected infants exposed to low doses of maternal lamivudine, zidovudine and either nevirapine or nelfinavir ingested through breast milk, using specimens collected from the Kisumu Breastfeeding Study., Methods: Plasma specimens with viral load ≥1000 copies/mL collected from HIV-infected infants at 0-1, 2, 6, 14, 24 and 36 weeks of age and maternal samples at delivery were tested for HIV drug resistance using Sanger sequencing of the polymerase gene. Factors associated with K65R emergence were assessed using Fisher's exact test and the Wilcoxon rank-sum test., Results: K65R was detected in samples from 6 of the 24 infants (25%) who acquired HIV-1 infection by the age of 6 months. K65R emerged in half of the infants by 6 weeks and in the rest by 14 weeks of age. None of the mothers at delivery or the infants with a positive genotype at first time of positivity had the K65R mutation. Infants with K65R had low baseline CD4 cell counts (P = 0.014), were more likely to have DRMs earlier (≤6 weeks versus ≥14 weeks, P = 0.007) and were more likely to have multiclass drug resistance (P = 0.035). M184V was the most common mutation associated with K65R emergence. K65R had reverted by 3 months after cessation of breastfeeding., Conclusions: A high rate of K65R emergence may suggest that ingesting low doses of lamivudine via breast milk could select for this mutation. The presence of this mutation may have a negative impact on future responses to NRTI-based ART. More in vitro studies are, however, needed to establish the molecular mechanism for this selection., (Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

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., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes., Methods and Findings: We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling., Conclusions: Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Background: Earlier antiretroviral therapy initiation can reduce the incidence of HIV-1. This benefit can be offset by increased transmitted drug resistance (TDR). We compared the preventive benefits of reducing incident infections with the potential TDR increase in East Africa., Methods: A mathematical model was constructed to represent Kampala, Uganda, and Mombasa, Kenya. We predicted the effect of initiating treatment at different immunological thresholds (<350, <500 CD4 cells/μl) on infections averted and mutation-specific TDR prevalence over 10 years compared to initiating treatment at CD4 cell count below 200 cells/μl., Results: When initiating treatment at CD4 cell count below 350 cells/μl, we predict 18 [interquartile range (IQR) 11-31] and 46 (IQR 30-83) infections averted for each additional case of TDR in Kampala and Mombasa, respectively, and 22 (IQR 17-35) and 32 (IQR 21-57) infections averted when initiating at below 500. TDR is predicted to increase most strongly when initiating treatment at CD4 cell count below 500 cells/μl, from 8.3% (IQR 7.7-9.0%) and 12.3% (IQR 11.7-13.1%) in 2012 to 19.0% (IQR 16.5-21.8%) and 19.2% (IQR 17.1-21.5%) in 10 years in Kampala and Mombasa, respectively. The TDR epidemic at all immunological thresholds was comprised mainly of resistance to non-nucleoside reverse transcriptase inhibitors. When 80-100% of individuals with virological failure are timely switched to second-line therapy, TDR is predicted to decline irrespective of treatment initiation threshold., Conclusion: Averted HIV infections due to the expansion of antiretroviral treatment eligibility offset the risk of transmitted drug resistance, as defined by more infections averted than TDR gained. The effectiveness of first-line non-nucleoside reverse transcriptase inhibitor-based therapy can be preserved by improving switching practices to second-line therapy.

In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01&#95;AE (CRF01&#95;AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01&#95;AE, and CRF02&#95;AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples.

Abstract In view of the recent antiretroviral therapy (ART) scale-up in Kenya, surveillance of transmitted HIV drug resistance (TDR) is important. A cross-sectional survey was conducted among newly HIV-1 diagnosed, antiretroviral-naive adults in Mombasa, Kenya. Surveillance drug resistance mutations (SDRMs) were identified according to the 2009 WHO list. HIV-1 subtypes were determined using REGA and SCUEAL subtyping tools. Genotypic test results were obtained for 68 of 81 participants, and SDRMs were identified in 9 samples. Resistance to nonnucleoside reverse transcriptase inhibitors (K103N) occurred in five participants, yielding a TDR prevalence of 7.4% (95% confidence interval 2.4-16.3%). Frequencies of HIV-1 subtypes were A (70.6%), C (5.9%), D (2.9%), and unique recombinant forms (20.6%). The TDR prevalence found in this survey is higher than previously reported in different regions in Kenya. These findings justify increased vigilance with respect to TDR surveillance in African regions where ART programs are scaled-up in order to inform treatment guidelines.

Background: Human immunodeficiency virus type 1 (HIV-1) drug resistance may limit the benefits of antiretroviral therapy (ART). This cohort study examined patterns of drug-resistance mutations (DRMs) in individuals with virological failure on first-line ART at 13 clinical sites in 6 African countries and predicted their impact on second-line drug susceptibility., Methods: A total of 2588 antiretroviral-naive individuals initiated ART consisting of different nucleoside reverse transcriptase inhibitor (NRTI) backbones (zidovudine, stavudine, tenofovir, or abacavir, plus lamivudine or emtricitabine) with either efavirenz or nevirapine. Population sequencing after 12 months of ART was retrospectively performed if HIV RNA was >1000 copies/mL. The 2010 International Antiviral Society-USA list was used to score major DRMs. The Stanford algorithm was used to predict drug susceptibility., Results: HIV-1 sequences were generated for 142 participants who virologically failed ART, of whom 70% carried ≥1 DRM and 49% had dual-class resistance, with an average of 2.4 DRMs per sequence (range, 1-8). The most common DRMs were M184V (53.5%), K103N (28.9%), Y181C (15.5%), and G190A (14.1%). Thymidine analogue mutations were present in 8.5%. K65R was frequently selected by stavudine (15.0%) or tenofovir (27.7%). Among participants with ≥1 DRM, HIV-1 susceptibility was reduced in 93% for efavirenz/nevirapine, in 81% for lamivudine/emtricitabine, in 59% for etravirine/rilpivirine, in 27% for tenofovir, in 18% for stavudine, and in 10% for zidovudine., Conclusions: Early failure detection limited the accumulation of resistance. After stavudine failure in African populations, zidovudine rather than tenofovir may be preferred in second-line ART. Strategies to prevent HIV-1 resistance are a global priority.

Objective: To assess the emergence of transmitted HIV-1 drug resistance (TDR) in Kampala, Uganda, 10 years after the scale-up of antiretroviral treatment (ART) and to compare with a previous survey among antenatal clinic attendees in 2007 (reporting 0% TDR)., Design: A cross-sectional survey was conducted among newly HIV-1 diagnosed, antiretroviral-naive young adults attending two large voluntary counseling and testing centers within the geographic area of Kampala., Methods: Proxy criteria for recent HIV-1 infection were used as defined by the WHO. Population sequencing of the pol gene was performed on plasma samples with HIV-1 RNA at least 1000 copies/ml. Surveillance drug resistance mutations (SDRMs) were identified according to the 2009 WHO list for surveillance of TDR. HIV-1 subtypes were designated using maximum likelihood phylogenetic reconstruction., Results: : Genotypic test results were obtained for 70 of 77 (90.9%) participants. SDRMs were identified in six samples yielding a prevalence of TDR of 8.6% (95% confidence interval 3.2-17.7%). Two had SDRMs to nucleoside reverse-transcriptase inhibitors (D67G and L210W), three had SDRMs to nonnucleoside reverse transcriptase inhibitors (G190A, G190S, and K101E), and one had SDRMs to protease inhibitors (N88D). Frequencies of HIV-1 subtypes were A (36/70, 51.4%), C ( two of 70; 2.9%), D (23/70, 32.9%), and unique recombinant forms (nine of 70, 12.9%)., Conclusion: This repeated survey suggests an increase in TDR in Kampala, compared with a previous survey. This finding justifies increased vigilance with respect to surveillance of TDR in areas in Africa where ART programs are rolled-out.

Objective: To assess the mutational patterns and factors associated with baseline drug-resistant HIV-1 present at initiation of first-line antiretroviral therapy (ART) at 3 sites in Lusaka, Zambia, in 2007-2008., Methods: Population sequencing of the HIV-1 pol gene was performed in the PharmAccess African Studies to Evaluate Resistance Monitoring cohort. Drug resistance-associated mutations (DRMs) were identified using the WHO 2009 Surveillance DRM list. Multiple logistic regression was used to assess factors associated with baseline resistance., Results: The overall prevalence of baseline resistance was 5.7% [31 of 548 participants; 95% confidence interval (CI): 3.9 to 7.9]; the prevalence of DRMs associated with nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors was 1.1%, 4.0%, and 1.1%, respectively. Resistance prevalence was 5.2% (27 of 523) in antiretroviral-naive and 16.0% (4 of 25) in antiretroviral-experienced (ie, previous use of ART or antiretroviral prophylaxis for prevention of mother-to-child transmission) participants (P = 0.022). Dual-class resistance to NRTIs and NNRTIs was observed in 0.6% of participants. HIV-1 subtype C was identified in 98.0% (537 of 548) of participants. Prior antiretroviral experience (odds ratio: 4.32, CI: 1.34 to 14.0, P = 0.015) and hemoglobin level (highest tertile versus lowest tertile odds ratio: 2.74, CI: 1.09 to 6.89, P = 0.033) were independently associated with baseline resistance., Conclusions: Baseline resistance may compromise the response to standard NNRTI-based first-line ART in 6% of patients in Lusaka, Zambia. Continuous resistance monitoring is warranted to maintain individual and population-level ART effectiveness.

Background: Dried spots on filter paper made of whole blood (dried blood spots; DBS), plasma (dried plasma spots; DPS) or serum (dried serum spots) hold promise as an affordable and practical alternative specimen source to liquid plasma for HIV type-1 (HIV-1) viral load determination and drug resistance genotyping in the context of the rapidly expanding access to antiretroviral therapy (ART) for HIV-1-infected individuals in low- and middle-income countries. This report reviews the current evidence for their utility., Methods: We systematically searched the English language literature published before 2009 on Medline, the websites of the World Health Organization and US Centers for Disease Control and Prevention, abstracts presented at relevant international conferences and references from relevant articles., Results: Data indicate that HIV-1 viral load determination and resistance genotyping from DBS and DPS is feasible, yielding comparable test performances, even after storage. Limitations include reduced analytical sensitivity resulting from small analyte volumes (approximately 3.5 log(10) copies/ml at 50 microl sample volume), nucleic acid degradation under extreme environmental conditions, impaired efficiency of nucleic acid extraction, potential interference of archived proviral DNA in genotypes obtained from DBS and the excision of spots from the filters in high-volume testing., Conclusions: This technology offers the advantages of a stable specimen matrix, ease of sample collection and shipment. The current sensitivity in drug resistance testing is appropriate for public health surveillance among pretreatment populations. However, consistently improved analytical sensitivity is needed for their routine application in the therapeutic monitoring of individuals receiving ART, particularly at the onset of treatment failure.

Access to highly active antiretroviral therapy (HAART) for persons infected with HIV in sub-Saharan Africa has greatly improved over the past few years. However, data on long-term clinical outcomes of Africans receiving HAART, patterns of HIV resistance to antiretroviral drugs and implications of HIV type-1 (HIV-1) subtype diversity in Africa for resistance, are limited. In resource-limited settings, concerns have been raised that deficiencies in health systems could create the conditions for accelerated development of resistance. Coordinated surveillance systems are being established to assess the emergence of resistance and the factors associated with resistance development, and to create the possibility for adjusting treatment guidelines as necessary. The purpose of this report is to review the literature on HIV-1 resistance to antiretroviral drugs in sub-Saharan Africa, in relation to the drug regimens used in Africa, HIV-1 subtype diversity and overall prevalence of resistance. The report focuses on resistance associated with treatment, prevention of mother-to-child transmission and transmitted resistance. It also outlines priorities for public health action and research.