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. 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. 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. About 35 million people are currently infected with HIV, the virus that causes AIDS by destroying immune system cells and leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected individuals died within ten years of infection. Then, in 1996, effective antiretroviral (ARV) therapy—drug combinations that suppress HIV replication by inhibiting reverse transcriptase and other essential viral enzymes—became available. For people living in affluent countries, HIV/AIDS became a chronic condition, but because ARV therapy was expensive, HIV/AIDS remained fatal in low- and middle-income countries (LMICs). In 2003, the international community began to work towards achieving universal access to ARV therapy. Now, more than 10 million HIV-positive individuals in LMICs receive ARV therapy, usually as a fixed-dose combination of two nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir and lamivudine, plus a non-nucleoside reverse transcriptase inhibitor (NNRTI), such as efavirenz or nevirapine. The global scale-up of ARV therapy has reduced deaths from HIV/AIDS and the incidence of HIV infection in LMICs, but the development of resistance to ARV therapy is threatening these advances. HIV rapidly accumulates genetic changes (mutations), some of which make HIV resistant to ARV therapy. Up to 30% of patients receiving a fixed-dose NRTI/NNRTI combination develop virological failure, and a high proportion of these patients develop mutations associated with resistance to the ARVs in their regimen. Moreover, the proportion of newly infected, ARV-naïve individuals with transmitted drug resistance (TDR) is also increasing. Organizations involved in HIV/AIDS control need to understand the regional and temporal mutational patterns of TDR to inform the development of guidelines for first-line ARV therapy and of inexpensive resistance mutation assays for use in LMICs. Here, using a statistical approach called meta-analysis to combine information from individual patients about the resistance mutations they carry, the researchers investigate the molecular epidemiology of TDR (the patterns of molecular changes underlying TDR in populations) and identify the HIV drug-resistance mutations most responsible for TDR in different world regions. The researchers identified 287 studies published between 2000 and 2013 from 111 countries...

The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzedin vitrosusceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs.

Characterizing the mutations selected by the integrase strand transfer inhibitor (INSTI) dolutegravir and their effects on susceptibility is essential for identifying viruses less likely to respond to dolutegravir therapy and for monitoring persons with virological failure (VF) on dolutegravir therapy. We systematically reviewed dolutegravir resistance studies to identify mutations emerging under dolutegravir selection pressure, the effect of INSTI resistance mutations on in vitro dolutegravir susceptibility, and the virological efficacy of dolutegravir in antiretroviral-experienced persons. We analysed 14 studies describing 84 in vitro passage experiments, 26 studies describing 63 persons developing VF plus INSTI resistance mutations on a dolutegravir-containing regimen, 41 studies describing dolutegravir susceptibility results, and 22 clinical trials and 16 cohort studies of dolutegravir-containing regimens. The most common INSTI resistance mutations in persons with VF on a dolutegravir-containing regimen were R263K, G118R, N155H and Q148H/R, with R263K and G118R predominating in previously INSTI-naive persons. R263K reduced dolutegravir susceptibility ∼2-fold. G118R generally reduced dolutegravir susceptibility >5-fold. The highest levels of reduced susceptibility occurred in viruses containing Q148 mutations in combination with G140 and/or E138 mutations. Dolutegravir two-drug regimens were highly effective for first-line therapy and for virologically suppressed persons provided dolutegravir’s companion drug was fully active. Dolutegravir three-drug regimens were highly effective for salvage therapy in INSTI-naive persons provided one or more of dolutegravir’s companion drugs was fully active. However, dolutegravir monotherapy in virologically suppressed persons and functional dolutegravir monotherapy in persons with active viral replication were associated with a non-trivial risk of VF plus INSTI resistance mutations.

The introduction of two new non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the past 5 years and the identification of novel NNRTI-associated mutations have made it necessary to reassess the extent of phenotypic NNRTI cross-resistance. We analysed a dataset containing 1975, 1967, 519 and 187 genotype–phenotype correlations for nevirapine, efavirenz, etravirine and rilpivirine, respectively. We used linear regression to estimate the effects of RT mutations on susceptibility to each of these NNRTIs. Sixteen mutations at 10 positions were significantly associated with the greatest contribution to reduced phenotypic susceptibility (≥10-fold) to one or more NNRTIs, including: 14 mutations at six positions for nevirapine (K101P, K103N/S, V106A/M, Y181C/I/V, Y188C/L and G190A/E/Q/S); 10 mutations at six positions for efavirenz (L100I, K101P, K103N, V106M, Y188C/L and G190A/E/Q/S); 5 mutations at four positions for etravirine (K101P, Y181I/V, G190E and F227C); and 6 mutations at five positions for rilpivirine (L100I, K101P, Y181I/V, G190E and F227C). G190E, a mutation that causes high-level nevirapine and efavirenz resistance, also markedly reduced susceptibility to etravirine and rilpivirine. K101H, E138G, V179F and M230L mutations, associated with reduced susceptibility to etravirine and rilpivirine, were also associated with reduced susceptibility to nevirapine and/or efavirenz. The identification of novel cross-resistance patterns among approved NNRTIs illustrates the need for a systematic approach for testing novel NNRTIs against clinical virus isolates with major NNRTI-resistance mutations and for testing older NNRTIs against virus isolates with mutations identified during the evaluation of a novel NNRTI.

HIV-1 integrase is the third enzymatic target of antiretroviral (ARV) therapy. However, few data have been published on the distribution of naturally occurring amino acid variation in this enzyme. We therefore characterized the distribution of integrase variants among more than 1,800 published group M HIV-1 isolates from more than 1,500 integrase inhibitor (INI)-naïve individuals. Polymorphism rates equal or above 0.5% were found for 34% of the central core domain positions, 42% of the C-terminal domain positions, and 50% of the N-terminal domain positions. Among 727 ARV-naïve individuals in whom the completepolgene was sequenced, integrase displayed significantly decreased inter- and intra-subtype diversity and a lower Shannon's entropy than protease or RT. All primary INI-resistance mutations with the exception of E157Q – which was present in 1.1% of sequences – were nonpolymorphic. Several accessory INI-resistance mutations including L74M, T97A, V151I, G163R, and S230N were also polymorphic with polymorphism rates ranging between 0.5% to 2.0%.

HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.IMPORTANCEMost antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.