Preexisting Drug Resistance Mutations Research Articles

Molecular Epidemiology of HIV-1 in Eastern Europe and Russia.

The HIV epidemic in Eastern Europe and Russia is large and not well-controlled. To describe the more recent molecular epidemiology of HIV-1, transmitted drug resistance, and the relationship between the epidemics in this region, we sequenced the protease and reverse transcriptase genes of HIV-1 from 812 people living with HIV from Ukraine (n = 191), Georgia (n = 201), and Russia (n = 420) before the initiation of antiretroviral therapy. In 190 Ukrainian patients, the integrase gene sequence was also determined. The most reported route of transmission was heterosexual contact, followed by intravenous drug use, and men having sex with men (MSM). Several pre-existing drug resistance mutations were found against non-nucleoside reverse transcriptase inhibitors (RTIs) (n = 103), protease inhibitors (n = 11), and nucleoside analogue RTIs (n = 12), mostly polymorphic mutations or revertants. In the integrase gene, four strains with accessory integrase strand transfer inhibitor mutations were identified. Sub-subtype A6 caused most of the infections (713/812; 87.8%) in all three countries, including in MSM. In contrast to earlier studies, no clear clusters related to the route of transmission were identified, indicating that, within the region, the exchange of viruses among the different risk groups may occur more often than earlier reported.

Short Communication: HIV-DRLink: A Tool for Reporting Linked HIV-1 Drug Resistance Mutations in Large Single-Genome Data Sets Using the Stanford HIV Database.

The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy. It is likely that pre-existing drug resistance mutations linked on the same viral genomes are predictive of treatment failure. Because of the large number of sequences generated by ultrasensitive single-genome sequencing (uSGS) and other similar next-generation sequencing methods, it is difficult to assess each sequence individually for linked drug resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large data sets, but they provide no information on linkage of resistance mutations. In this study, we report the HIV-DRLink program, a research tool that provides resistance mutation frequencies as well as their genetic linkage by parsing and summarizing the Sierra output from the Stanford HIV Database. The HIV-DRLink program should only be used on data sets generated by methods that eliminate artifacts due to polymerase chain reaction recombination, for example, standard single-genome sequencing or uSGS. HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

Naturally occurring antiviral drug resistance in HIV patients who are mono-infected or co-infected with HBV or HCV in China.

Drug resistance mutations (DRMs) may reduce the efficacy of antiviral therapy. However, the studies focused on naturally occurring, pre-existing DRMs among co-infected patients in China are limited. To investigate DRMs prevalence in treatment-naïve human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) mono- and co-infected patients in China, a total of 570 patients were recruited for this study. DRMs sequences were amplified and successfully sequenced in 481 of these patients, who were grouped into three cohorts: (i) The HBV cohort included 100 HIV/HBV co-infected and 110 HBV mono-infected patients who were sequenced for HBV; (ii) The HCV cohort included 91 patients who were HIV/HCV co-infected and 72 who were HCV mono-infected for HCV sequencing; and (iii) The HIV cohort included 39 HIV mono-infected, 22 HIV/HCV, and 47 HIV/HBV co-infected patients for HIV sequencing. Next-generation sequencing and Sanger sequencing were used in this study. The results showed that in the HCV cohort, HCV genotypes 6a (P < 0.001) and 3b (P = 0.004) were more prevalent in HIV/HCV co-infected patients, however, the prevalence of HBV and HIV genotypes were similar within the HBV and HIV cohorts. HBV DRMs prevalence was significantly higher in HIV/HBV co-infected than HBV mono-infected patients (8.0% vs 0.9%, P = 0.015), whereas HCV and HIV DRMs did not differ within the HCV and HIV cohort (P > 0.05). This study revealed that HBV DRMs were more prevalent in HIV/HBV co-infected patients in China, while DRMs in HCV and HIV patients did not differ. Further dynamic surveillance of DRMs may be needed.

HIV Antiretroviral Resistance and Transmission in Mother–Infant Pairs Enrolled in a Large Perinatal Study

BackgroundDetection of antiretroviral (ARV) resistance in HIV-infected individuals is not uncommon and may be particularly problematic in HIV-infected pregnant women as it can lead to infant infection with resistant strains. To better evaluate the effect of drug resistance mutations (DRMs) on HIV mother-to-child transmission (MTCT), we determined the prevalence of DRMs in a subset of mother–infant pairs enrolled in a multi-center trial of infant prophylaxis among women not receiving ARVs during the current pregnancy.MethodsA case–control design of 1:4 (1 transmitter to 4 nontransmitters) was utilized to evaluate ARV resistance as a predictor of HIV MTCT in specimens obtained from mother–infant pairs. Secondary objectives included identification of potential risk factors associated with the presence of DRMs. Viroseq HIV-1 Genotyping System was performed on mother–infant specimens to assess for mutations that might result in a substantial reduction in drug susceptibility and clinical outcome, as determined by the Stanford HIV Drug Resistance Database.ResultsOne hundred and forty infants were infected. Of these, 123 HIV infected mother–infant pairs and 483 of 560 women who did not transmit HIV had amplifiable HIV nucleic acid enabling ARV resistance testing. A wide variety of DRMs were detected (Figure 1). Sixty (10%) of 606 women had clinically relevant DRMs; 12 (2%) had DRMs against more than 1 ARV class. Among 123 HIV− infected infants, 13 (11%) had clinically relevant DRMs with 3 (2%) harboring DRMs against more than 1 ARV class. Of 13 infants with DRMs, 10 (77%) were infected in utero. In univariate and multivariate analyses, DRMs in mothers were not associated with increased risk of HIV MTCT (AOR 0.79, 95% CI 0.38–1.5). Log HIV viral load was the only predictor of MTCT (OR 1.4, 95% CI 1.2−1.6). The presence of DRMs in mothers who transmitted was strongly associated with the presence of DRMs in infants (P < 0.001).ConclusionIn infected pregnant women without ARV exposure during their current gestation, the presence of pre-existing DRMs with a wide diversity was noted. DRMs do not increase the risk of HIV MTCT. However, if women with DRMs are not virologically suppressed they are likely to transmit resistant mutations even without selective ARV pressure, thus complicating treatment options.Disclosures All authors: No reported disclosures.

Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the SPIRIT Study

Objectives: Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF).Methods: SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48.Results: Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed.Discussion: Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance data are unavailable.

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set.

Effect of Natural Polymorphisms in the HIV-1 CRF02_AG Protease on Protease Inhibitor Hypersusceptibility

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.

Genetic Diversity and Antiretroviral Drug Resistance among Drug-Na&amp;#239;ve HIV-1 Infected Pregnant Women Attending Antenatal Clinics in Abidjan, C&amp;#244;te d'Ivoire

To clarify the distribution of HIV-1 subtypes and drug resistance-related mutations, we collected and analysed serum from pregnant women who are ARV drug-naive in Abidjan. The prevalence of HIV-1 subtypes and mutations associated with antiretroviral drug resistance among drug-na?ve HIV-1 infected pregnant women was investigated from plasma of 90 young pregnant primigravida. The HIV-1 pol and env genes were amplified by using primers recognizing conserved viral sequences and sequenced by employing BigDye chemistry. Positions 1 - 99 of the PR and 1 - 350 of the RT genes were analyzed for mutations based on the international AIDS society USA panel. In 39 strains which both genes were sequenced including CFR02_AG 30 (76.9%), subtype A 3 (7.7%), CFR06_cpx 2 (5.1%), CFR09_cpx 1 (2.6%), and discordant sequences suggesting the presence of a few number of recombinant involving CRF02-AG and subtype A 3 (7.7%). None of the major drug resistance mutations was detected. The frequent minor mutations associated drug resistance observed were M36I (52%/96.3%), L10I/R/V (19%/35.2%) and L63P (7%/12.9%). The M36I mutation was widespread in all subtypes. Our result demonstrated first a significant level of viral heterogeneity and then only the presence of minor resistance associated mutations. Our study emphasizes the need of HIV sentinel survey in C?te d'Ivoire and shows that pregnant women who are candidates for receiving antiretroviral drug therapies do not contain naturally occurring or preexisting drug resistance mutations. So such drug therapies are likely to be highly effective in this setting.

Minority K65R Variants and Early Failure of Antiretroviral Therapy in HIV-1–infected Eritrean Immigrant

Minority K65R Variants and Early Failure of Antiretroviral Therapy in HIV-1–infected Eritrean Immigrant

Genetic diversity of simian immunodeficiency virus encoding HIV-1 reverse transcriptase persists in macaques despite antiretroviral therapy.

The impact of antiretroviral therapy (ART) on the genetics of simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) populations has been incompletely characterized. We analyzed SIV genetic variation before, during, and after ART in a macaque model. Six pigtail macaques were infected with an SIV/HIV chimeric virus, RT-SHIV(mne), in which SIV reverse transcriptase (RT) was replaced by HIV-1 RT. Three animals received a short course of efavirenz (EFV) monotherapy before combination ART was started. All macaques received 20 weeks of tenofovir, emtricitabine, and EFV. Plasma virus populations were analyzed by single-genome sequencing. Population diversity was measured by average pairwise difference, and changes in viral genetics were assessed by phylogenetic and panmixia analyses. After 20 weeks of ART, viral diversity was not different from pretherapy viral diversity despite more than 10,000-fold declines in viremia, indicating that, within this range, there is no relationship between diversity and plasma viremia. In two animals with consistent SIV RNA suppression to <15 copies/ml during ART, there was no evidence of viral evolution. In contrast, in the four macaques with viremias >15 copies/ml during therapy, there was divergence between pre- and during-ART virus populations. Drug resistance mutations emerged in two of these four animals, resulting in virologic failure in the animal with the highest level of pretherapy viremia. Taken together, these findings indicate that viral diversity does not decrease with suppressive ART, that ongoing replication occurs with viremias >15 copies/ml, and that in this macaque model of ART drug resistance likely emerges as a result of incomplete suppression and preexisting drug resistance mutations.

Monitoring of hepatitis B virus surface antigen escape mutations and concomitantly nucleos(t)ide analog resistance mutations in Turkish patients with chronic hepatitis B

The hepatitis B virus (HBV) polymerase gene completely overlaps with the envelope gene. In the present study we aimed to monitor the prevalence and pattern of the typical mutations for hepatitis B surface antigen (HBsAg) escape, and concomitantly nucleos(t)ide analog (NUC) resistance mutations, in Turkish patients undergoing different antiviral therapies and in treatment-naïve patients with chronic hepatitis B (CHB). The investigation was undertaken between March 2007 and August 2009 and involved a total of 142 patients under NUC therapy (88 males; mean age 42 years (range 13-68); hepatitis B e antigen (HBeAg) negativity in 94 patients; HBV DNA median log 4.3 log(10) IU/ml (range 2.0->6.0); alanine aminotransferase (ALT) median level 76.1 IU/ml (range 12-1082)) and 185 treatment-naïve CHB patients (120 males; mean age 39 years (range 1-76 years); HBeAg negativity in 132 patients; HBV DNA median log 3.5 log(10) IU/ml (range 2.0-6.0); ALT median level 60.7 IU/l (range 8-874)). The overall prevalence of typical HBsAg escape mutations found in the CHB patients was 8.3% (27/327). In the NUC therapy group the prevalence was 8.5% (12/142), with the following patterns: sY100C+sI110V, sL109I, sP120T, sP127T, sG130R+sG145X, sS132A+sY134N, sY134N+sG145R, sC137G, sD144E, sG145R. In the treatment-naïve group the prevalence was 8.1% (15/185), with the following patterns: sL109I, sI110V, sS117INST, sP120T, sP127T, sM133I, sC137L+sG145R, sS143L. However, NUC resistance mutations were found in 7.7% (11/142) of the patients on NUC therapy and 3.8% (7/185) of the treatment-naïve group patients. Interestingly, the treatment-naïve patients had preexisting drug resistance mutations related to lamivudine (rtL180M+rtM204I), adefovir (rtA181V, rtQ215S, rtI233V), entecavir (intermediate susceptibility with rtL180M+rtM204IHBV variant), telbivudine (rtL180M+rtM204I), and tenofovir (rtA194T). The findings of this study show preexisting typical HBsAg escape and NUC resistance mutations are possible. The genetic arrangement of the HBV genome with polymerase and surface genes overlapping has substantial public health and diagnostic implications and relevance.

Efficient Suppression of Minority Drug‐Resistant HIV Type 1 (HIV‐1) Variants Present at Primary HIV‐1 Infection by Ritonavir‐Boosted Protease Inhibitor–Containing Antiretroviral Therapy

Selection of preexisting minority variants of drug-resistant human immunodeficiency virus type 1 (HIV-1) can lead to virological failure in patients who receive antiretroviral therapy (ART) with low genetic resistance barriers. We studied treatment response and dynamics of minority variants during the first weeks of ART containing a ritonavir-boosted protease inhibitor (PI) and 2 nucleoside reverse-transcriptase inhibitors (NRTIs), which is a regimen with a high genetic resistance barrier. Plasma samples obtained prior to initiation of ART from 109 patients with primary HIV infection and samples obtained during viral decay during early ART from 17 of these 109 patients were tested by allele-specific polymerase chain reaction for K103N and M184V variants. K103N and/or M184V mutations were detected in 15 (13.8%) of 109 patients prior to ART as minority variants. No selection of these variants was observed within the first weeks of ART in 7 of 15 patients with preexisting drug resistance mutations, nor was any selection observed in 10 patients without preexisting drug resistance mutations. Most patients received ART immediately after diagnosis of HIV-1 infection, showed a rapid decrease in viral load, and experienced sufficient suppression of viremia for 48 months. Minority variants, in particular viruses harboring the M184V mutation, were efficiently suppressed in patients with acute infection who received a ritonavir-boosted PI and 2 NRTIs (most regimens included lamivudine). Under this high genetic resistance barrier regimen, the M184V was not further selected.

Prevalence of hepatitis B virus DNA polymerase mutations in treatment‐naïve patients with chronic hepatitis B

One of the most important factors in treatment failure using nucleos(t)ide analogues in chronic hepatitis B is anti-viral resistance. Primary drug resistance refers to amino acid changes in the hepatitis B virus polymerase/reverse transcriptase (rt) that result in reduced susceptibility to anti-viral agents. Pre-existing drug resistance mutations may occur in untreated patients and may affect their treatment outcomes. To determine the prevalence of hepatitis B DNA polymerase mutations in treatment-naïve patients. We used a direct PCR sequencing test to detect DNA polymerase mutations in 472 consecutive treatment-naïve patients at two community gastroenterology clinics in Northern California. A majority of patients were Asians (>95%), had either genotype B or C (95%) and had no evidence of cirrhosis or liver cancer (94%). Mean age was 45 +/- 13 and mean hepatitis B virus DNA was 5.3 +/- 1.8 log(10) IU/mL. Most patients did not have any detectable mutations (82.4%). Some (16.7%) had mutations of unknown clinical significance (rtV207M/L/I) and only 4 patients had rtA181A/S, rtA194S or M250I. No rtM204V/I or rtN236T mutations were observed in our study. Less than 1% of our patients had mutations that can be associated with primary resistance to existing anti-viral therapies for hepatitis B virus.

HIV-1 Subtype C Reverse Transcriptase Sequences from Drug-Naive Pregnant Women in South Africa

The HIV-1 reverse transcriptase genes from 37 HIV-1-positive pregnant women attending an antenatal clinical in Soweto, South Africa were sequenced and analyzed for the presence of drug resistance mutations. All women were antiretroviral drug naive, but were being screened as potential participants in clinical trials of antiretroviral drugs aimed at preventing mother-to-child transmission. Sequence analysis revealed that all belonged to HIV-1 subtype C, the predominant subtype among heterosexual populations in South Africa. Twenty-three amino acid loci associated with resistance to zidovudine, lamivudine, didanosine, stavudine, and nevirapine were examined and found not to encode mutations that would confer resistance to these drugs. Polymorphisms at these loci occurred infrequently, with three patients harboring the A98S and V179I polymorphisms. An additional three patients harbored V118I, which can function as an accessory resistance mutation, but in this context is also likely to be a polymorphism. These data show that pregnant women who are candidates for receiving antiretroviral drug therapies do not contain naturally occurring or preexisting drug resistance mutations and that such drug therapies are likely to be highly effective in this setting.