Low-frequency Drug Resistance Mutations Research Articles

Low-frequency HIV-1 drug resistance mutations in antiretroviral naïve individuals in Botswana.

Individuals living with human immunodeficiency virus (HIV) who experience virological failure (VF) after combination antiretroviral therapy (cART) initiation may have had low-frequency drug resistance mutations (DRMs) at cART initiation. There are no data on low-frequency DRMs among cART-naïve HIV-positive individuals in Botswana. We evaluated the prevalence of low-frequency DRMs among cART-naïve individuals previously sequenced using Sanger sequencing. The generated pol amplicons were sequenced by next-generation sequencing. We observed low-frequency DRMs (detected at <20% in 33/103 (32%) of the successfully sequenced individuals, of whom four also had mutations detected at >20%. K65R was the most common low-frequency DRM detected in 8 individuals. Eighty-two of the 103 individuals had follow-up viral load data while on cART. Twenty-seven of the 82 individuals harbored low-frequency DRMs. Only 12 of 82 individuals experienced VF. The following low-frequency DRMs were observed in four individuals experiencing VF: K65R, K103N, V108I, and Y188C. No statistically significant difference was observed in the prevalence of low-frequency DRMs between individuals experiencing VF (4/12) and those not experiencing VF (23/70) (P = .97). However, individuals with non-nucleoside reverse transcriptase inhibitors-associated low-frequency DRMs were 2.68 times more likely to experience VF (odds ratio, 2.68; 95% confidential interval, 0.4-13.9) compared with those without (P = .22). Next-generation sequencing was able to detect low-frequency DRMs in this cohort in Botswana, but these DRMs did not contribute significantly to VF.

Validation of publicly-available software used in analyzing NGS data for HIV-1 drug resistance mutations and transmission networks in a Washington, DC, Cohort.

The DC Cohort is an ongoing longitudinal observational study of persons living with HIV. To better understand HIV-1 drug resistance and potential transmission clusters among these participants, we performed targeted, paired-end next-generation sequencing (NGS) of protease, reverse transcriptase and integrase amplicons. We elected to use free, publicly-available software (HyDRA Web, Stanford HIVdb and HIV-TRACE) for data analyses so that laboratory personnel without extensive bioinformatics expertise could use it; making the approach accessible and affordable for labs worldwide. With more laboratories transitioning away from Sanger-based chemistries to NGS platforms, lower frequency drug resistance mutations (DRMs) can be detected, yet their clinical relevance is uncertain. We looked at the impact choice in cutoff percentage had on number of DRMs detected and found an inverse correlation between the two. Longitudinal studies will be needed to determine whether low frequency DRMs are an early indicator of emerging resistance. We successfully validated this pipeline against a commercial pipeline, and another free, publicly-available pipeline. RT DRM results from HyDRA Web were compared to both SmartGene and PASeq Web; using the Mantel test, R2 values were 0.9332 (p<0.0001) and 0.9097 (p<0.0001), respectively. PR and IN DRM results from HyDRA Web were then compared with PASeq Web only; using the Mantel test, R2 values were 0.9993 (p<0.0001) and 0.9765 (p<0.0001), respectively. Drug resistance was highest for the NRTI drug class and lowest for the PI drug class in this cohort. RT DRM interpretation reports from this pipeline were also highly correlative compared to SmartGene pipeline; using the Spearman’s Correlation, rs value was 0.97757 (p<0.0001). HIV-TRACE was used to identify potential transmission clusters to better understand potential linkages among an urban cohort of persons living with HIV; more individuals were male, of black race, with an HIV risk factor of either MSM or High-risk Heterosexual. Common DRMs existed among individuals within a cluster. In summary, we validated a comprehensive, easy-to-use and affordable NGS approach for tracking HIV-1 drug resistance and identifying potential transmission clusters within the community.

HIV drug resistance: problems and perspectives

Access to combination antiretroviral treatment (ART) has improved greatly over recent years. At the end of 2011, more than eight million HIV-infected people were receiving ART in low-income and middle-income countries. ART generally works well in keeping the virus suppressed and the patient healthy. However, treatment only works as long as the virus is not resistant against the drugs used. In the last decades, HIV treatments have become better and better at slowing down the evolution of drug resistance, so that some patients are treated for many years without having any resistance problems. However, for some patients, especially in low-income countries, drug resistance is still a serious threat to their health. This essay will review what is known about transmitted and acquired drug resistance, multi-class drug resistance, resistance to newer drugs, resistance due to treatment for the prevention of mother-to-child transmission, the role of minority variants (low-frequency drug-resistance mutations), and resistance due to pre-exposure prophylaxis.

Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA

Background454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.ResultsWe constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions.ConclusionStandard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.

Low-Frequency HIV-1 Drug Resistance Mutations and Risk of NNRTI-Based Antiretroviral Treatment Failure

Presence of low-frequency, or minority, human immunodeficiency virus type 1 (HIV-1) drug resistance mutations may adversely affect response to antiretroviral treatment (ART), but evidence regarding the effects of such mutations on the effectiveness of first-line ART is conflicting. To evaluate the association of preexisting drug-resistant HIV-1 minority variants with risk of first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral virologic failure. Systematic review of published and unpublished studies in PubMed (1966 through December 2010), EMBASE (1974 through December 2010), conference abstracts, and article references. Authors of all studies were contacted for detailed laboratory, ART, and adherence data. Studies involving ART-naive participants initiating NNRTI-based regimens were included. Participants were included if all drugs in their ART regimen were fully active by standard HIV drug resistance testing. Cox proportional hazard models using pooled patient-level data were used to estimate the risk of virologic failure based on a Prentice weighted case-cohort analysis stratified by study. Individual data from 10 studies and 985 participants were available for the primary analysis. Low-frequency drug resistance mutations were detected in 187 participants, including 117 of 808 patients in the cohort studies. Low-frequency HIV-1 drug resistance mutations were associated with an increased risk of virologic failure (hazard ratio (HR], 2.3 [95% confidence interval {CI}, 1.7-3.3]; P < .001) after controlling for medication adherence, race/ethnicity, baseline CD4 cell count, and plasma HIV-1 RNA levels. Increased risk of virologic failure was most strongly associated with minority variants resistant to NNRTIs (HR, 2.6 [95% CI, 1.9-3.5]; P < .001). Among participants from the cohort studies, 35% of those with detectable minority variants experienced virologic failure compared with 15% of those without minority variants. The presence of minority variants was associated with 2.5 to 3 times the risk of virologic failure at either 95% or greater or less than 95% overall medication adherence. A dose-dependent increased risk of virologic failure was found in participants with a higher proportion or quantity of drug-resistant variants. In a pooled analysis, low-frequency HIV-1 drug resistance mutations, particularly involving NNRTI resistance, were significantly associated with a dose-dependent increased risk of virologic failure with first-line ART.

Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of approximately 0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population.