Prevalence and Clinical Significance of HIV Drug Resistance Mutations by Ultra-Deep Sequencing in Antiretroviral-Naïve Subjects in the CASTLE Study

Lataillade, Max; Chiarella, Jennifer; Yang, Rong; Schnittman, Steven M.; Wirtz, Victoria; Uy, Jonathan; Seekins, Daniel; Krystal, Mark; Mancini, Marco; McGrath, Donnie; Simen, Birgitte B.; Egholm, Michael; Kozal, Michael J.

doi:10.1371/journal.pone.0010952

Cited by 114 publications

(103 citation statements)

References 27 publications

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“…According to the SPREAD Program, subtype B infection was strongly associated with transmitted drug resistance (23). However, some studies regarding primary resistance in ART-naive subjects determined that the B and non-B HIV-1 subtypes had a similar overall infection rate (25)(26)(27). Therefore, future prospective studies are warranted to determine the prevalence of primary antiretroviral resistance in different HIV-1 subtypes in Turkey.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 Subtypes and Primary Antiretroviral Resistance Mutations in Antiretroviral Therapy Naive HIV-1 Infected Individuals in Turkey

et al. 2013

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Section: Discussionmentioning

confidence: 99%

HIV-1 Subtypes and Primary Antiretroviral Resistance Mutations in Antiretroviral Therapy Naive HIV-1 Infected Individuals in Turkey

et al. 2013

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“…Using more-sensitive genotypic assays, different research groups (15,30,32,37,48,58,65) have reported higher proportions of transmitted DRM in ART-naive individuals. The clinical importance of these low-level DRM remains unclear, as they have been associated with clinical consequences in some (21,24,32,36,37,40,46,54,55,63,66,71) but not all (30, 47, 58) studies.Highly sensitive assays for detecting low-frequency DRM include point mutation assays and high-resolution sequencing techniques. Point mutation assays, such as allele-specific PCR, can detect DRM at frequencies as low as 0.01% of the sampled viral population (31,45,53,54), but they do not provide information about the sequence context surrounding a given DRM and may be prone to false positives at the lower level of detection (22).…”

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confidence: 99%

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Gianella

Delport

Pacold

et al. 2011

J Virol

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Reports of a high frequency of the transmission of minority viral populations with drug-resistant mutations (DRM) are inconsistent with evidence that HIV-1 infections usually arise from mono-or oligoclonal transmission. We performed ultradeep sequencing (UDS) of partial HIV-1 gag, pol, and env genes from 32 recently infected individuals. We then evaluated overall and per-site diversity levels, selective pressure, sequence reproducibility, and presence of DRM and accessory mutations (AM). To differentiate biologically meaningful mutations from those caused by methodological errors, we obtained multinomial confidence intervals (CI) for the proportion of DRM at each site and fitted a binomial mixture model to determine background error rates for each sample. We then examined the association between detected minority DRM and the virologic failure of first-line antiretroviral therapy (ART). Similar to other studies, we observed increased detection of DRM at low frequencies (average, 0.56%; 95% CI, 0.43 to 0.69; expected UDS error, 0.21 ؎ 0.08% mutations/site). For 8 duplicate runs, there was variability in the proportions of minority DRM. There was no indication of increased diversity or selection at DRM sites compared to other sites and no association between minority DRM and AM. There was no correlation between detected minority DRM and clinical failure of first-line ART. It is unlikely that minority viral variants harboring DRM are transmitted and maintained in the recipient host. The majority of low-frequency DRM detected using UDS are likely errors inherent to UDS methodology or a consequence of error-prone HIV-1 replication.Using standard population-based genotypic assays of HIV from individuals not yet treated with antiretroviral drugs, several studies (43,64,69,76) have estimated the rate of transmitted drug resistance to be between 8 and 27% in countries with the highest rates of antiretroviral therapy (ART) use. In resource-limited settings, in which the introduction of ART has been more recent, the estimated frequency of transmitted drug resistance mutations (DRM) is substantially lower (41). It appears, however, to be increasing in these settings as well (2,14,51). Using more-sensitive genotypic assays, different research groups (15,30,32,37,48,58,65) have reported higher proportions of transmitted DRM in ART-naive individuals. The clinical importance of these low-level DRM remains unclear, as they have been associated with clinical consequences in some (21,24,32,36,37,40,46,54,55,63,66,71) but not all (30, 47, 58) studies.Highly sensitive assays for detecting low-frequency DRM include point mutation assays and high-resolution sequencing techniques. Point mutation assays, such as allele-specific PCR, can detect DRM at frequencies as low as 0.01% of the sampled viral population (31,45,53,54), but they do not provide information about the sequence context surrounding a given DRM and may be prone to false positives at the lower level of detection (22). High-resolution sequencing techniques, such as single ge...

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“…HCV has already been reported to be a highly variable virus with a quasispecies distribution, large viral populations, and very rapid turnover in individual patients (6,26). Previous studies using metagenomic sequencing of other viruses from human clinical samples mostly employed pyrosequencing (11,12,23,30,46). The longer reads from pyrosequencing (250 to 450 bp) facilitate the assembly of individual reads into contigs, which facilitates the classification of the sequence data by homology-based BLAST alignment.…”

Section: Discussionmentioning

confidence: 99%

“…In recent years, new technologies have been developed that are able to sequence viruses from environmental samples without using specific primers, cloning, and resorting by recombinant DNA techniques and thus can obtain the sequence information for the complete virome in an unbiased way. Metagenomic approaches such as deep sequencing have proven increasingly successful at identifying variants or mutations of the nucleotide sequence (23,42,45). …”

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confidence: 99%

Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants

et al. 2012

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Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus that shows diverse viral populations even in one individual. Though Sanger sequencing has been used to determine viral sequences, deep sequencing technologies are much faster and can perform large-scale sequencing. We demonstrate the successful use of Illumina deep sequencing technology and subsequent analyses to determine the genetic variants and amino acid substitutions in both treatment-naïve (patient 1) and treatmentexperienced (patient 7) isolates from HCV-infected patients. As a result, almost the full nucleotide sequence of HCV was detectable for patients 1 and 7. The reads were mapped to the HCV reference sequence. The coverage was 99.8% and the average depth was 69.5؋ for patient 7, with values of 99.4% (coverage) and 51.1؋ (average depth) for patient 1. In patient 7, amino acid (aa) 70 in the core region showed arginine, with methionine at aa 91, by Sanger sequencing. Major variants showed the same amino acid sequence, but minor variants were detectable in 18% ( . The single open reading frame encodes a polyprotein of 3,011 amino acids (aa) that is cleaved by viral and cellular proteases into 10 different proteins. The three structural proteins, which constitute the viral particle, include the core protein and the envelope glycoproteins E1 and E2. Two regions in E2, known as hypervariable regions 1 and 2, are reported to have extreme sequence variability. The seven nonstructural components include the p7 polypeptide, the NS2-3 protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide, the NA4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (RdRp) (29). At both ends of the open reading frame lie the 5=-and 3=-untranslated regions (5=-UTR and 3=-UTR). The nucleotide sequence of the 5=-UTR is relatively well conserved among different HCV genotypes. The HCV 5=-UTR contains an internal ribosome entry site (IRES) that directs the cap-independent initiation of virus translation and forms on four characteristic stem-loop structures (17, 18). HCV displays very high genetic variability both in populations and within infected individuals, where it exists as a cluster of closely related but distinct variants, termed "quasispecies," as occurs in many other RNA viruses with a polymerase enzyme lacking proofreading ability (6,8,26).Current standard treatment of chronic HCV infection is based on the combination of pegylated alpha interferon (peg-IFN-␣) and ribavirin (RBV). However, patients with a high load of genotype 1b virus (Ͼ1 ϫ 10 5 log IU/ml) do not achieve high sustained virological response (SVR) rates (Ͻ50%), even when the most effective combination treatment (IFN plus RBV) is administered for 48 weeks (14,25). Some investigations concerning therapeutic prediction based on virological features revealed that substitutions of arginine for glutamine at amino acid (aa) 70 and/or leucine for methionine at aa 91 in the core region are independent and significant factors associated with SVR or that patients whose viruses ...

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Prevalence and Clinical Significance of HIV Drug Resistance Mutations by Ultra-Deep Sequencing in Antiretroviral-Naïve Subjects in the CASTLE Study

Cited by 114 publications

References 27 publications

HIV-1 Subtypes and Primary Antiretroviral Resistance Mutations in Antiretroviral Therapy Naive HIV-1 Infected Individuals in Turkey

HIV-1 Subtypes and Primary Antiretroviral Resistance Mutations in Antiretroviral Therapy Naive HIV-1 Infected Individuals in Turkey

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants

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