Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

Johnson, Jeffrey A.; Jin-fen, LI; Wei, Xierong; Lipscomb, Jonathan; Bennett, Diane; Brant, A. W.; Cong, Mian‐er; Spira, Thomas J.; Shafer, Robert W.; Heneine, Walid

doi:10.1371/journal.pone.0000638

Cited by 67 publications

(80 citation statements)

References 17 publications

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“…The fitness cost of individual RT resistance mutations is well established, particularly for 215Y, 184V, and 65R (5). The transmission of any of the resistant variants more frequently associated with a fitness impairment might be associated with a more rapid reversion to a more "fit," WT genotype that might fail to be detected by conventional sequencing (17). Further studies are needed to determine whether these traditionally less fit variants are uniformly replaced at a significantly higher rate than other variants associated with higher RC values.…”

Section: Discussionmentioning

confidence: 99%

“…The spontaneous appearance of drug resistance mutations in 3 of 14 patients (01-0512, 01-0550, and 01-0575) in the absence of selective drug pressure was most likely related to the presence of these particular resistant variants as relatively minor populations at the time of transmission, below the threshold of assay detection. More sensitive real-time PCR methods for detecting lowfrequency minor variants among treatment-naive individuals have shown that resistant variants identified by real-time PCR and missed by conventional sequencing may represent 0.7% to 11% of the population by clonal sequencing (17). Potential selective advantages may have resulted in the emergence of these resistant isolates over time to detectable levels despite the absence of selective drug pressure.…”

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Persistence of Transmitted Drug Resistance among Subjects with Primary Human Immunodeficiency Virus Infection

Little¹,

Frost

Wong

et al. 2008

J Virol

198

179

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Following interruption of antiretroviral therapy among individuals with acquired drug resistance, preexisting drug-sensitive virus emerges relatively rapidly. In contrast, wild-type virus is not archived in individuals infected with drug-resistant human immunodeficiency virus (HIV) and thus cannot emerge rapidly in the absence of selective drug pressure. Fourteen recently HIV-infected patients with transmitted drug-resistant virus were followed for a median of 2.1 years after the estimated date of infection (EDI) without receiving antiretroviral therapy. HIV drug resistance and pol replication capacity (RC) in longitudinal plasma samples were assayed. Resistance mutations were characterized as pure populations or mixtures. The mean time to first detection of a mixture of wild-type and drug-resistant viruses was 96 weeks (1.8 years) (95% confidence interval, 48 to 192 weeks) after the EDI. The median time to loss of detectable drug resistance using population-based assays ranged from 4.1 years (conservative estimate) to longer than the lifetime of the individual (less conservative estimate). The transmission of drugresistant virus was not associated with virus with reduced RC. Sexual transmission of HIV selects for highly fit drug-resistant variants that persist for years. The prolonged persistence of transmitted drug resistance strongly supports the routine use of HIV resistance genotyping for all newly diagnosed individuals.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Persistence of Transmitted Drug Resistance among Subjects with Primary Human Immunodeficiency Virus Infection

Little¹,

Frost

Wong

et al. 2008

J Virol

198

179

View full text Add to dashboard Cite

show abstract

“…Various ligation-mediated methods were consequently developed to detect point mutations in infectious and noninfectious circumstances, including Oligonucleotide Ligation Assay 14,15 and Ligation Amplification assay. 16,17 Other quantitative PCR-based detection techniques, such as allele-specific PCR (ASPCR) 18 and variations of multiplexing PCR assays, 19 depend on the retarded activity of DNA polymerase to extend on templates when there is a primer mismatch at the 3 0 end. However, all ligation-mediated methods and ASPCR are DNA based, requiring initial reverse transcription of RNA to cDNA.…”

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One-Step Ligation on RNA Amplification for the Detection of Point Mutations

Wang

Coetzer

Angione

et al. 2015

The Journal of Molecular Diagnostics

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The detection of point mutations is required in the diagnosis of many human diseases. The conformal specificity of DNA ligases was elegantly used to distinguish single-nucleotide mismatches. However, to detect point mutations in RNA retroviruses, conventional ligase-mediated approaches require the reverse transcription of viral genomes before separate ligation and amplification steps. We developed one-step ligation on RNA amplification (LRA) for the direct detection of RNA point mutations. The process combines the ligase-mediated joining of two oligonucleotides and subsequent hot start amplification into a singletube reaction. We report that modifications to the structure of the oligonucleotide ligation probes improve the rate of ligation and the specificity of mutation detection on RNA. We applied LRA to the detection of a common, clinically relevant HIV-1 reverse transcriptase drug-resistant point mutation, K103N, and compared it with allele-specific PCR and pyrosequencing. LRA achieved a limit of specific quantitation of 1:100 (1%), and a limit of specific detection for mutant K103N RNA transcripts among excess wild-type strands of 1:10,000 (0.01%). LRA also exhibited good detection threshold of 5 Â 10 2 copies/mL K103N RNA transcripts. LRA is a novel point mutation detection method, with potential utilization in HIV drug resistance detection and early diagnostics of genetic disorders associated with other infectious diseases and cancer. (J Mol Diagn 2015, 17: 679e688; http://dx

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“…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 genome sequencing (SGS) and ultradeep sequencing (UDS), permit analyses of DRM in the context of the surrounding genetic sequence and this allows the investigation of accessory mutations (AM) that are often associated with DRM during DRM selection (50).…”

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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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Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

Cited by 67 publications

References 17 publications

Persistence of Transmitted Drug Resistance among Subjects with Primary Human Immunodeficiency Virus Infection

Persistence of Transmitted Drug Resistance among Subjects with Primary Human Immunodeficiency Virus Infection

One-Step Ligation on RNA Amplification for the Detection of Point Mutations

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

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