Performance of HIV-1 Drug Resistance Testing at Low-Level Viremia and Its Ability to Predict Future Virologic Outcomes and Viral Evolution in Treatment-Naive Individuals

González‐Serna, Alejandro; Min, Jeong Eun; Woods, Conan K.; Chan, David Yiu Leung; Lima, VD; Montaner, Julio S. G.; Harrigan, P. Richard; Swenson, Luke C.

doi:10.1093/cid/ciu019

Cited by 85 publications

(73 citation statements)

References 31 publications

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“…For example, the Sanger assay was able to amplify and to sequence successfully only ϳ40% of samples with pVLs of Ͻ1,000 copies/ml in this experiment. In contrast, our laboratory typically achieves a Ͼ85% success rate for samples with low pVLs if samples that initially fail to produce a PCR product are retested with a backup protocol (34). Furthermore, the sequencing success rates for the internal controls (pNL4-3 and POS08) were comparable to those of the clinical samples for both the Sanger (86%) and MiSeq (93%) assays.…”

Section: Discussionmentioning

confidence: 97%

“…Depending on the sample source, one of three PCR products was produced by nested PCR. Briefly, an amplicon spanning complete protease and RT codons 1 to 400 was produced for Canadian samples (34). In cases in which amplification of Canadian samples failed, backup amplification of a product covering protease and RT codons 1 to 240 was attempted (34).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, an amplicon spanning complete protease and RT codons 1 to 400 was produced for Canadian samples (34). In cases in which amplification of Canadian samples failed, backup amplification of a product covering protease and RT codons 1 to 240 was attempted (34). For UARTO samples, a smaller amplicon, spanning RT codons 35 to 234, was generated using PCR primers designed to target regions conserved among HIV-1 subtypes A, B, C, and D. PCR primers are listed in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

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HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Lapointe

Dong

Lee

et al. 2015

Antimicrob Agents Chemother

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f Limited access to HIV drug resistance testing in low-and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n ‫؍‬ 759) and from a Ugandan study cohort (n ‫؍‬ 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log 10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.A dvances in highly active antiretroviral therapy (HAART) in recent decades have resulted in sustained decreases in HIVrelated morbidity and mortality rates. HIV-infected individuals who receive treatment now have nearly normal life expectancies, such that HIV is now considered a manageable chronic disease (1, 2); antiretroviral therapy (ART) not only provides benefits at the individual patient level but also results in a population-level advantage through HAART-induced suppression of HIV replication and the inherent prevention of onward transmission of the virus, termed "treatment as prevention" (3-6).Drug resistance testing is an essential complement to HAART, enabling clinicians to identify patients infected with drug-resistant HIV and to prescribe the appropriate antiretroviral regimens (7). Lack of access to HIV drug resistance testing acts as a major barrier to long-term treatment success, either through prescription of ineffective regimens in the case of transmitted resistance or through decreased ability of physicians to identify causes of treatment failure (7-9). Cases of unsuppressed viremia allow continued transmission and can compromise the management of HIV on both the patient and population levels. These challenges are particula...

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Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Lapointe

Dong

Lee

et al. 2015

Antimicrob Agents Chemother

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“…Reporting based on a more sensitive assay can potentially have significant repercussions on pharmacy costs and clinical outcomes. The clinical relevance of detecting these variants in HBV is still unclear (20)(21)(22), but experience in resistance testing for HIV patients with low viral loads (and rare mutations) has suggested that they may have long-term clinical impacts (23). With increased adoption of NGS for clinical applications, further study is required to understand the potential clinical impact of the additional information identified by NGS, such as low-level mutant subpopulations.…”

Section: Discussionmentioning

confidence: 99%

Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory

et al. 2016

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“…When economically feasible, resistance markers can be used to identify resistance before treatment and drugs chosen accordingly (personalized medicine). This is already the standard practice in the treatment of various bacterial 35,36 and HIV infections 37,38 in many countries, and could conceivably become so in malaria, at least in rich country or military settings. We contend that a fundamental challenge for this sort of personalized medicine will ultimately prove to be the amount of pathogen biomass that can be sampled and the ability of the molecular assay to detect very rare genotypes in that sample.…”

Section: Discussionmentioning

confidence: 99%

Relevance of Undetectably Rare Resistant Malaria Parasites in Treatment Failure: Experimental Evidence from Plasmodium chabaudi

Huijben

Chan

Read

2015

The American Society of Tropical Medicine and Hygiene

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Abstract. Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants can be extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance.

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Performance of HIV-1 Drug Resistance Testing at Low-Level Viremia and Its Ability to Predict Future Virologic Outcomes and Viral Evolution in Treatment-Naive Individuals

Abstract: Routine HIV-1 genotyping of LLV samples can be performed with a reasonably high success rate, and the results appear predictive of future virologic outcomes.

Cited by 85 publications

References 31 publications

HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory

Relevance of Undetectably Rare Resistant Malaria Parasites in Treatment Failure: Experimental Evidence from Plasmodium chabaudi

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