scholarly journals Limited Marginal Utility of Deep Sequencing for HIV Drug Resistance Testing in the Age of Integrase Inhibitors

2018 ◽  
Vol 56 (12) ◽  
Author(s):  
Ronit R. Dalmat ◽  
Negar Makhsous ◽  
Gregory G. Pepper ◽  
Amalia Magaret ◽  
Keith R. Jerome ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Virological Failure ◽  
Sanger Sequencing ◽  
Low Frequency ◽  
Resistance Testing ◽  
Antiviral Resistance ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing

ABSTRACTHIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect low-frequency alleles. However, the clinical value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping in 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high-frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers HyDRA and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low-frequency mutations (1 to 20% frequency) associated with higher levels of drug resistance in 30/105 (29%) protease-reverse transcriptase tests and 4/39 (10%) integrase tests. In clinical follow-up of 69 individuals for a median of 674 days, we did not find a difference in rates of virological failure between individuals with and without low-frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low-frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during the preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points, consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.

scholarly journals Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

2018 ◽  
Author(s):  
Ronit Dalmat ◽  
Negar Makhsous ◽  
Gregory Pepper ◽  
Amalia Magaret ◽  
Keith R. Jerome ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Virological Failure ◽  
Sanger Sequencing ◽  
Low Frequency ◽  
Resistance Testing ◽  
Antiviral Resistance ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Next Generation Sequencing Ngs

AbstractHIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.ImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

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

2015 ◽  
Vol 59 (11) ◽  
pp. 6824-6833 ◽  
Author(s):  
H. R. Lapointe ◽  
W. Dong ◽  
G. Q. Lee ◽  
D. R. Bangsberg ◽  
J. N. Martin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Sanger Sequencing ◽  
Illumina Miseq ◽  
Quality Data ◽  
Resistance Testing ◽  
Study Cohort ◽  
Miseq Sequencing ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing

ABSTRACTLimited 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 log10HIV 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.

HIV Drug Resistance Testing in a Resource Limited Setting with High Viral Diversity: The First Twenty Eight Months Experience

2017 ◽  
Vol 15 (4) ◽  
Author(s):  
Elodie Teclaire Ngo-Malabo ◽  
Paul Alain Ngoupo ◽  
Serge Alain Sadeuh-Mba ◽  
Emmanuel Akongnwi ◽  
Robert Banaï ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resource Limited Setting ◽  
Resistance Testing ◽  
Viral Diversity ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing ◽  
Resource Limited ◽  
Limited Setting

Clinically relevant thresholds for ultrasensitive HIV drug resistance testing: a multi-country nested case-control study

2018 ◽  
Vol 5 (11) ◽  
pp. e638-e646 ◽  
Author(s):  
Seth C Inzaule ◽  
Raph L Hamers ◽  
Marc Noguera-Julian ◽  
Maria Casadellà ◽  
Mariona Parera ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Case Control Study ◽  
Case Control ◽  
Resistance Testing ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing ◽  
Nested Case Control ◽  
Control Study

scholarly journals Evaluation of the HIV-1 drug resistance to elsulfavirine and the effectiveness of it among Russian treatment-naïve patients

2021 ◽  
Vol 12 (5) ◽  
pp. 29-39
Author(s):  
A. A. Kirichenko ◽  
D. E. Kireev ◽  
A. V. Kravchenko ◽  
A. V. Pokrovskaya ◽  
U. A. Kuimova ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Antiretroviral Therapy ◽  
Virological Failure ◽  
Resistance Testing ◽  
Study Group ◽  
Resistance Mutations ◽  
Drug Resistance Testing ◽  
The Third ◽  
Treatment Naïve ◽  
Hiv 1

The aim of the study: to analyze the prevalence of resistance mutations to elsulfavirine and to evaluate the effectiveness of it among HIV-infected treatment-naïve patients in real clinical practice.Materials and methods. The study included 578 patients with HIV infection, which divided into 3 groups. The first group is 354 HIV-infected treatment-naïve patients for whom HIV-1 nucleotide sequences were obtained as part of routine drug resistance testing. The second study group included 111 HIV-infected treatment-naïve patients, tested for drug resistance before the antiretroviral therapy containing elsulfavirine. The third study group included 113 HIV-infected treatment-naïve patients, each of whom was assigned a treatment regimen containing elsulfavirine without prior drug resistance testing. The observation period for patients of the second and third groups who received treatment was 24 weeks. To assess the effectiveness of antiretroviral therapy in patients, viral load, CD4+ T-cell counts, and adherence to therapy were assessed. HIV-1 subtypes and drug resistance mutations were determined using the Stanford HIV Resistance Database (v. 8.9-1). To clarify the results of subtyping, phylogenetic analysis of nucleotide sequences was carried out using the MEGA program (v. 6.0).Results. The prevalence of mutations associated with decreased susceptibility to elsulfavirine among HIV-infected treatment-naïve patients was 1.7% and 4.5% for the first and second groups of patients, respectively. All of the patients have only single resistance mutations which, according to the results of preclinical studies, cannot cause drug resistance. The use of elsulfavirine in real clinical practice among treatment-naïve patients has demonstrated good virological and immunological efficacy of the drug. As a result of 24 weeks of therapy in patients of the second group, no treatment ineffectiveness, and the development of drug resistance were observed. Among the patients of the third group, 6 patients (5.3%) have the virological failure of therapy associated with the resistance to the used drugs. All patients with virological failure had a resistance mutation profile associated with a high level of drug resistance to one of the drugs in the treatment regimen, lamivudine. Additionally, 1 patient had a combination of mutations that reduce susceptibility to elsulfavirine, and 4 patients had mutations that can reduce susceptibility to elsulfavirine in combination with other mutations.Conclusion. The low prevalence of mutations associated with a decrease in susceptibility to elsulfavirine and the absence of combinations of mutations make it possible to predict the successful use of this drug for Russian treatment-naïve patients. Reported cases of virological failure of antiretroviral therapy are difficult to interpret in the context of elsulfavirine due to the lack of an exact list of mutations and their combinations, and associations with the degree of resistance to it. This study describes for the first time the mutation profiles in patients with virological failure of therapy containing elsulfavirine and demonstrates the necessity of the further study of drug resistance profile to drug in vitro and in vivo.

scholarly journals Evidence of virological failure in patients on second-line anti-retroviral therapy in Southwestern Nigeria: An indication for HIV drug resistance testing

2021 ◽  
Vol 22 (3) ◽  
pp. 415-419
Author(s):  
S.O. Usman ◽  
O.M. Ajayi ◽  
O. Ebiekura ◽  
N. Egbonrelu ◽  
G. Ebhojie ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Viral Load ◽  
Virological Failure ◽  
Resistance Testing ◽  
Second Line ◽  
Southwestern Nigeria ◽  
Drug Resistance Testing ◽  
The Mean ◽  
Traitement Antirétroviral ◽  
Charge Virale

Background: In sub-Saharan Africa where genotypic anti-retroviral (ARV) drug resistance testing is rarely performed and poor adherence is blamed for the inability to achieve viral suppression and treatment failure, programmatic approaches to preventing and handling these are essential. This study was aimed at assessing the virological outcomes among HIV patients receiving second-line anti-retroviral therapy (ART) in Southwestern Nigeria.Methodology: This was a 5-year observational retrospective study of randomly selected people living with HIV (PLWHIV) who have been switched to second-line ART for at least six months before the commencement of the study in multiple comprehensive ART sites across the three levels of care, in Ondo and Ekiti States, Southwestern Nigeria, from January 2015 to December 2019. Quantitative viral load analysis was done using polymerase chain reaction (PCR) assay. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 24.0.Results: A total of 249 (71 males and 178 females) subjects eligible for the study were recruited using simple random sampling technique. The mean age (± SD) of the subjects was 44.21 ± 11.45 years. The mean number of years the patients have been on ART regimen was 7.92 ± 2.68 years. The mean number of years the patients were on first line ART regimen before being switched to second line was 4.27 ± 2.63 years. Patients with viral load <1000 RNA copies/ml (suppressed viral load) were 216 (86.7%) out of which 113 (45.4%) had viral load <20 RNA copies/ml while 33 (13.3%) had viral load >1000 RNA copies/ml (unsuppressed viral load or virological failure).Conclusion: About 13% of the patients on second line ART had unsuppressed viral load of more than 1000 RNA copies/ml indicating virological failure. Thus, critical factors such as poor adherence to ART and drug resistance chiefly contributing to virological failure have to be routinely checked. Keywords: suppression, ART, resistance, virological, failure, Nigeria   French title: Preuve d'échec virologique chez les patients sous traitement antirétroviral de deuxième intention dans le sud-ouest du Nigeria: une indication pour le test de résistance aux médicaments contre le VIH   Contexte: En Afrique subsaharienne, où les tests génotypiques de résistance aux antirétroviraux (ARV) sont rarement effectués et où une mauvaise observance est imputée à l'incapacité d'obtenir la suppression virale et l'échec du traitement, des approches programmatiques pour les prévenir et les gérer sont essentielles. Cette étude visait à évaluer les résultats virologiques chez les patients VIH recevant un traitement antirétroviral (TAR) de deuxième intention dans le sud-ouest du Nigeria. Méthodologie: Il s'agissait d'une étude rétrospective d'observation de 5 ans portant sur des personnes vivant avec le VIH (PVVIH) sélectionnées au hasard et passées à un TAR de deuxième intention pendant au moins six mois avant le début de l'étude dans plusieurs sites de TAR complets aux trois niveaux. de soins, dans les États d'Ondo et d'Ekiti, dans le sud-ouest du Nigéria, de janvier 2015 à décembre 2019. L'analyse quantitative de la charge virale a été effectuée à l'aide d'un test de réaction en chaîne par polymérase (PCR). Les données ont été analysées à l'aide du logiciel Paquet statistique pour les sciences sociales (SPSS) version 24.0. Résultats: Un total de 249 (71 hommes et 178 femmes) sujets éligibles à l'étude ont été recrutés à l'aide d'une technique  d'échantillonnage aléatoire simple. L'âge moyen (± ET) des sujets était de 44,21±11,45 ans. Le nombre moyen d'années pendant lesquelles les patients ont été sous traitement antirétroviral était de 7,92±2,68 ans. Le nombre moyen d'années pendant lesquelles les patients étaient sous traitement antirétroviral de première ligne avant de passer en deuxième ligne était de 4,27 ± 2,63 ans. Les patients avec une charge virale <1000 copies d'ARN/ml (charge virale supprimée) étaient 216 (86,7%) dont 113 (45,4%) avaient une charge virale <20 copies d'ARN/ml tandis que 33 (13,3%) avaient une charge virale >1000 ARN copies/ml (charge virale non supprimée ou échec virologique). Conclusion: Environ 13 % des patients sous TAR de deuxième ligne avaient une charge virale non supprimée de plus de 1000 copies d'ARN/ml indiquant un échec virologique. Ainsi, les facteurs critiques tels qu'une mauvaise adhésion au TARV et la résistance aux médicaments contribuant principalement à l'échec virologique doivent être systématiquement vérifiés. Mots clés: suppression, TAR, résistance, virologique, échec, Nigeria  

scholarly journals PhyloPi: An affordable, purpose built phylogenetic pipeline for the HIV drug resistance testing facility

PLoS ONE ◽  
2019 ◽  
Vol 14 (3) ◽  
pp. e0213241
Author(s):  
Phillip Armand Bester ◽  
Andrie De Vries ◽  
Stephanus Riekert ◽  
Kim Steegen ◽  
Gert van Zyl ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Testing ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing ◽  
Testing Facility
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