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Pretreatment drug resistance in a large countrywide Ethiopian HIV-1C cohort: a comparison of Sanger and high-throughput sequencing.

ABSTRACT: Baseline plasma samples of 490 randomly selected antiretroviral therapy (ART) naïve patients from seven hospitals participating in the first nationwide Ethiopian HIV-1 cohort were analysed for surveillance drug resistance mutations (sDRM) by population based Sanger sequencing (PBSS). Also next generation sequencing (NGS) was used in a subset of 109 baseline samples of patients. Treatment outcome after 6- and 12-months was assessed by on-treatment (OT) and intention-to-treat (ITT) analyses. Transmitted drug resistance (TDR) was detected in 3.9% (18/461) of successfully sequenced samples by PBSS. However, NGS detected sDRM more often (24%; 26/109) than PBSS (6%; 7/109) (p?=?0.0001) and major integrase strand transfer inhibitors (INSTI) DRMs were also found in minor viral variants from five patients. Patients with sDRM had more frequent treatment failure in both OT and ITT analyses. The high rate of TDR by NGS and the identification of preexisting INSTI DRMs in minor wild-type HIV-1 subtype C viral variants infected Ethiopian patients underscores the importance of TDR surveillance in low- and middle-income countries and shows added value of high-throughput NGS in such studies.

SUBMITTER: Telele NF

PROVIDER: S-EPMC5954158 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Pretreatment drug resistance in a large countrywide Ethiopian HIV-1C cohort: a comparison of Sanger and high-throughput sequencing.

Telele Nigus Fikrie NF Kalu Amare Worku AW Gebre-Selassie Solomon S Fekade Daniel D Abdurahman Samir S Marrone Gaetano G Neogi Ujjwal U Tegbaru Belete B Sönnerborg Anders A

Scientific reports 20180515 1

Baseline plasma samples of 490 randomly selected antiretroviral therapy (ART) naïve patients from seven hospitals participating in the first nationwide Ethiopian HIV-1 cohort were analysed for surveillance drug resistance mutations (sDRM) by population based Sanger sequencing (PBSS). Also next generation sequencing (NGS) was used in a subset of 109 baseline samples of patients. Treatment outcome after 6- and 12-months was assessed by on-treatment (OT) and intention-to-treat (ITT) analyses. Trans ...[more]

PMID: 29765082

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Project description:BackgroundThe emergence, accumulation and spread of HIV-1 drug resistance strains in Africa could compromise the effectiveness of HIV treatment programs. This study was aimed at determining the incidence of virological failure and acquired drug resistance mutations overtime and identifying the most common mutational pathways of resistance in a well characterized HIV-1C infected Ethiopian cohort.MethodsA total of 320 patients (220 ART naïve and 100 on first lines ART) were included and followed. ART initiation and patients' monitoring was based on the WHO clinical and immunological parameters. HIV viral load measurement and genotypic drug resistance testing were done at baseline (T0-2008) and after on average at a median time of 30 months on ART at three time points (T1-2011, T2-2013, T3-2015).FindingsThe incidence of virological failure has increased overtime from 11 at T1 to 17 at T2 and then to 30% at T3. At all time point's almost all of the patients with virological failure and accumulated drug resistance mutations had not met the WHO clinical and immunologic failure criteria and continued the failing regimen. A steep increase in the incidence and accumulation of major acquired NRTI and NNRTI drug resistance mutations have been observed (from 40% at T1 to 64% at T2 and then to 66% at T3). The most frequent NRTIs drug resistance associated mutations are mainly the lamivudine-induced mutation M184V which was detected in 4 patients at T1 and showed a 2 fold increase in the following time points (T2: n = 8) and at (T3: n = 12) and the thymidine analogue mutations (such as D67N, K70R and K219E) which were not-detected at baseline T0 and T1 but were increased progressively to 10 at T2 and to 17 at T3. The most frequent NNRTIs associated mutations were K103N, V106M and Y188C.ConclusionsAn upward trend in the incidence of virological failure and accumulation of NRTI and NNRTI associated acquired antiretroviral drug resistance mutations are observed. The data suggest the need for virological monitoring, resistance testing for early detection of failure and access for TDF and PI containing drugs. Population-level and patient targeted interventions to prevent the spread of mutant variants is warranted.

Project description:Next-generation sequencing (NGS)'s crucial role in supporting genetic diagnosis and personalized medicine leads to the definition of Guidelines for Diagnostic NGS by the European Society of Human Genetics. Factors of different nature producing false-positive/negative NGS data together with the paucity of internationally accepted guidelines providing specified NGS quality metrics to be followed for diagnostics purpose made the Sanger validation of NGS variants still mandatory. We reported the analysis of three cases of discrepancy between NGS and Sanger sequencing in a cohort of 218 patients. NGS was performed by Illumina MiSeq® and Haloplex/SureSelect protocols targeting 97 or 57 or 10 gene panels usually applied for diagnostics. Variants called following guidelines suggested by the Broad Institute and identified according to MAF <0.01 and allele balance >0.2 were Sanger validated. Three out of 945 validated variants showed a discrepancy between NGS and Sanger. In all three cases, a deep evaluation of the discrepant gene variant results and methodological approach allowed to confirm the NGS datum. Allelic dropout (ADO) occurrence during polymerase chain or sequencing reaction was observed, mainly related to incorrect variant zygosity. Our study extends literature data in which almost 100% "high quality" NGS variants are confirmed by Sanger; moreover, it demonstrates that in case of discrepancy between a high-quality NGS variant and Sanger validation, NGS call should not be a priori assumed to represent the source of the error. Actually, difficulties (i.e., ADO, unpredictable presence of private variants on primer-binding regions) of the so-called gold standard direct sequencing should be considered especially in light of the constantly implemented and accurate high-throughput technologies. Our data along with literature raise a discussion on the opportunity to establish a standardized quality threshold by International Guidelines for clinical NGS in order to limit Sanger confirmation to borderline conditions of variant quality parameters and verification of correct gene variant call/patient coupling on a different blood sample aliquot.

Dataset Information

Pretreatment drug resistance in a large countrywide Ethiopian HIV-1C cohort: a comparison of Sanger and high-throughput sequencing.

Publications

Pretreatment drug resistance in a large countrywide Ethiopian HIV-1C cohort: a comparison of Sanger and high-throughput sequencing.

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