Dataset Information

Detection of cytomegalovirus drug resistance mutations by next-generation sequencing.

ABSTRACT: Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations.

SUBMITTER: Sahoo MK

PROVIDER: S-EPMC3889754 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Detection of cytomegalovirus drug resistance mutations by next-generation sequencing.

Sahoo Malaya K MK Lefterova Martina I MI Yamamoto Fumiko F Waggoner Jesse J JJ Chou Sunwen S Holmes Susan P SP Anderson Matthew W MW Pinsky Benjamin A BA

Journal of clinical microbiology 20130828 11

Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for ...[more]

PMID: 23985916

Similar Datasets

Project description:Patients with antiretroviral therapy interruption have a high risk of virological failure when re-initiating antiretroviral therapy (ART), especially those with HIV drug resistance. Next-generation sequencing may provide close scrutiny on their minority drug resistance variant. A cross-sectional study was conducted in patients with ART interruption in five regions in China in 2016. Through Sanger and next-generation sequencing in parallel, HIV drug resistance was genotyped on their plasma samples. Rates of HIV drug resistance were compared by the McNemar tests. In total, 174 patients were included in this study, with a median 12 (interquartile range (IQR), 6-24) months of ART interruption. Most (86.2%) of them had received efavirenz (EFV)/nevirapine (NVP)-based first-line therapy for a median 16 (IQR, 7-26) months before ART interruption. Sixty-one (35.1%) patients had CRF07_BC HIV-1 strains, 58 (33.3%) CRF08_BC and 35 (20.1%) CRF01_AE. Thirty-four (19.5%) of the 174 patients were detected to harbor HIV drug-resistant variants on Sanger sequencing. Thirty-six (20.7%), 37 (21.3%), 42 (24.1%), 79 (45.4%) and 139 (79.9) patients were identified to have HIV drug resistance by next-generation sequencing at 20% (v.s. Sanger, p = 0.317), 10% (v.s. Sanger, p = 0.180), 5% (v.s. Sanger, p = 0.011), 2% (v.s. Sanger, p < 0.001) and 1% (v.s. Sanger, p < 0.001) of detection thresholds, respectively. K65R was the most common minority mutation, of 95.1% (58/61) and 93.1% (54/58) in CRF07_BC and CRF08_BC, respectively, when compared with 5.7% (2/35) in CRF01_AE (p < 0.001). In 49 patients that followed-up a median 10 months later, HIV drug resistance mutations at >20% frequency such as K103N, M184VI and P225H still existed, but with decreased frequencies. The prevalence of HIV drug resistance in ART interruption was higher than 15% in the survey. Next-generation sequencing was able to detect more minority drug resistance variants than Sanger. There was a sharp increase in minority drug resistance variants when the detection threshold was below 5%.

Project description:IntroductionTransmitted drug-resistance mutations (TDRM) may hamper successful anti-HIV-1 therapy and impact future control of the HIV-1 epidemic. Recently infected, therapy-naïve individuals are best suited for surveillance of such TDRM. In this study, TDRM, detected by next-generation sequencing (NGS) were compared to those identified by Sanger-based population sequencing (SBS) in recently infected HIV-1 patients.MethodsHistorical samples from 80 recently infected HIV-1 patients, diagnosed between 2000 and 2014, were analysed by MiSeq (NGS) and ABI (SBS). DeepChek-HIV (ABL) was used for interpretation of the results.ResultsMost patients were males (80%); Men who have sex with men (MSM) was the major transmission group (58.8%). Overall, TDRM were detected in 31.3% of patients by NGS and 8.8% by SBS, with SBS TDRM restricted to persons infected with subtype B. All SBS-detected TDRM were identified by NGS. The prevalence of TDRM impacting protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) was 11.3, 26.2 7.5%, respectively, in NGS analyses and 0, 3.8 and 5%, respectively, in SBS analyses. More patients with NGS and SBS TDRM were identified in 2008-2014 (37.2% or 13.9%, respectively) compared to 2000-2007 (24.3% or 2.7%, respectively), and a significantly greater number of these patients had multiple NGS TDRM. The most abundant, albeit, minor-frequency RT TDRM, were the K65R and D67N, while K103N, M184V and T215S were high-frequency mutations. Minor TDRM did not become a major variant in later samples and did not hinder successful treatment.ConclusionsNGS can replace SBS for mutation detection and allows for the detection of low-frequency TDRM not identified by SBS. Although rates of TDRM in Israel continued to increase from 2000 to 2014, minor TDRM did not become major species. The need for ongoing surveillance of low-frequency TDRM should be revisited in a larger study.

Dataset Information

Detection of cytomegalovirus drug resistance mutations by next-generation sequencing.

Publications

Detection of cytomegalovirus drug resistance mutations by next-generation sequencing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets