A16 Next-generation sequencing to detect transmitted drug resistance mutations in Romanian people who inject drugs (original) (raw)
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Journal of the International AIDS Society, 2017
Transmitted 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. Historical 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. Most 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...
Journal of the International Association of Providers of AIDS Care (JIAPAC)
Introduction: With the rapid scale-up of antiretroviral therapy (ART) to treat HIV infection, there are ongoing concerns regarding probable emergence and transmission of HIV drug resistance (HIVDR) mutations. This scale-up has to lead to an increased need for routine HIVDR testing to inform the clinical decision on a regimen switch. Although the majority of wet laboratory processes are standardized, slow, labor-intensive data transfer and subjective manual sequence interpretation steps are still required to finalize and release patient results. We thus set out to validate the applicability of a software package to generate HIVDR patient results from raw sequence data independently. Methods: We assessed the performance characteristics of Hyrax Bioscience’s Exatype (a sequence data to patient result, fully automated sequence analysis software, which consolidates RECall, MEGA X and the Stanford HIV database) against the standard method (RECall and Stanford database). Exatype is a web-b...
Genotyping in HIV drug resistance mutations: epidemiology in 145 patients
International Journal of Antimicrobial Agents, 2000
The epidemiology of HIV-1 genomic mutations causing antiretroviral drug resistance, in 145 HIV-1 infected patients were obtained using a new sequencing technique. All patients were in a follow up treatment for at least 4 months with all drugs available in clinical practice in accordance to international guidelines. The percentage of the mutations were calculated both on the number of all participants and on the number of patients who received the drugs selecting for that specific resistance. It was possible then to evaluate patients who showed the mutation without receiving the drug. We consider this new sequencing method reliable and hopeful in clinical practice, giving the opportunity for a guided therapy for the single patient and in detecting and monitoring the antiretroviral drug resistance mutations in the pertinent geographic area following a periodic surveillance program.
Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting
Viruses
The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As exp...
Introduction: With the rapid scale-up of antiretroviral therapy (ART) to treat HIV infection, there are ongoing concerns regarding probable emergence and transmission of HIV drug resistance (HIVDR) mutations. This scaleup has to lead to an increased need for routine HIVDR testing to inform the clinical decision on a regimen switch. Although the majority of wet laboratory processes are standardized, slow, labor intensive data transfer and subjective manual sequence interpretation steps are still required to finalize and release patient results. We thus set out to validate the applicability of a software package to generate HIVDR patient results from raw sequence data independently. Methods: We assessed the performance characteristics of Hyrax Bioscience Exatype (a sequence data to patient result, fully automated sequence analysis software, which consolidates RECall, MEGA X and the Stanford HIV database) against the standard method (RECall and Stanford database). Exatype is a web-base...
HIV-1 Drug Resistance Genotyping
PharmacoEconomics, 2000
Conventional HIV drug resistance (HIVDR) genotyping utilizes sanger sequencing (ss) methods, which are limited by low data throughput and the inability of detecting low abundant drug resistant variants (LADRVs). Here we present a next generation sequencing (NGs)-based HIVDR typing platform that leverages the advantages of Illumina Miseq and HyDRA Web. the platform consists of a fully validated sample processing protocol and HyDRA web, an open web portal that allows automated customizable NGs-based HIVDR data processing. this platform was characterized and validated using a panel of HIVspiked plasma representing all major HIV-1 subtypes, pedigreed plasmids, HIVDR proficiency specimens and clinical specimens. All examined major HIV-1 subtypes were consistently amplified at viral loads of ≥1,000 copies/ml. The gross error rate of this platform was determined at 0.21%, and minor variations were reliably detected down to 0.50% in plasmid mixtures. All HIVDR mutations identifiable by SS were detected by the MiSeq-HyDRA protocol, while LADRVs at frequencies of 1~15% were detected by Miseq-HyDRA only. As compared to ss approaches, the Miseq-HyDRA platform has several notable advantages including reduced cost and labour, and increased sensitivity for LADRVs, making it suitable for routine HIVDR monitoring for both patient care and surveillance purposes. Successful antiretroviral therapy (ART) serves to suppress HIV viral load (VL), reduce the incidence of new infections, and increase the life expectancy of infected individuals 1,2. HIV-infected patients adhering to a prescribed ART regimen now have close to normal life expectancies and maintain viral suppression at levels that mitigate transmission of the virus 3. However, HIV drug resistance (HIVDR) can emerge as a result of poor proofreading during HIV replication and/or unsuccessful ART due to ineffective ART regimens, poor adherence to treatment, or prolonged usage of a failing regimen 4. The presence of drug resistance (DR) variants, including low-abundance drug-resistant variants (LADRVs), can significantly reduce ART efficacy and present a risk for treatment failure 5-10. At the population level, transmitted HIVDR variants can have a considerable impact on the effectiveness of currently available antiretroviral drugs. Effective HIVDR monitoring and surveillance programs are essential for informing clinical care of individual patients and for strategic ART recommendations at population levels 4,11. Conventional HIVDR genotyping employs Sanger sequencing (SS) technology, which reliably detects DR mutations present at frequencies of ≥20% in HIV quasispecies 6,10. However, SS is limited by its low throughput, failure to resolve codons with multiple mixed bases, and limited ability to accurately detect variants present at frequencies below 20% 12. Recently, there has been an increased focus on the clinical significance of LADRVs on the effectiveness of ART 10,13. It has been shown previously that patients with treatment failure had been infected with viruses that harboured pre-existing drug resistance variants ranging from 0.07 to 2.0%, as measured by allele-specific real-time polymerase chain reaction 14. Furthermore, Pennings estimated that the probability of drug resistance evolving due to pre-existing genetic variation to be as high as 39%, depending on the antiviral treatment 15. Recent studies demonstrated that lowering drug-resistance mutations (DRM) detection threshold from 20% to 5% improved the identification of patients at risk of virological failure 16,17 and, depending on a drug
Viruses
Affordable, sensitive, and scalable technologies are needed for monitoring antiretroviral treatment (ART) success with the goal of eradicating HIV-1 infection. This review discusses use of Sanger sequencing and next generation sequencing (NGS) methods for HIV-1 drug resistance (HIVDR) genotyping, focusing on their use in resource limited settings (RLS). Sanger sequencing remains the gold-standard method for detecting HIVDR mutations of clinical relevance but is mainly limited by high sequencing costs and low-throughput. NGS is becoming a more common sequencing method, with the ability to detect low-abundance drug-resistant variants and reduce per sample costs through sample pooling and massive parallel sequencing. However, use of NGS in RLS is mainly limited by infrastructure costs. Given these shortcomings, our review discusses sequencing technologies for HIVDR genotyping, focusing on common in-house and commercial assays, challenges with Sanger sequencing in keeping up with change...
Antiviral Therapy, 2011
Background HIV drug-resistance (DR) surveillance in resource-limited settings can be performed using dried blood spots (DBS) because of ease of collection, transportation and storage. Analysis of pooled specimens on next-generation sequencing (NGS)-based platforms, such as the 454 pyrosequencing, is an efficient sequencing method for determining HIV DR rates. In this study, we conducted HIV DR surveillance on DBS using NGS and identified minority variants in individual patients. Methods A total of 48 extracts of DBS from an HIV DR surveillance study in Mexico City were re-amplified using primers tagged with multiplex identifiers, pooled and pyrosequenced. Consensus sequences were generated for each specimen with mixtures identified at positions where >20% of the reads contained a variant. Individual consensus sequences were then analysed for DR mutations and compared with those derived from Sanger sequencing. Results DBS analysed with tagged pooled pyrosequencing (TPP) were highl...
Journal of Virological Methods, 1998
The performance to detect drug resistence mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT™. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.
HIV-1 genotypic drug resistance testing: digging deep, reaching wide?
Current opinion in virology, 2015
For many years, population-based Sanger sequencing has been the golden standard for drug resistance testing within the routine follow-up of HIV-1 infected patients in resource-rich settings. Often, the data generated within this framework were subsequently used for research and surveillance purposes: to understand therapy response and to gain insights into epidemiological processes. Sanger sequencing was however ill suited for diagnostic and prognostic use in resource-limited settings (RLS) and therefore not broadly implemented. Next-generation sequencing (NGS) technologies provide high-throughput approaches by the rapid acquisition of thousands to millions of short nucleotide sequences. Depending on the experimental design, the roll-out of NGS drug resistance testing at a larger scale is feasible, providing better characterization and understanding of the evolving population of viral variants within a patient and potentially improving the prognostic value of drug resistance testing...