Dataset Information

Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing.

ABSTRACT: Genotypic antiretroviral drug resistance testing is a critical component of the global efforts to control the HIV-1 epidemic. This study investigates the semiautomated, next-generation sequencing (NGS)-based Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay in a prospective cohort of HIV-1-infected patients. Two-hundred sixty-nine samples were successfully sequenced by both NGS and Sanger sequencing. Among the 261 protease/reverse transcriptase (PR/RT) sequences, a mean of 0.37 drug resistance mutations were identified by both Sanger and NGS, 0.08 by NGS alone, and 0.03 by Sanger alone. Among the 50 integrase sequences, a mean of 0.3 drug resistance mutations were detected by both Sanger and NGS, and 0.08 by NGS alone. NGS estimated higher levels of drug resistance to one or more antiretroviral drugs for 6.5% of PR/RT sequences and 4.0% of integrase sequences, whereas Sanger estimated higher levels of drug resistance for 3.8% of PR/RT sequences. Although the samples successfully sequenced by the Sentosa SQ HIV Genotyping Assay demonstrated similar predicted resistance compared with Sanger, 44% of Sentosa runs failed quality control requiring 17 additional runs. This semi-automated NGS-based assay may aid in HIV-1 genotypic drug resistance testing, though numerous quality control issues were observed when this platform was used in a clinical laboratory setting. With additional refinement, the Sentosa SQ HIV-1 Genotyping Assay may contribute to the global efforts to control HIV-1.

SUBMITTER: Weber J

PROVIDER: S-EPMC7152740 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing.

Weber Jenna J Volkova Ilona I Sahoo Malaya K MK Tzou Philip L PL Shafer Robert W RW Pinsky Benjamin A BA

The Journal of molecular diagnostics : JMD 20190802 6

Genotypic antiretroviral drug resistance testing is a critical component of the global efforts to control the HIV-1 epidemic. This study investigates the semiautomated, next-generation sequencing (NGS)-based Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay in a prospective cohort of HIV-1-infected patients. Two-hundred sixty-nine samples were successfully sequenced by both NGS and Sanger sequencing. Among the 261 protease/reverse transcriptase (PR/RT) sequences, a mean of 0.37 drug resistance ...[more]

PMID: 31382033

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Project description:BackgroundRecently, a growing number of novel genetic defects underlying primary immunodeficiencies (PIDs) have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS) technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome.ObjectiveTo evaluate the role of targeted NGS and whole exome sequencing (WES) in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures.MethodsWe retrospectively analyzed genetic variants identified through targeted NGS or WES in 45 patients with complex PID of unknown etiology.ResultsForty-seven variants were identified using targeted NGS, while 5 were identified using WES. Newly identified genetic variants were classified into four groups: (I) variations associated with a well-defined PID, (II) variations associated with atypical features of a well-defined PID, (III) functionally relevant variations potentially involved in the immunological features, and (IV) non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%). Among them, four patients presented with a typical well-defined PID. In the remaining three cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causative per se of the disorder.ConclusionNGS technologies represent a cost-effective and rapid first-line genetic approach for the evaluation of complex PIDs. WES, despite a moderate higher cost compared to targeted, is emerging as a valuable tool to reach in a timely manner, a PID diagnosis with a considerable potential to draw genotype-phenotype correlation. Nevertheless, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants might be proven informative in future studies with larger cohorts of patients.

Project description:Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

Project description:IntroductionSepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting.MethodsThe Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists.DiscussionNeonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis.Trial registrationDRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.

Project description:BackgroundSepsis remains a major challenge, even in modern intensive care medicine. The identification of the causative pathogen is crucial for an early optimization of the antimicrobial treatment regime. In this context, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care, although they are associated with relevant limitations. Accordingly, culture-independent molecular diagnostic procedures might be of help for the identification of the causative pathogen in infected patients. The concept of an unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) has recently been identified to be a promising diagnostic platform for critically ill patients suffering from bloodstream infections. Although this new approach might be more sensitive and specific than culture-based state-of-the-art technologies, additional clinical trials are needed to exactly define the performance as well as clinical value of a NGS-based approach.MethodsNext GeneSiS is a prospective, observational, noninterventional, multicenter study to assess the diagnostic performance of a NGS-based approach for the detection of relevant infecting organisms in patients with suspected or proven sepsis [according to recent sepsis definitions (sepsis-3)] by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard (culture-based) microbiological diagnostics. The clinical value of this NGS-based approach will be estimated by a panel of independent clinical specialists, retrospectively identifying potential changes in patients' management based on NGS results. Further subgroup analyses will focus on the clinical value especially for patients suffering from a failure of empiric treatment within the first 3 days after onset [as assessed by death of the patient or lack of improvement of the patient's clinical condition (in terms of an inadequate decrease of SOFA-score) or persistent high procalcitonin levels].DiscussionThis prospective, observational, noninterventional, multicenter study for the first time investigates the performance as well as the clinical value of a NGS-based approach for the detection of bacteremia in patients with sepsis and may therefore be a pivotal step toward the clinical use of NGS in this indication.Trial registrationDRKS-ID: DRKS00011911 (registered October 9, 2017) https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00011911; ClinicalTrials.gov Identifier: NCT03356249 (registered November 29, 2017) https://clinicaltrials.gov/ct2/show/NCT03356249.

Dataset Information

Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing.

Publications

Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing.

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