Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays.

Background: Genomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS.

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory.

Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability.

Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy.

Background: The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) can decrease the use of personal protective equipment (PPE) and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort.

Methods: Self-collected anterior nasal swabs from employee return-to-work programs were tested using the Quest Diagnostics Emergency Use Authorization SARS-CoV-2 RT-PCR.

Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing.

Background: Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real-time reverse transcription polymerase chain reaction (RT-PCR) test authorized by the US Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens.

Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2 by Use of Real-Time Reverse Transcription-PCR.

The global coronavirus (CoV) disease 2019 (COVID-19) pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) RNA in specimen transport media under various storage conditions. Transport media tested included UTM, UTM-RT, ESwab, M4, and saline (0.

Trends in HIV-1 Drug Resistance Mutations from a U.S. Reference Laboratory from 2006 to 2017.

Trends in resistance to antiretroviral drugs for HIV-1 may inform clinical support and drug development. We evaluated drug resistance mutation (DRM) trends for nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor (PI), and integrase strand transfer inhibitor (INSTI) in a large U.S.

Detailed Transmission Network Analysis of a Large Opiate-Driven Outbreak of HIV Infection in the United States.

In January 2015, an outbreak of undiagnosed human immunodeficiency virus (HIV) infections among persons who inject drugs (PWID) was recognized in rural Indiana. By September 2016, 205 persons in this community of approximately 4400 had received a diagnosis of HIV infection. We report results of new approaches to analyzing epidemiologic and laboratory data to understand transmission during this outbreak.

Antiretroviral Drug Resistance Among Children and Youth in the United States With Perinatal HIV.

Among 234 US youths with perinatal human immunodeficiency virus, 75% had antiretroviral resistance, substantially higher than that of the reference laboratory overall (36%-44%). Resistance to newer antiretrovirals and to all antiretrovirals in a class was uncommon. The only factor independently associated with future resistance was a higher peak viral load.

Determination of HIV-1 coreceptor tropism using proviral DNA in women before and after viral suppression.

Background: An HIV-1 tropism test is recommended prior to CCR5 antagonist administration to exclude patients harboring non-R5 virus from treatment with this class of antiretrovirals. HIV-1 tropism determination based on proviral DNA (pvDNA) may be useful in individuals with plasma viral RNA suppression. We developed a genotypic tropism assay for pvDNA and assessed its performance in a retrospective analysis of samples collected longitudinally.

A genotypic HIV-1 proviral DNA coreceptor tropism assay: characterization in viremic subjects.

Background: HIV-1 coreceptor tropism testing is used to evaluate eligibility for CCR5 antagonist therapy. However, HIV-1 RNA-based tests are not suitable for virologically suppressed patients, therefore the use of proviral DNA tropism testing has been investigated. We describe a novel proviral DNA-based genotypic tropism assay and compare its performance to that of a sensitive HIV-1 RNA-based genotypic test.

Comparison of genotypic and phenotypic HIV type 1 tropism assay: results from the screening samples of Cenicriviroc Study 202, a randomized phase II trial in treatment-naive subjects.

Cenicriviroc is a once-daily oral CCR5/CCR2 antagonist in development for treatment of HIV infection. CVC Study 202 (652-2-202; NCT01338883) excluded treatment-naive subjects demonstrated to harbor non-R5 (CXCR4-tropic or dual-mixed) tropic HIV-1 by either genotypic or phenotypic tropism testing. Here we compare the results of genotypic and phenotypic tropism testing in Study 202.

A genotypic test for HIV-1 tropism combining Sanger sequencing with ultradeep sequencing predicts virologic response in treatment-experienced patients.

A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno([coreceptor]) and PSSM(x4r5).

Hepatitis B virus reverse transcriptase sequence variant database for sequence analysis and mutation discovery.

Drug resistance resulting from reverse transcriptase (RT) mutations is one of the main obstacles to successful hepatitis B virus (HBV) therapy. Indeed, HBV treatment guidelines recommend HBV genotypic resistance testing for patients receiving nucleos(t)ide RT inhibitors (N(t)RTIs) who develop virological failure. N(t)RTI-resistance mutations at 10 RT positions have been well characterized in phenotypic studies, however, data are lacking on the relative frequency of these mutations in N(t)RTI-treated and untreated individuals.

Molecular characterization of clinical isolates of human immunodeficiency virus resistant to the protease inhibitor darunavir.

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir.

Additional HIV-1 mutation patterns associated with reduced phenotypic susceptibility to etravirine in clinical samples.

We investigated the phenotypic impact of a number of uncommon amino acid substitutions at HIV-1 reverse transcriptase positions 103 and 138, which are not part of the etravirine resistance score and were found in combination with the high-impact mutation K101P. Etravirine phenotypic fold changes were 380-1400 for K101P + E138A/G/Q + K103N/S/T + V179I and 12-130 for K101P + (K103S +/- V179I) in the absence of E138A/G/Q. Although the effect of K103S is unclear, additional position 138 substitutions seem important for etravirine susceptibility.

Enzymatic and structural analysis of the I47A mutation contributing to the reduced susceptibility to HIV protease inhibitor lopinavir.

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR.

Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region.

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years.

Identification of a novel resistance (E40F) and compensatory (K43E) substitution in HIV-1 reverse transcriptase.

Background: HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) have been used in the clinic for over twenty years. Interestingly, the complete resistance pattern to this class has not been fully elucidated. Novel mutations in RT appearing during treatment failure are still being identified.

The probable source of both the primary multidrug-resistant (MDR) HIV-1 strain found in a patient with rapid progression to AIDS and a second recombinant MDR strain found in a chronically HIV-1-infected patient.

Background: Rapid progression to AIDS after acute infection with a multidrug-resistant (MDR), dual-tropic strain of human immunodeficiency virus type 1 (HIV-1) was reported in a New York City man (hereafter referred to as "NYC") who has sex with men. The probable source of this HIV-1 (hereafter referred to as "CT01") and the development of a recombinant MDR HIV-1 in the source's partner (hereafter referred to as "CT02") are described.

Methods: After identification of the epidemiological link of CT01 and CT02 to NYC, viral sequences and phenotypic analyses were compared.

New two-amino acid insertion near codon 70 of the HIV type 1 protease gene.

A two-amino acid insertion near codon 70 of the HIV-1 protease gene was found in an individual failing protease inhibitor therapy. Susceptibility of this strain to protease inhibitors was similar to that of non-insert-containing strains with comparable resistance mutations and one or more other major PI mutations.

Structural analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor lopinavir.

We have identified a rare HIV-1 protease (PR) mutation, I47A, associated with a high level of resistance to the protease inhibitor lopinavir (LPV) and with hypersusceptibility to the protease inhibitor saquinavir (SQV). The I47A mutation was found in 99 of 112,198 clinical specimens genotyped after LPV became available in late 2000, but in none of 24,426 clinical samples genotyped from 1998 to October 2000. Phenotypic data obtained for five I47A mutants showed unexpected resistance to LPV (86- to >110-fold) and hypersusceptibility to SQV (0.

Rare one and two amino acid inserts adjacent to codon 103 of the HIV-1 reverse transcriptase (RT) affect susceptibility to non-nucleoside RT inhibitors.

HIV-1 strains that possess a one or two amino acid insert between codons 102 and 103 of the reverse transcriptase (RT) gene were identified in three HIV-1-infected individuals. Each strain also had one or more known mutations associated with nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). Recombinant viruses from these strains had reduced susceptibility to efavirenz and nevirapine, and homology modelling predicted a loss of binding contacts with efavirenz.