Plasma SARS-CoV-2 RNA Levels as a Biomarker of Lower Respiratory Tract SARS-CoV-2 Infection in Critically Ill Patients With COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in serially collected samples from mechanically ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (n = 33, r = 0.

Plasma SARS-CoV-2 RNA levels as a biomarker of lower respiratory tract SARS-CoV-2 infection in critically ill patients with COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in simultaneously collected longitudinal samples from mechanically-ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (r=0.

CpG Methylation Profiles of HIV-1 Pro-Viral DNA in Individuals on ART.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART).

Short Communication: HIV-DRLink: A Tool for Reporting Linked HIV-1 Drug Resistance Mutations in Large Single-Genome Data Sets Using the Stanford HIV Database.

The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy. It is likely that pre-existing drug resistance mutations linked on the same viral genomes are predictive of treatment failure. Because of the large number of sequences generated by ultrasensitive single-genome sequencing (uSGS) and other similar next-generation sequencing methods, it is difficult to assess each sequence individually for linked drug resistance mutations.

Linked dual-class HIV resistance mutations are associated with treatment failure.

We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population before initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single-genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) control (nonfailure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine/tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/Optimal Combined Therapy After Nevirapine Exposure (OCTANE) Trial 1 among women who had received prior single-dose nevirapine.

No evidence of HIV replication in children on antiretroviral therapy.

It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low.

Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA.

Background: Although next generation sequencing (NGS) offers the potential for studying virus populations in unprecedented depth, PCR error, amplification bias and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. Here we report a method, termed ultrasensitive Single-Genome Sequencing (uSGS), for NGS library construction and analysis that eliminates PCR errors and recombinants, and generates single-genome sequences of the same quality as the "gold-standard" of HIV-1 single-genome sequencing assay but with more than 100-fold greater depth.

Results: Primer ID tagged cDNA was synthesized from mixtures of cloned BH10 wild-type and mutant HIV-1 transcripts containing ten drug resistance mutations.

PAPNC, a novel method to calculate nucleotide diversity from large scale next generation sequencing data.

Estimating viral diversity in infected patients can provide insight into pathogen evolution and emergence of drug resistance. With the widespread adoption of deep sequencing, it is important to develop tools to accurately calculate population diversity from very large datasets. Current methods for estimating diversity that are based on multiple alignments are not practical to apply to such data.

Low-frequency nevirapine (NVP)-resistant HIV-1 variants are not associated with failure of antiretroviral therapy in women without prior exposure to single-dose NVP.

Background: Low-frequency nevirapine (NVP)-resistant variants have been associated with virologic failure (VF) of initial NVP-based combination antiretroviral therapy (cART) in women with prior exposure to single-dose NVP (sdNVP). We investigated whether a similar association exists in women without prior sdNVP exposure.

Methods: Pre-cART plasma was analyzed by allele-specific polymerase chain reaction to quantify NVP-resistant mutants in human immunodeficiency virus-infected African women without prior sdNVP who were starting first-line NVP-based cART in the OCTANE/A5208 trial 2.

Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA.

Background: 454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.

Ultrasensitive allele-specific PCR reveals rare preexisting drug-resistant variants and a large replicating virus population in macaques infected with a simian immunodeficiency virus containing human immunodeficiency virus reverse transcriptase.

It has been proposed that most drug-resistant mutants, resulting from a single-nucleotide change, exist at low frequency in human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) populations in vivo prior to the initiation of antiretroviral therapy (ART). To test this hypothesis and to investigate the emergence of resistant mutants with drug selection, we developed a new ultrasensitive allele-specific PCR (UsASP) assay, which can detect drug resistance mutations at a frequency of ≥0.001% of the virus population.

Short-course Combivir after single-dose nevirapine reduces but does not eliminate the emergence of nevirapine resistance in women.

Background: In the Treatment Options Preservation Study (TOPS) trial, 4 or 7 days of Combivir (CBV; zidovudine/lamivudine) with maternal single-dose nevirapine (sdNVP) significantly reduced the emergence of NVP resistance as determined by virus population genotyping. To detect NVP resistance with greater sensitivity, we analysed TOPS samples by allele-specific real-time PCR (ASP).

Methods: In a random subset of women from each arm of the trial, plasma samples from before and 6 weeks after sdNVP were analysed using ASP at codons 103, 181, 184 and 190.

Role of low-frequency HIV-1 variants in failure of nevirapine-containing antiviral therapy in women previously exposed to single-dose nevirapine.

In the OCTANE/A5208 study of initial antiretroviral therapy (ART) in women exposed to single-dose nevirapine (sdNVP) ≥ 6 mo earlier, the primary endpoint (virological failure or death) was significantly more frequent in the NVP-containing treatment arm than in the lopinavir/ritonavir-containing treatment arm. Detection of NVP resistance in plasma virus at study entry by standard population genotype was strongly associated with the primary endpoint in the NVP arm, but two-thirds of endpoints occurred in women without NVP resistance. We hypothesized that low-frequency NVP-resistant mutants, missed by population genotype, explained excess failure in the NVP treatment arm.

Low frequency nonnucleoside reverse-transcriptase inhibitor-resistant variants contribute to failure of efavirenz-containing regimens in treatment- experienced patients.

Background: The contribution of low frequency drug-resistant human immunodeficiency virus type 1 (HIV-1) variants to failure of antiretroviral therapy is not well defined in treatment-experienced patients. We sought to detect minor nonnucleoside reverse-transcriptase inhibitor (NNRTI)-resistant variants at the initiation of multidrug efavirenz-containing therapy in both NNRTI-naive and NNRTI-experienced patients and to determine their association with virologic response.

Methods: Plasma samples at entry and at time of virologic failure from patients enrolled in the AIDS Clinical Trials Group study 398 were analyzed by standard genotype, single-genome sequencing and allele-specific polymerase chain reaction (K103N and Y181C) to detect and quantify minor NNRTI-resistant variants.

Optimization of allele-specific PCR using patient-specific HIV consensus sequences for primer design.

Allele-specific PCR based on subtype consensus sequences is a powerful technique for detecting low frequency drug resistant mutants in HIV-1 infected patients. However, this approach can be limited by genetic variation in the region complementary to the primers, leading to variability in allele detection. The goals of this study were to quantify this effect and then to improve assay performance.

Suppression of viremia and evolution of human immunodeficiency virus type 1 drug resistance in a macaque model for antiretroviral therapy.

Antiretroviral therapy (ART) in human immunodeficiency virus type 1 (HIV-1)-infected patients does not clear the infection and can select for drug resistance over time. Not only is drug-resistant HIV-1 a concern for infected individuals on continual therapy, but it is an emerging problem in resource-limited settings where, in efforts to stem mother-to-child-transmission of HIV-1, transient nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy given during labor can select for NNRTI resistance in both mother and child. Questions of HIV-1 persistence and drug resistance are highly amenable to exploration within animals models, where therapy manipulation is less constrained.

Blinded, multicenter comparison of methods to detect a drug-resistant mutant of human immunodeficiency virus type 1 at low frequency.

We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.