[Show abstract][Hide abstract]ABSTRACT: Importance:
Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.
Full-text Article · Apr 2016 · Journal of Virology
[Show abstract][Hide abstract]ABSTRACT: Prevalence of Nucleoside RT Inhibitor (NRTI) Drug-Resistance Mutations (DRMs) in Antiretroviral (ARV)-Naïve and -Treated Individuals and Their Estimated Contributions to Reduced NRTI Susceptibility.
[Show abstract][Hide abstract]ABSTRACT: Absolute and Cumulative Percent of Each Major Nucleoside (NRTI) Drug-Resistance Mutation (DRM) in 712 Children with Virological Failure and Intermediate or High-Level Acquired NRTI Drug Resistance while Receiving a First-Line NRTI/NNRTI Regimen.
[Show abstract][Hide abstract]ABSTRACT: Prevalence of Non-Nucleoside RT Inhibitor (NNRTI) Drug-Resistance Mutations (DRMs) in Antiretroviral (ARV)-Naïve and -Treated Individuals and Their Estimated Contributions to Reduced NNRTI Susceptibility.
[Show abstract][Hide abstract]ABSTRACT: The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.
[Show abstract][Hide abstract]ABSTRACT: Absolute and Cumulative Percent of Each Major Nucleoside (NRTI) Drug-Resistance Mutation (DRM) in 467 Individuals with Virological Failure and Intermediate or High-Level Acquired NRTI Drug Resistance while Receiving a First-Line TDF Containing Regimen.
[Show abstract][Hide abstract]ABSTRACT: Proportion of Different 24 Nucleotide Sequences Flanking Position 103 in HIV-1 RT Sequences from 26,358 Individuals in the Stanford HIV Drug Resistance Database From Six Low- and Middle-Income Country Regions.
[Show abstract][Hide abstract]ABSTRACT: Prevalence of Protease Inhibitor (PI) Drug-Resistance Mutations (DRMs) in PI-Naïve and -Treated Individuals and Their Estimated Contributions to Reduced PI Susceptibility.
[Show abstract][Hide abstract]ABSTRACT: Absolute and Cumulative Percent of Each Major Nonnucleoside (NNRTI) Drug-Resistance Mutation (DRM) in 721 Children with Virological Failure and Intermediate or High-Level Acquired NNRTI Drug Resistance while Receiving a First-Line NRTI/NNRTI Regimen.
[Show abstract][Hide abstract]ABSTRACT: Proportion of Different Codons at Position 103 in HIV-1 RT Sequences from 26,358 Individuals in the Stanford HIV Drug Resistance Database From Six Low- and Middle-Income Country Regions.
[Show abstract][Hide abstract]ABSTRACT: Background:
Sofosbuvir is a chain-terminating nucleotide analogue inhibitor of the hepatitis C virus (HCV) NS5B RNA polymerase that is efficacious in subjects with HCV genotype 1-6 infection. Sofosbuvir resistance is primarily conferred by the S282T substitution in NS5B.
NS5B sequencing and susceptibility testing of HCV from subjects infected with genotypes 1-6 who participated in phase 2 and 3 sofosbuvir clinical trials was performed.
No NS5B variants present at baseline among 1645 sofosbuvir-treated subjects were associated with treatment failure; sofosbuvir susceptibility was within 2-fold of reference. Among 282 subjects who did not achieve sustained virologic response, no novel sofosbuvir resistance-associated variants were identified, and the NS5B changes observed did not confer significant reductions in sofosbuvir susceptibility. In 1 subject with S282T observed at relapse 4 weeks after sofosbuvir monotherapy, the resistant variant (13.5-fold reduced sofosbuvir susceptibility, replication capacity <2% of control) became undetectable by deep sequencing 12 weeks after treatment. L159F and V321A were identified as treatment-emergent variants but did not confer resistance to sofosbuvir in the replicon system.
These data demonstrate a uniform susceptibility of subject-derived HCV to sofosbuvir, and also show that selection of sofosbuvir-resistant HCV is exceedingly rare and is associated with a significant reduction in viral fitness.
[Show abstract][Hide abstract]ABSTRACT: World Health Organization-recommended surveys of acquired HIV-1 drug resistance include assessment of HIV-1 viral load suppression to levels below 1,000 copies/ml and drug resistance-associated mutation patterns in subjects on antiretroviral therapy. Surveys are being conducted in regions of the world that cannot support the collection, storage, and shipping of frozen plasma. Therefore, dried blood spots are often the specimen type of choice for both genotyping and viral load measurement. Furthermore, viral load testing for individual patient management in these regions is being scaled-up in accordance with WHO 2013 Guidelines for Antiretroviral Treatment. Technical issues related to the adaptation of viral load assays to dried blood spots, especially with respect to sensitivity (limit of detection), specificity (cell-free RNA vs. cell-associated DNA or RNA), and assay method, affect the interpretation of a viral load result from dried blood spots. Amongst published studies of commercial assay performance with dried blood spots, the bioMérieux EasyQ® and Abbott RealTime assays tended to show high (> 90%) specificity and sensitivity; the Biocentric Generic or Roche TaqMan® assays tended to show high sensitivity but lower specificity, using a 1,000 copies/ml threshold. The relative contribution of cell-associated DNA or RNA to a viral load measurement is likely to vary between patients, depending on clinical parameters and treatment status. A model was developed that predicts that in patients on antiretroviral therapy with low plasma viral load, cellular DNA is the predominant source of non-plasma virus-derived nucleic acid in dried blood spots. The extent of viral load overestimation from dried blood spots becomes less important when plasma viral load is over about 5,000 copies/ml. To avoid misclassifying subjects with plasma viral load suppression, the World Health Organization-recommended threshold of 1,000 copies/ml can be applied only when an assay that can distinguish between DNA and RNA is used (e.g. bioMérieux EasyQ® or Abbott RealTime). There is a need for additional affordable technologies with the ability to discriminate between cell-free (plasma) and cell-associated nucleic acids.
[Show abstract][Hide abstract]ABSTRACT: The nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV) has been co-formulated with emtricitabine and tenofovir disoproxil fumarate for initial therapy of HIV-1-infected individuals. RPV, formulated as a long-acting nanosuspension, will also be assessed for its ability to prevent HIV-1 infection in resource limited settings. In this study, we determined whether any pre-existing genetic differences occurred among different HIV-1 subtypes at residues in RT associated with decreased virologic response to RPV. We found that the E138A substitution occurs more frequently in subtype C (range: 5.9-7.5%) than B (range: 0-2.3%) sequences from both treatment-naïve and -experienced individuals (p<0.01) in 4 independent genotype databases. In one of the databases (Stanford University), E138K and E138Q were also more common in RTI-experienced subtype C sequences (1.0% and 1.1%, respectively) than in subtype B sequences (0.3% and 0.6%, respectively). E138A/K/Q in subtype C decreased RPV susceptibility 2.9-, 5.8-, and 5.4-fold, respectively. Taken together, these data suggest that E138A could impact treatment or prevention strategies that include RPV in geographic areas where subtype C infection is prevalent.
[Show abstract][Hide abstract]ABSTRACT: The Abbott RealTime (RT) HCV assay targets the 5′ untranslated region (UTR) of the HCV genome. Here, we analyzed the sequence variability of
the assay target regions from 1,092 specimens. Thermodynamic modeling of the percentage of primers/probes bound at the assay
annealing temperature was performed to assess the potential effect of sequence variability. An analysis of this large data
set revealed that the primer and probe binding sites of the RealTime HCV viral load assay are highly conserved and that naturally occurring sequence polymorphisms are not expected to discernibly
impact assay performance.
Full-text Article · Jan 2014 · Journal of clinical microbiology
[Show abstract][Hide abstract]ABSTRACT: The Abbott RealTime (ART) HIV-1 assay targets the integrase region and is designed to tolerate mismatches. Variability in integrase sequences comprising the assay target regions from>1000 clinical specimens submitted for phenotypic and genotypic raltegravir resistance testing were analyzed. In this large collection of sequences from clinical specimens, the number and location of raltegravir resistance associated mutations did not differ from those tested previously and shown not to result in under-estimation of viral loads.
Full-text Article · Jul 2013 · Journal of virological methods
[Show abstract][Hide abstract]ABSTRACT: The Abbott RealTime HBV assay targets the N-terminal region of the S gene. Here we analyzed the sequence variability of the
assay target region from >2,100 clinical specimens. Thermodynamic modeling of the percentage of bound primer/probe at the
assay annealing temperature was performed to assess the potential effect of sequence variability.
Full-text Article · Jan 2013 · Journal of clinical microbiology
[Show abstract][Hide abstract]ABSTRACT: Durable suppression of HIV-1 replication requires the establishment of antiretroviral drug concentrations that exceed the
susceptibility of the virus strain(s) infecting the patient. Minimum plasma drug concentrations (Ctrough) are correlated with response, but determination of target Ctrough values is hindered by a paucity of in vivo concentration-response data. In the absence of these data, in vitro susceptibility measurements, adjusted for serum protein binding, can provide estimations of suppressive in vivo drug concentrations. We derived serum protein binding correction factors (PBCF) for protease inhibitors, nonnucleoside reverse
transcriptase inhibitors, and an integrase inhibitor by measuring the effect of a range of human serum concentrations on in vitro drug susceptibility measured with the PhenoSense HIV assay. PBCFs corresponding to 100% HS were extrapolated using linear
regression and ranged from 1.4 for nevirapine to 77 for nelfinavir. Using the mean 95% inhibitory concentration (IC95) for ≥1,200 drug-susceptible viruses, we calculated protein-bound IC95 (PBIC95) values. PBIC95 values were concordant with the minimum effective Ctrough values that were established in well-designed pharmacodynamic studies (e.g., indinavir, saquinavir, and amprenavir). In other
cases, the PBIC95 values were notably lower (e.g., darunavir, efavirenz, and nevirapine) or higher (nelfinavir and etravirine) than existing
target recommendations. The establishment of PBIC95 values as described here provides a convenient and standardized approach for estimation of the minimum drug exposure that
is required to maintain viral suppression and prevent the emergence of drug-resistant variants, particularly when in vivo concentration-response relationships are lacking.
Full-text Article · Sep 2012 · Antimicrobial Agents and Chemotherapy