[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: 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.
No preview · Article · Apr 2014 · Antiviral research
[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.
No preview · 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.
No preview · 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.
[Show abstract][Hide abstract] ABSTRACT: The World Health Organization (WHO) has developed a global laboratory network to support human immunodeficiency virus drug
resistance genotyping for public health surveillance in resource-limited countries. Blinded proficiency panels are an essential
part of a genotyping quality-assurance program and are used to monitor the reliability of genotyping data in the WHO laboratory
network. Laboratories in Europe, North America, Asia, Africa, and the Caribbean have tested panels annually since 2007; 103
of 131 submissions (79%) had >99% nucleotide sequence identity and resistance mutation concordance, compared with consensus.
Most errors were associated with mixtures in the test specimen, leading to subjectivity in base-calling or amplification bias.
Overall, genotyping assays used by the WHO laboratory network are reliable.
Full-text · Article · May 2012 · Clinical Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: In resource-limited settings, there is increased demand for human immunodeficiency virus type 1 drug resistance testing. Because
preservation of plasma specimens is often not feasible in resource-limited settings, use of dried blood spots (DBSs) is being
adopted. We used 2 panels of DBSs for genotyping assay validation and proficiency testing in selected laboratories in the
World Health Organization laboratory network in 14 countries. An amplification sensitivity of 1000 copies/mL was achieved
by 2 laboratories. Reproducibility and accuracy of nucleotide sequence determination and resistance-associated mutation identification
from DBSs was similar to that previously determined for plasma. International shipping at ambient temperature had no significant
effect on amplification success. These studies indicate that DBS-based genotyping is equally reproducible and reliable, although
slightly less sensitive, compared with plasma.
Full-text · Article · May 2012 · Clinical Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: In resource-limited settings, there is increased demand for human immunodeficiency virus type 1 drug resistance testing. Because preservation of plasma specimens is often not feasible in resource-limited settings, use of dried blood spots (DBSs) is being adopted. We used 2 panels of DBSs for genotyping assay validation and proficiency testing in selected laboratories in the World Health Organization laboratory network in 14 countries. An amplification sensitivity of 1000 copies/mL was achieved by 2 laboratories. Reproducibility and accuracy of nucleotide sequence determination and resistance-associated mutation identification from DBSs was similar to that previously determined for plasma. International shipping at ambient temperature had no significant effect on amplification success. These studies indicate that DBS-based genotyping is equally reproducible and reliable, although slightly less sensitive, compared with plasma. Development and transmission of drug-resistant human immunodeficiency virus (HIV) in resource-limited settings is a potential negative consequence of the international effort to provide antiretroviral treat-ment to millions of persons living with HIV infection . HIV drug resistance (HIVDR) is associated with increased risk of therapeutic failures; transmission of drug-resistant virus; and decreased therapeutic options, treatment program effectiveness, and survival. The World Health Organization (WHO) global strategy for prevention and assessment of HIVDR  is a coordinated effort to assess HIVDR worldwide. The laboratory testing component of this strategy promotes
[Show abstract][Hide abstract] ABSTRACT: Dried blood spots (DBS) may be a promising alternative specimen type to plasma for measuring the viral load (VL) in HIV-infected individuals in resource-limited settings. However, characterization of assay performance using DBS is incomplete. In this prospective study, the VL was measured in parallel using plasma and DBS specimens collected at the same time from 157 HIV-1-infected individuals. DBS were prepared by dispensing 50 μl of blood onto filter paper cards and were stored desiccated at -20°C. Nucleic acid extraction from plasma and DBS was performed automatically using the Abbott m2000sp instrument, and the VL was measured using the RealTime HIV-1 VL assay, which has a lower limit of detection of 40 HIV RNA copies/ml. The correlation between plasma and DBS results was good (R = 0.91; P < 0.001). The mean difference in the VL (DBS minus plasma) was 0.35 log copies (standard deviation [SD], 0.47 log copies). A total of 40 (26%) paired specimens had a difference of >0.5 log copy, and in 12 (7.8%) it was >1 log copy. the VL from DBS was measurable in 95.7% of specimens with a plasma VL of >2.74 log copies (550 HIV RNA copies/ml). In summary, the VL can reliably be measured using DBS with the Abbott RealTime HIV-1 assay. The estimated lower limit of detection of this automated methodology on DBS is 550 copies/ml, a threshold that may be acceptable for periodic VL monitoring in patients on antiretroviral therapy in resource-limited settings, where early detection of virologic treatment failure is often problematic.
Full-text · Article · Dec 2011 · Journal of clinical microbiology
[Show abstract][Hide abstract] ABSTRACT: Resistance-associated mutations in the HIV-1 protease modify viral fitness through changes in the catalytic activity and altered binding affinity for substrates and inhibitors. In this report, we examine the effects of 31 mutations at 26 amino acid positions in protease to determine their impact on infectivity and protease inhibitor sensitivity. We found that primary resistance mutations individually decrease fitness and generally increase sensitivity to protease inhibitors, indicating that reduced virion-associated protease activity reduces virion infectivity and the reduced level of per virion protease activity is then more easily titrated by a protease inhibitor. Conversely, mutations at more variable positions (compensatory mutations) confer low-level decreases in sensitivity to all protease inhibitors with little effect on infectivity. We found significant differences in the observed effect on infectivity with a pseudotype virus assay that requires the protease to cleave the cytoplasmic tail of the amphotropic murine leukemia virus (MuLV) Env protein. Additionally, we were able to mimic the fitness loss associated with resistance mutations by directly reducing the level of virion-associated protease activity. Virions containing 50% of a D25A mutant protease were 3- to 5-fold more sensitive to protease inhibitors. This level of reduction in protease activity also resulted in a 2-fold increase in sensitivity to nonnucleoside inhibitors of reverse transcriptase and a similar increase in sensitivity to zidovudine (AZT), indicating a pleiotropic effect associated with reduced protease activity. These results highlight the interplay between enzyme activity, viral fitness, and inhibitor mechanism and sensitivity in the closed system of the viral replication complex.
Preview · Article · Nov 2011 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.
No preview · Article · Sep 2011 · AIDS research and human retroviruses
[Show abstract][Hide abstract] ABSTRACT: GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was
selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated
an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to
the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+ T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately
affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects.
When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374
showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic
boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent
with potential for clinical efficacy in both treatment-naïve and -experienced patients.
Full-text · Article · Mar 2011 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: The Abbott RealTime HIV-1 viral load assay uses primers and probes targeted to integrase, which is also the target of integrase inhibitors such
as raltegravir. Viral loads of 42 raltegravir-susceptible and 40 raltegravir-resistant specimens were determined using RealTime HIV-1 and Roche Monitor (v1.5). The differences in viral load measurements between assays were comparable in the two groups,
demonstrating that the RealTime HIV-1 assay can tolerate raltegravir-selected mutations.
Full-text · Article · Feb 2011 · Journal of clinical microbiology
[Show abstract][Hide abstract] ABSTRACT: Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated
with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was
alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold
decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility
to atazanavir and saquinavir.
Full-text · Article · Nov 2010 · Antimicrobial Agents and Chemotherapy