Context-dependent accuracy of the cobas plasma separation card for HCV RNA viral load measurement.

Collection and preservation of plasma are challenging in remote or under-resourced settings. The cobas® Plasma Separation Card (PSC) is an alternative specimen type for blood-borne pathogen nucleic acid quantitation. We assessed PSC as a specimen type for HCV RNA quantitation in Pakistan. Plasma from venous blood and PSC from finger prick blood were prepared at two sites: Site 1 (in Lahore, n = 199) consisted of laboratory-based outpatient clinics. Specimens were prepared in the same facility and stored frozen. Site 2 was a catchment area within a resource-limited, semi-urban locality of Islamabad with limited access to healthcare services (n = 151). Community public health outreach staff collected blood and prepared the PSC in the participants' homes. Specimens were transported to the central hepatitis laboratory in Lahore to be stored frozen until tested. HCV RNA testing was performed using the cobas HCV RNA test in a central laboratory. Concordance with respect to RNA detectability was high at Site 1 (97.4%), but lower at Site 2 (82.4%). At Site 1, HCV viral load in plasma and PSC were well correlated across the linear range with a 0.21 log10 IU/mL mean bias toward higher concentrations in PSC. At Site 2, HCV viral load in plasma and PSC were poorly correlated. There was a 0.11 log10 IU/mL mean bias toward higher concentrations in PSC. PSC performance can be excellent in underserved settings where refrigerated transport of traditional specimens is difficult. In very challenging field settings, extra support must be provided to ensure correct specimen collection and handling.

Low-Abundance Drug-Resistant HIV-1 Variants in Antiretroviral Drug-Naive Individuals: A Systematic Review of Detection Methods, Prevalence, and Clinical Impact.

BACKGROUND: The presence of high-abundance drug-resistant HIV-1 jeopardizes success of antiretroviral therapy (ART). Despite numerous investigations, the clinical impact of low-abundance drug-resistant HIV-1 variants (LA-DRVs) at levels <15%-25% of the virus population in antiretroviral (ARV) drug-naive individuals remains controversial. METHODS: We systematically reviewed 103 studies assessing prevalence, detection methods, technical and clinical detection cutoffs, and clinical significance of LA-DRVs in antiretroviral drug-naive adults. RESULTS: In total, 14 919 ARV drug-naive individuals were included. Prevalence of LA-DRVs (ie, proportion of individuals harboring LA-DRVs) was 0%-100%. Technical detection cutoffs showed a 4 log range (0.001%-10%); 42/103 (40.8%) studies investigating the impact of LA-DRVs on ART; 25 studies included only individuals on first-line nonnucleoside reverse transcriptase inhibitor-based ART regimens. Eleven of those 25 studies (44.0%) reported a significantly association between preexisting LA-DRVs and risk of virological failure whereas 14/25 (56.0%) did not. CONCLUSIONS: Comparability of the 103 studies is hampered by high heterogeneity of the studies' designs and use of different methods to detect LA-DRVs. Thus, evaluating clinical impact of LA-DRVs on first-line ART remains challenging. We, the WHO HIVResNet working group, defined central areas of future investigations to guide further efforts to implement ultrasensitive resistance testing in routine settings.

Kinetics of SARS-CoV-2 infection biomarkers in a household transmission study.

SARS-CoV-2 is the causative agent of COVID-19. Timely and accurate diagnostic testing is vital to contain the spread of infection, reduce delays in treatment and care, and inform patient management. Optimal specimen type (e.g. nasal swabs or saliva), timing of sampling, viral marker assayed (RNA or antigen), and correlation with viral infectivity and COVID-19 symptoms severity remain incompletely defined. We conducted a field study to evaluate SARS-CoV-2 viral marker kinetics starting from very early times after infection. We measured RNA and antigen levels in nasal swabs and saliva, virus outgrowth in cell culture from nasal swabs, and antibody levels in blood in a cohort of 30 households. Nine household contacts (HHC) became infected with SARS-CoV-2 during the study. Viral RNA was detected in saliva specimens approximately 1-2 days before nasal swabs in six HHC. Detection of RNA was more sensitive than of antigen, but antigen detection was better correlated with culture positivity, a proxy for contagiousness. Anti-nucleocapsid antibodies peaked one to three weeks post-infection. Viral RNA and antigen levels were higher in specimens yielding replication competent virus in cell culture. This study provides important data that can inform how to optimally interpret SARS-CoV-2 diagnostic test results.

Sequence conservation of the region targeted by the Abbott RealTime HBV viral load assay in clinical specimens.

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.

Novel method to assess antiretroviral target trough concentrations using in vitro susceptibility data.

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 (C(trough)) are correlated with response, but determination of target C(trough) 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 (IC(95)) for ≥1,200 drug-susceptible viruses, we calculated protein-bound IC(95) (PBIC(95)) values. PBIC(95) values were concordant with the minimum effective C(trough) values that were established in well-designed pharmacodynamic studies (e.g., indinavir, saquinavir, and amprenavir). In other cases, the PBIC(95) values were notably lower (e.g., darunavir, efavirenz, and nevirapine) or higher (nelfinavir and etravirine) than existing target recommendations. The establishment of PBIC(95) 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.

Performance of the Abbott RealTime HIV-1 viral load assay is not impacted by integrase inhibitor resistance-associated mutations.

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.

Prevalence, mutation patterns, and effects on protease inhibitor susceptibility of the L76V mutation in HIV-1 protease.

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.

In vivo fitness cost of the M184V mutation in multidrug-resistant human immunodeficiency virus type 1 in the absence of lamivudine.

Lamivudine therapy selects for the M184V mutation. Although this mutation reduces the replicative capacity of human immunodeficiency virus in vitro, its impact on viral fitness in vivo has not been well defined. We used quantitative allele-specific PCR to precisely calculate the fitness differences between the mutated M184V virus and one that had reverted to the wild type in a cohort of patients by selectively interrupting reverse transcriptase inhibitor therapy, and we found that the M184V variants were consistently 4 to 8% less fit than the wild type in the absence of drug. After a lag phase of variable duration, wild-type variants emerged due to continued evolution of pol and back mutation rather than through emergence of an archived wild-type variant.

Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing.

The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS. In collaboration with the World Health Organization (WHO) Global HIVDR Laboratory Network and the Public Health Agency of Canada, the Rush VQA program distributed archived proficiency testing panels to ten laboratories to evaluate internally developed NGS assays. Consensus FASTA files were submitted using 5%, 10%, and 20% variant detection thresholds, and scored based on the same criteria used for SS. This small study showed that the SS External Quality Assurance (EQA) approach can be used as a transitional strategy for using NGS to generate SS-like data and for ongoing performance while using NGS data from the same quality control materials to further evaluate NGS assay performance.

Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping.

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4-99; reverse transcriptase 38-247). The concordance among laboratories' sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1-100%), compared to 15% (99.4%, 88.5-100%), 10% (99.2%, 87.4-100%), or 5% (98.5%, 86.4-100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.

Persistence of transmitted drug resistance among subjects with primary human immunodeficiency virus infection.

Following interruption of antiretroviral therapy among individuals with acquired drug resistance, preexisting drug-sensitive virus emerges relatively rapidly. In contrast, wild-type virus is not archived in individuals infected with drug-resistant human immunodeficiency virus (HIV) and thus cannot emerge rapidly in the absence of selective drug pressure. Fourteen recently HIV-infected patients with transmitted drug-resistant virus were followed for a median of 2.1 years after the estimated date of infection (EDI) without receiving antiretroviral therapy. HIV drug resistance and pol replication capacity (RC) in longitudinal plasma samples were assayed. Resistance mutations were characterized as pure populations or mixtures. The mean time to first detection of a mixture of wild-type and drug-resistant viruses was 96 weeks (1.8 years) (95% confidence interval, 48 to 192 weeks) after the EDI. The median time to loss of detectable drug resistance using population-based assays ranged from 4.1 years (conservative estimate) to longer than the lifetime of the individual (less conservative estimate). The transmission of drug-resistant virus was not associated with virus with reduced RC. Sexual transmission of HIV selects for highly fit drug-resistant variants that persist for years. The prolonged persistence of transmitted drug resistance strongly supports the routine use of HIV resistance genotyping for all newly diagnosed individuals.

Prevalence, genotypic associations and phenotypic characterization of K65R, L74V and other HIV-1 RT resistance mutations in a commercial database.

BACKGROUND: Nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations (NAMs) can affect response to treatment with NRTIs and might also result in HIV-1 with reduced replication capacity. METHODS: A large commercial HIV-1 database (n=60,487) was analysed for the prevalence of NAMs, antiviral drug susceptibilities and viral replication capacity. RESULTS: Thymidine analogue mutations (TAMs) and M184V were the most commonly observed NAMs (>25%). L74V/I was detected in 11% of isolates. K65R was detected in 3.3% of isolates and its frequency remained stable from 2003 to 2006, similar to trends observed for other NAMs. TAMs were rarely observed in combination with K65R, but frequently associated with L74V/I. HIV-1 with K65R or L74V/I alone were fully susceptible to zidovudine and stavudine. K65R was associated with reduced susceptibility to tenofovir, didanosine, abacavir and lamivudine; L74V/I was associated with reduced susceptibility to abacavir and didanosine. The addition of M184V to either K65R or L74V/I improved susceptibility to tenofovir, zidovudine and stavudine, but reduced susceptibility to abacavir, didanosine and lamivudine. Other NAMs commonly associated with K65R were A62V, S68G and Y115F; their NRTI susceptibilities were similar to those of viruses containing K65R alone. The replication capacity for HIV-1 with M184V/I or K65R was significantly reduced compared with wild-type (median 68%/ and 72%, respectively; P<0.0001), whereas replication capacity for HIV-1 with L74V or TAMs was not significantly reduced (88% and 97%, respectively). CONCLUSIONS: These results demonstrate a relative stability in the prevalence of HIV-1 clinical isolates with NAMs from 2003 to 2006. Differences between the genotypic patterns, phenotype and replication capacity associated with common NAMs are described.

Identification of alternative amino acid substitutions in drug-resistant variants of the HIV-1 reverse transcriptase.

OBJECTIVE/DESIGN: To identify new drug-resistance-associated mutations in the HIV-1 reverse transcriptase (RT) protein, we screened the RT sequence database of our hospital for alternative amino acid substitutions at known RT drug-resistance positions. METHOD: The genotypic database used for this analysis contained 1322 RT sequences from 1015 patients. We analysed this RT database with a focus on alternative mutations at RT positions known to be involved in drug resistance. The patterns of drug resistance associated with these alternative mutations were investigated in a separate database containing genotype and drug-susceptibility results. RESULTS: We identified multiple alternative resistance-associated mutations at amino acid positions 44, 62, 67, 69, 70, 74, 75, 103, 181, 190, 210, and 219 in RT. Phenotypic analysis indicated that drug-resistance properties of the alternative Y181V and L74I mutants are similar, but not identical, to that of the well-known Y181C and L74V mutations. CONCLUSION: This initial survey indicates that many resistance-associated phenomena can be distilled from existing data. These findings endorse a more extensive analysis by computerized methods.

HIV type 1 fitness evolution in antiretroviral-experienced patients with sustained CD4+ T cell counts but persistent virologic failure.

BACKGROUND: Over recent years, treatment guidelines for human immunodeficiency virus (HIV) infection have evolved from monotherapy to combination regimens that include > or = 3 active drugs, resulting in a sharp decrease in morbidity and mortality. In the present article, we evaluated changes in HIV type 1 viral fitness associated with the sequential introduction of antiretroviral treatment strategies in 4 chronically infected patients with sustained CD4 cell count despite having a persistently detectable viral load. METHODS: Plasma samples were obtained before and during treatment to construct recombinant virus containing the 3'-end of gag, the protease and the reverse-transcriptase coding region. Drug susceptibility phenotype was evaluated with a panel of multiple reverse-transcriptase and protease inhibitors. Replicative capacity (RC) and infectivity were measured, and production of p24 was monitored after transfection. RESULTS: Multidrug-resistant (MDR) viruses selected during long-term antiretroviral therapy were less fit and infectious than their wild-type or monotherapy-selected counterparts, with the exception of viruses recovered from patient B. In 3 of 4 cases, p24 kinetics after transfection showed a delay in viral production of recombinant viruses containing MDR mutations. Data from the RC and infectivity assays showed good correlation (P < .03) and corroborated the p24 kinetics data. CONCLUSIONS: This study shows that accumulation of MDR mutations during long-term antiretroviral treatment results, albeit not in all cases, in reductions of viral fitness.

In vitro activity of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor (NRTI), against 215 HIV-1 isolates resistant to other NRTIs.

SPD754 (also known as AVX-754) is a deoxycytidine analogue nucleoside reverse transcriptase inhibitor (NRTI) with antiretroviral activity against HIV-1 and HIV-2 in vitro and against recombinant viruses containing thymidine analogue mutations (TAMs). In order to better establish the activity of SPD754 against HIV-1 containing TAMs, twelve panels of up to twenty clinical isolates with defined TAM combinations were selected from the ViroLogic database. Phenotypic viral susceptibility to SPD754 and five other NRTIs was tested using the PhenoSense HIV assay and expressed as median fold-change compared with a reference strain. In total, 215 isolates were selected, representing four TAM patterns in both pathways by which TAMs accumulate clinically. The presence of five TAMs in the 41, 215 pathway, at codons 41, 67, 210, 215, and 219 of reverse transcriptase (RT), produced a median 1.8-fold reduction in SPD754 susceptibility, compared with fold reductions to zidovudine, lamivudine, abacavir, didanosine and tenofovir of 438, 4.8, 4.5, 1.4 and 3.6, respectively. Five TAMs in the 67, 70, 219 pathway (at codons 41, 67, 70, 215 and 219) reduced SPD754 susceptibility by a median 1.3-fold, compared with fold reductions for the aforementioned NRTIs of 108, 3.2, 3.0, 1.3 and 2.5, respectively. M184V addition reduced SPD754 susceptibility by 1.8-fold in the presence or absence of TAMs. SPD754 retains a substantial proportion of its antiviral activity against HIV-1 containing multiple TAMs, with or without the M184V mutation. These data suggest that SPD754 is a promising new NRTI for the treatment of NRTI-experienced HIV-infected patients.

Improving lopinavir genotype algorithm through phenotype correlations: novel mutation patterns and amprenavir cross-resistance.

BACKGROUND: Current genotypic algorithms suggest that the HIV-1 protease inhibitors (PI) lopinavir (LPV) and amprenavir (APV) have distinct resistance profiles. However, phenotypic data indicate that cross-resistance is more common than expected. METHODS: Protease genotype (GT) and phenotype (PT) from 1418 patient viruses with reduced PI susceptibility and/or resistance-associated mutations (training data) were analyzed. Samples were classified as LPV resistant by GT (GT-R) if six or more LPV mutations were present, and by PT (PT-R) if the 50% inhibitory concentration (IC(50)) fold-change (FC) was over 10. RESULTS: There were 182 samples (13%) that were GT-S but PT-R for LPV. A comparison of the mutation prevalence in PT-R/GT-S samples with that in PT-S/GT-S samples identified mutations associated with LPV PT-R. Several previously defined LPV mutations were found to have a stronger than average effect (e.g., M46I/L, I54V/T, V82A/F), and new variants at known positions (e.g., I54A/M/S, V82S) were identified. Other mutations, including known APV resistance mutations, were found to contribute to reduced LPV susceptibility. A new LPV genotypic interpretation algorithm was constructed that improved overall genotypic/phenotypic concordance from 80% to 91%. The algorithm demonstrated a concordance rate of 90% when tested on 523 new samples. Cross-resistance between APV and LPV was greater in samples with primary APV resistance mutations than in those lacking them. CONCLUSIONS: The current LPV mutation score does not fully account for many resistant viruses. Consequently, cross-resistance between LPV and APV is underappreciated. Phenotypic results from large and diverse patient virus populations should be used to guide the development of more accurate GT interpretation algorithms.

Antiretroviral drug resistance in non-subtype B HIV-1, HIV-2 and SIV.

Patients infected with HIV-1 of subtype other than B ('non-subtype B') or with HIV-2 are being treated with antiretroviral drugs in increasing numbers. In addition, healthcare providers and laboratory workers working with clinical specimens or animals infected with HIV, SIV or SHIV are at risk of being exposed to the virus and might require post-exposure prophylactic treatment. Thus, it is important to understand the inherent antiviral susceptibility of non-subtype B HIV-1, HIV-2 and SIV to currently available antiretroviral drugs, which have been developed with subtype B HIV-1-infected patients as the primary target population. In addition, knowledge about the consequences of treatment failure in non-subtype B HIV-1- and HIV-2-infected patients, with respect to the development of drug resistance, is crucial for designing optimal treatment strategies. This review summarizes the current state of knowledge in these areas. Non-subtype B group M HIV-1 appears to be susceptible to available agents, but follows several unique pathways to resistance to some drugs that have important clinical implications. Group O HIV-1 is naturally resistant to the non-nucleoside reverse transcriptase inhibitors (NNRTIs). HIV-2 and SIVsm are also naturally resistant to the NNRTIs as well as the protease inhibitor amprenavir. More research into the clinical responses to existing drugs and interpretation of genotypic information is needed, as well as development of diagnostic assays specific for non-subtype B HIV-1 and HIV-2.

Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA.

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.

E138A in HIV-1 reverse transcriptase is more common in subtype C than B: implications for rilpivirine use in resource-limited settings.

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.

Minimal sequence variability of the region of HIV-1 integrase targeted by the Abbott RealTime HIV-1 viral load assay in clinical specimens with reduced susceptibility to raltegravir.

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.