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 (0.9% NaCl). Specimen types tested included nasopharyngeal/oropharyngeal swabs in the above-named transport media, bronchoalveolar lavage (BAL) fluid, and sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled SARS-CoV-2 RNA-negative specimen remnants for the various medium types. Aliquots of samples were stored at 18°C to 26°C, 2°C to 8°C, and -10°C to -30°C and then tested at time points up to 14 days. Specimens consistently yielded amplifiable RNA with mean cycle threshold differences of <3 over the various conditions assayed, thus supporting the use and transport of alternative collection media and specimen types under a variety of temperature storage conditions.

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. HIV genetic distances were calculated using the polymerase region. Networks were generated using data about reported high-risk contacts, viral genetic similarity, and their most parsimonious combinations. Sample collection dates and recency assay results were used to infer dates of infection. Epidemiologic and laboratory data each generated large and dense networks. Integration of these data revealed subgroups with epidemiologic and genetic commonalities, one of which appeared to contain the earliest infections. Predicted infection dates suggest that transmission began in 2011, underwent explosive growth in mid-2014, and slowed after the declaration of a public health emergency. Results from this phylodynamic analysis suggest that the majority of infections had likely already occurred when the investigation began and that early transmission may have been associated with sexual activity and injection drug use. Early and sustained efforts are needed to detect infections and prevent or interrupt rapid transmission within networks of uninfected PWID.

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. RESULTS: We randomly selected paired plasma/PBMC samples from the Women's Interagency HIV Study with plasma viral load ≥5,000 cp/mL at time 1 (T1), undetectable viral load maintained for ≥1 year and CD4+ >200 cells/µL at time 2 (T2). pvDNA was isolated from cryopreserved PBMCs. Sequences were analyzed in triplicate from 49/50 women, with tropism assigned using the geno2pheno (g2p) algorithm. The median time between T1 and T2 was 4.1 years. CXCR4-using virus was detected in 24% of the RNA samples and 33% of the pvDNA samples at T1, compared to 37% of the pvDNA samples at T2. Concordance between plasma RNA and pvDNA tropism was 88% at T1 and 80% at T2. The g2p scores for RNA (T1) vs DNA (T1, T2) were strongly correlated (Spearman rho: 0.85 (T1); 0.78 (T2)). In women with evidence of tropism switch at T2 (either R5 to non-R5 or non-R5 to R5), there was a correlation between change in tropism and time. Mean pvDNA viral load decreased by 0.4 log10 copies/106 cells between T1 and T2 (p < 0.0001), but this decrease was not significantly associated with tropism status. CONCLUSIONS: We demonstrated that pvDNA tropism in women with HIV-1 suppression is concordant with prior RNA tropism results, even after a median time of >4 years. pvDNA tropism testing may be useful to determine eligibility of patients with viral suppression to switch to a CCR5-antagonist based regimen as well as for research purposes.

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. METHODS: Tropism was determined using HIV-1 plasma RNA and proviral DNA from 42 paired samples from patients with plasma viral loads ≥1000 HIV-1 RNA copies/mL. Proviral DNA sample types included whole blood, separated peripheral blood mononuclear cells resuspended in phosphate-buffered saline and peripheral blood mononuclear cells resuspended in spun plasma. The HIV-1 envelope V3 region was PCR-amplified, sequenced in triplicate, and analyzed for tropism with the geno2pheno algorithm using a 10% false-positive rate (FPR). RESULTS: Amplicons were obtained from proviral DNA and plasma RNA in 41/42 samples. Tropism predictions were highly concordant (93%-98%) between proviral DNA and plasma RNA, regardless of the proviral DNA isolation method. Non-R5 proviral DNA results were obtained for 100% of patients with detectable non-R5 plasma HIV-1 RNA results. Geno2pheno FPRs for proviral DNA and plasma RNA were highly correlated (Spearman rho = 0.86). CONCLUSIONS: Our findings demonstrate that proviral DNA tropism determinations from whole blood or peripheral blood mononuclear cells were highly concordant with plasma HIV-1 RNA tropism determinations. This assay may be useful for screening virologically suppressed patients for CCR5-antagonist eligibility and for research purposes.

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. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants. Methods: SARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant. Results: A total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results. Conclusions: These data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.

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. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.

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. The cycle threshold (Ct) values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of health care provider-collected specimens from patients with and without coronavirus disease 2019 symptoms. RESULTS: Among 47 923 participants, 1.8% were positive. RP failed to amplify for 13/115 435 (0.011%) specimens. The median (interquartile range) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1268 (5.2%) specimens but only a single false-negative result. CONCLUSIONS: Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population, as the likelihood of false-negative results is very low when using a sensitive, dual-target methodology.

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. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

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. METHODS: Positive specimens were selected from 3 prevalence groups, 1%-3%, >3%-6%, and >6%-10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with 3 negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3091 positive specimens. RESULTS: PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r = 0.96-0.99; slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90, respectively, for the N1 and N3 targets. The median Cts for 3091 positive specimens were 25.9 (N1) and 24.7 (N3). The percentage of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). CONCLUSIONS: Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement, which demonstrates the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2 with a low risk of false-negative results.

Human immunodeficiency virus type 1 isolates with the reverse transcriptase (RT) mutation Q145M retain nucleoside and nonnucleoside RT inhibitor susceptibility.

Q145M, a mutation in a conserved human immunodeficiency virus type 1 reverse transcriptase (RT) region, was reported to decrease susceptibility to multiple RT inhibitors. We report that Q145M and other Q145 mutations do not emerge with RT inhibitors nor decrease RT inhibitor susceptibility. Q145M should not, therefore, be considered an RT inhibitor resistance mutation.

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. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

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. reference laboratory database. DRMs with a Stanford HIV Drug Resistance Database mutation score ≥10 from deidentified subtype B NRTI/NNRTI/PI specimens (2006-2017; >10,000/year) and INSTI specimens (2010-2017; >1,000/year) were evaluated. Sequences with NRTI, NNRTI, or PI single- or multiclass DRMs declined from 48.9% to 39.3%. High-level dual- and triple-class resistance declined from 43.3% (2006) to 17.1% (2017), while sequences with only single-class DRMs increased from 40.0% to 52.9%. The prevalence of DRMs associated with earlier treatment regimens declined, while prevalence of some DRMs associated with newer regimens increased. M184V/I decreased from 48.3% to 29.4%. K103N/S/T declined from 42.5% in 2012 to 36.4% in 2017. Rilpivirine and etravirine DRMs E138A/Q/R and E138K increased from 4.9% and 0.4% to 9.7% and 1.7%, respectively. Sequences with ≥1 darunavir DRM declined from 18.1% to 4.8% by 2017. INSTI DRM Q148H/K/R declined from 39.3% (2010) to 13.8% (2017). Prevalence of elvitegravir-associated DRMs T66A/I/K, E92Q, S147G, and the dolutegravir-associated DRM R263K increased. For a subset of patients with serial testing, 50% (2,646/5,290) of those who initially had no reportable DRM subsequently developed ≥1 DRM for NRTI/NNRTI/PI and 49.7% (159/320) for INSTI. These trends may inform the need for baseline genotypic resistance testing. The detection of treatment-emergent DRMs in serially tested patients confirms the value of genotypic testing following virologic failure.

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. To unravel the role of two of these newly identified changes, E40F and K43E, we investigated their effect on viral drug susceptibility and replicative capacity. RESULTS: A large database (Quest Diagnostics database) was analysed to determine the associations of the E40F and K43E changes with known resistance mutations. Both amino acid changes are strongly associated with the well known NRTI-resistance mutations M41L, L210W and T215Y. In addition, a strong positive association between these changes themselves was observed. A panel of recombinant viruses was generated by site-directed mutagenesis and phenotypically analysed. To determine the effect on replication capacity, competition and in vitro evolution experiments were performed. Introduction of E40F results in an increase in Zidovudine resistance ranging from nine to fourteen fold depending on the RT background and at the same time confers a decrease in viral replication capacity. The K43E change does not decrease the susceptibility to Zidovudine but increases viral replication capacity, when combined with E40F, demonstrating a compensatory role for this codon change. CONCLUSION: In conclusion, we have identified a novel resistance (E40F) and compensatory (K43E) change in HIV-1 RT. Further research is indicated to analyse the clinical importance of these changes.

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 (0.1- to 0.7-fold). Molecular modeling and energy calculations for these mutants using our structural phenotyping methodology showed an increase in the binding energy of LPV by 1.9-3.1 kcal/mol with respect to the wild type complex, corresponding to a 20- to >100-fold decrease in binding affinity, consistent with the observed high levels of LPV resistance. In the WT PR-LPV complex, the Ile 47 side chain is positioned close to the phenoxyacetyl moiety of LPV and its van der Waals interactions contribute significantly to the ligand binding. These interactions are lost for the smaller Ala 47 residue. Calculated binding energy changes for SQV ranged from -0.4 to -1.2 kcal/mol. In the mutant I47A PR-SQV complexes, the PR flaps are packed more tightly around SQV than in the WT complex, resulting in the formation of additional hydrogen bonds that increase binding affinity of SQV consistent with phenotypic hypersusceptibility. The emergence of mutations at PR residue 47 strongly correlates with increasing prescriptions of LPV (Spearman correlation r(s) = 0.96, P < .0001).

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. Mutagenesis studies indicated that replication of insert-containing strains was dependent on RT gene mutations and polymorphisms that co-evolved with the insert. These results suggest that inserts in the NNRTI-binding pocket contribute to NNRTI resistance, but are tolerated only under specific genetic conditions.

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.

Structure-based phenotyping predicts HIV-1 protease inhibitor resistance.

Mutations in HIV-1 drug targets lead to resistance and consequent therapeutic failure of antiretroviral drugs. Phenotypic resistance assays are time-consuming and costly, and genotypic rules-based interpretations may fail to predict the effects of multiple mutations. We have developed a computational procedure that rapidly evaluates changes in the binding energy of inhibitors to mutant HIV-1 PR variants. Models of WT complexes were produced from crystal structures. Mutant complexes were built by amino acid substitutions in the WT complexes with subsequent energy minimization of the ligand and PR binding site residues. Accuracy of the models was confirmed by comparison with available crystal structures and by prediction of known resistance-related mutations. PR variants from clinical isolates were modeled in complex with six FDA-approved PIs, and changes in the binding energy (DeltaE(bind)) of mutant versus WT complexes were correlated with the ratios of phenotypic 50% inhibitory concentration (IC(50)) values. The calculated DeltaE(bind) of five PIs showed significant correlations (R(2) = 0.7-0.8) with IC(50) ratios from the Virco Antivirogram assay, and the DeltaE(bind) of six PIs showed good correlation (R(2) = 0.76-0.85) with IC(50) ratios from the Virologic PhenoSense assay. DeltaE(bind) cutoffs corresponding to a four-fold increase in IC(50) were used to define the structure-based phenotype as susceptible, resistant, or equivocal. Blind predictions for 78 PR variants gave overall agreement of 92% (kappa = 0.756) and 86% (kappa = 0.666) with PhenoSense and Antivirogram phenotypes, respectively. The structural phenotyping predicted drug resistance of clinical HIV-1 PR variants with an accuracy approaching that of frequently used cell-based phenotypic assays.

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 total of 304 subjects screened had paired genotypic and phenotypic results. Genotypic tropism testing (GTT) incorporated triplicate population sequencing using the geno2pheno algorithm and the PSSM algorithm, followed by ultradeep sequencing (UDS) for samples with R5 results. All samples were further evaluated with a phenotypic test, the enhanced-sensitivity Trofile assay (ESTA). Concordance between GTT and ESTA was 80% and increased to 84% when only geno2pheno was used for triplicate population sequencing. GTT (geno2pheno) classified 18% of the samples as non-R5 compared to 16% by ESTA. Only one-third of samples with non-R5 results by either test were classified as non-R5 by both tests. Median CD4((+)) cell counts were lower in patients with concordant non-R5 results by UDS and ESTA than in subjects with an R5 result by either assay (p=0.0004). UDS detected non-R5 virus in an additional 27/304 subjects (median 15% non-R5, interquartile range: 3.7-62%) with R5 results by ESTA. In conclusion, the geno2pheno algorithm improves concordance of GTT with a clinically validated phenotypic tropism assay as does the use of UDS. These findings provide support for recent guidelines indicating that genotypic tropism testing may be considered as an alternative to phenotypic testing.