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.

A genotypic test for HIV-1 tropism combining Sanger sequencing with ultradeep sequencing predicts virologic response in treatment-experienced patients.

A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno([coreceptor]) and PSSM(x4r5). Virologic response as a function of tropism readout was retrospectively assessed using blinded samples from treatment-experienced subjects who received maraviroc (N = 327) in the MOTIVATE and A4001029 clinical trials. MOTIVATE patients were classified as R5 and A4001029 patients were classified as non-R5 by the original Trofile test. Virologic response was compared between the R5 and non-R5 groups determined by TPS, UDS alone, the reflex strategy and the Trofile Enhanced Sensitivity (TF-ES) test. UDS had greater sensitivity than TPS to detect minority non-R5 variants. The median log(10) viral load change at week 8 was -2.4 for R5 subjects, regardless of the method used for classification; for subjects with non-R5 virus, median changes were -1.2 for TF-ES or the Reflex Test and -1.0 for UDS. The differences between R5 and non-R5 groups were highly significant in all 3 cases (p<0.0001). At week 8, the positive predictive value was 66% for TF-ES and 65% for both the Reflex test and UDS. Negative predictive values were 59% for TF-ES, 58% for the Reflex Test and 61% for UDS. In conclusion, genotypic tropism testing using UDS alone or a reflex strategy separated maraviroc responders and non-responders as well as a sensitive phenotypic test, and both assays showed improved performance compared to TPS alone. Genotypic tropism tests may provide an alternative to phenotypic testing with similar discriminating ability.