The genotypic false positive rate determined by V3 population sequencing can predict the burden of HIV-1 CXCR4-using species detected by pyrosequencing

Abstract

Objective: The false-positive rate (FPR) is a percentage-score provided by Geno2Pheno-algorithm indicating the likelihood that a V3-sequence is falsely predicted as CXCR4-using. We evaluated the correlation between FPR obtained by V3 population-sequencing and the burden of CXCR4-using variants detected by V3 ultra-deep sequencing (UDPS) and Enhanced-Sensitivity Trofile assay (ESTA).

Methods: 54 HIV-1 B-subtype infected-patients (all maraviroc-naïve), with viremia >10,000copies/ml, were analyzed. HIV-tropism was assessed by V3 population-sequencing, UDPS (considering variants with >0.5% prevalence), and ESTA.

Results: By UDPS, CCR5-using variants were detected in 53/54 patients, irrespective of FPR values, and their intra-patient prevalence progressively increased by increasing the FPR obtained by V3 population-sequencing (rho = 0.75, p = 5.0e-8). Conversely, the intra-patient prevalence of CXCR4-using variants in the 54 patients analyzed progressively decreased by increasing the FPR (rho = -0.61; p = 9.3e-6). Indeed, no CXCR4-using variants were detected in 13/13 patients with FPR>60. They were present in 7/18 (38.8%) patients with FPR 20-60 (intra-patient prevalence range: 2.1%-18.4%), in 5/7 (71.4%) with FPR 10-20, in 4/6 (66.7%) with FPR 5-10, and in 10/10(100%) with FPR<5 (intra-patient prevalence range: 12.1%-98.1%).

Conclusions: FPR by V3 population-sequencing can predict the burden of CXCR4-using variants. This information can be used to optimize the management of tropism determination in clinical practice. Due to its low cost and short turnaround time, V3 population-sequencing may represent the most feasible test for HIV-1 tropism determination. More sensitive methodologies (as UDPS) might be useful when V3 population-sequencing provides a FPR >20 (particularly in the range 20-60), allowing a more careful identification of patients harboring CXCR4-using variants.

Figure 1. Box plot reporting the distribution of FPR values of V3 sequences obtained by UDPS, sorted according to the FPR value at population sequencing.

The medians, interquartile ranges, upper and lower whiskers, and outlier values are shown. P-value was calculated through Kruskal-Wallis Test.

Figure 2. The graphs report the proportion of R5 (A) and X4 (B) variants per patient according to the values of FPR at population V3 sequencing.

Distribution of R5 and X4 variants in relationship to the False Positive Rate (FPR) detected by population V3 sequencing. The graphs report the proportion of R5 (A) and X4 (B) variants per patient according to the values of FPR at population V3 sequencing. P-values were calculated by Spearman test. A FPR of 5.75 has been used as cut-off to infer HIV-1 co-receptor usage.

Figure 3. The graph reports the distribution of FPR values of all the V3 variants detected by UDPS in each patient according to FPR ranges at population V3 sequencing.

The relative dimension of green and red dots represents the prevalence of R5 and X4 species detected by UDPS. Yellow dots represent the FPR determined by population sequencing and letters within dots indicate the phenotypic tropism determined by ESTA (R =  pure CCR5 tropism, X = pure CXCR4 tropism, D = dual/mixed tropism. For blank yellow dots, ESTA result was not available. A FPR of 5.75 has been used as cut-off to infer HIV-1 co-receptor usage of V3 sequences obtained by both V3 population and ultra-deep sequencing.

Figure 4. Quasispecies heterogeneity.

The box plots represent the diversity of amino acid sequences (A), and Shannon entropy (B) among patients with X4 species detected by UDPS at a prevalence lower or higher than 1%. P-values were calculated by Mann-Whitney Test.