Determination Of HIV-1 Tropism Research Articles

Genotypic HIV-1 tropism determination might help to identify people with exhausted treatment options and advanced disease.

To evaluate HIV-1 tropism in 1382 combined antiretroviral therapy (cART)-experienced patients failing therapy to characterize those with exhausted therapeutic options. HIV-1 genotypic tropism was inferred through Geno2Pheno by estimating the false-positive-rate (FPR) values. Cumulative resistance and drug activity were evaluated by Stanford algorithm. Overall, median (IQR) CD4 count (cells/mm3) nadir and at last genotypic resistance test (GRT) available were 98 (33-211) and 312 (155-517), respectively. Considering HIV-1 tropism, 30.5% had X4/dual-mixed strains (FPR ≤5%: 22.2%; FPR 5%-10%: 8.3%). By stratifying according to tropism, by decreasing FPR, a significant decrease of CD4 nadir and at last GRT was observed. The proportion of individuals with CD4 count <200 cells/mm3, who were perinatally infected and with a long treatment history significantly increased as FPR levels decreased. Regarding resistance, 933 (67.5%) individuals accumulated at least one class resistance, with 52.7%, 48.2%, 23.5% and 13.2% of individuals showing resistance to NRTIs, NNRTIs, PIs and INIs; while 23.2%, 27.2%, 14.3% and 2.8% harboured resistance to 1, 2, 3 and 4 classes, respectively. Individuals with FPR ≤5% showed a significantly higher level of resistance to PIs, NRTIs and INIs compared with others. The proportion of individuals harbouring strains susceptible to ≤2 active drugs was only about 2%; nonetheless, this proportion doubled (4.6%) in patients infected with FPR ≤5%. Our findings showed that a small proportion of cART failing individuals have limited therapeutic options. However, tropism determination might help to identify people who have accumulated a high level of resistance and have a greater risk of advanced disease.

THETA: a new genotypic approach for predicting HIV-1 CRF02-AG coreceptor usage.

The circulating recombinant form of HIV-1 CRF02-AG is the most frequent non-B subtype in Europe. Anti-HIV therapy and pathophysiological studies on the impact of HIV-1 tropism require genotypic determination of HIV-1 tropism for non-B subtypes. But genotypic approaches based on analysis of the V3 envelope region perform poorly when used to determine the tropism of CRF02-AG. We, therefore, designed an algorithm based on information from the gp120 and gp41 ectodomain that better predicts the tropism of HIV-1 subtype CRF02-AG. We used a bio-statistical method to identify the genotypic determinants of CRF02-AG coreceptor use. Toulouse HIV Extended Tropism Algorithm (THETA), based on a Least Absolute Shrinkage and Selection Operator method, uses HIV envelope sequence from phenotypically characterized clones. Prediction of R5X4/X4 viruses was 86% sensitive and that of R5 viruses was 89% specific with our model. The overall accuracy of THETA was 88%, making it sufficiently reliable for predicting the tropism of subtype CRF02-AG sequences. Binaries are freely available for download at https://github.com/viro-tls/THETA. It was implemented in Matlab and supported on MS Windows platform. The sequence data used in this work are available from GenBank under the accession numbers MK618182-MK618417.

Performance of genotypic algorithms for predicting tropism for HIV-1 CRF01_AE recombinant

ObjectivesThere is no consensus about the performances of genotypic rules for predicting HIV-1 non-B subtype tropism. Three genotypic methods were compared for CRF01_AE HIV-1 tropism determination. MethodsThe V3 env region of 207 HIV-1 CRF01_AE and 178 B subtypes from 17 centers in France and 1 center in Switzerland was sequenced. Tropism was determined by Geno2Pheno algorithm with false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25, net charge rule and NXT/S mutations. ResultsOverall, 72.5%, 59.4%, 86.0%, 90.8% of the 207 HIV-1 CRF01_AE were R5-tropic viruses determined by Geno2pheno FPR5%, Geno2pheno FPR10%, the combined criteria and the 11/25 rule, respectively. A concordance of 82.6% was observed between Geno2pheno FPR5% and the combined criteria for CRF01_AE. The results were nearly similar for the comparison between Geno2pheno FPR5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR10%. Neither HIV viral load, nor current or nadir CD4 was associated with the discordance rate between the different algorithms. ConclusionGeno2pheno predicted more X4-tropic viruses for this set of CRF01_AE sequences than the combined criteria or the 11/25 rule alone. For a conservative approach, Geno2pheno FPR5% seems to be a good compromise to predict CRF01_AE tropism.

Performance of genotypic algorithms for predicting tropism of HIV-1CRF02_AG subtype

BackgroundSeveral genotypic rules for predicting HIV-1 non-B subtypes tropism are commonly used, but there is no consensus about their performances. ObjectivesThree genotypic methods were compared for CRF02_AG HIV-1 tropism determination. Study designV3 env region of 178HIV-1 CRF02_AG from Pitié-Salpêtrière and Saint-Antoine Hospitals was sequenced from plasma HIV-1 RNA. HIV-1 tropism was determined by Geno2Pheno algorithm, false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25 and net charge rule. ResultsA concordance of 91.6% was observed between Geno2pheno 5% and the combined criteria. The results were nearly similar for the comparison between Geno2pheno 5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR 10%. A lower nadir CD4 cell count was associated with a discordance of tropism prediction between Geno2pheno 5% and the combined criteria or the 11/25 rule (p=0.02 and p=0.03, respectively). A lower HIV-1 viral load was associated with some discordance for the comparison of Geno2pheno 10% and the combined rule (p=0.02). ConclusionGeno2pheno FPR 5% or 10% predicted more X4-tropic viruses for this set of CRF02_AG sequences than the combined criteria or the 11/25 rule alone. Furthermore, Geno2pheno FPR 5% was more concordant with the 11/25 rule and the combined rule than Geno2pheno 10% to predict HIV-1 tropism. Overall, Geno2pheno 5% could be used to predict CRF02_AG tropism as well as other genotypic rules.

METHODS FOR DETERMINATION OF HIV-1 TROPISM: THE CURRENT STATE OF THE PROBLEM

The procedures, advantages, and disadvantages of the phenotyping and genotyping methods for determination of HIV-1 tropism are reviewed. The results of a genotyping determination of HIV tropism and subtypes based on sequencing of the V3 region of the gene coding for HIV envelope gp120 glycoprotein from 98 patients are presented. Lifelong use of combined antiretroviral therapy results in the need to introduce novel classes of drugs, such as CCR5 antagonists, and novel test for HIV tropism. At present, the most convenient in the clinical practice are genotyping methods based on gp120 V3-loop sequencing, which are informative and affordable. One of advantages of genotyping is the opportunity to determine HIV tropism and subtype simultaneously. Accumulating the results of forecasting non-B HIV subtypes will make it possible, with time, to enhance the accuracy of using the method in patients infected with different HIV subtypes.

Determination of HIV-1 coreceptor tropism using proviral DNA in women before and after viral suppression.

BackgroundAn 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12981-015-0055-x) contains supplementary material, which is available to authorized users.

High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA

Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism.

Genotypic Tropism Prediction from Paired Cell and Plasma Using Single and Replicate Sequences

HIV-1 tropism determination is necessary prior to CCR5 antagonist use as antiretroviral therapy. Genotypic prediction of coreceptor use is a practical alternative to phenotypic tests. Cell DNA and plasma RNA-based prediction has shown discordance in many studies. We evaluate paired cell and plasma either as single or replicate V3 sequences to assess prediction comparability. The HIV-1 partial env region was sequenced and tropism was predicted using geno2pheno and position-specific scoring matrices (PSSM). Nucleotide ambiguities at V3 were quantified and genetic distance (Protdist) was determined using BioEdit. Wilcoxon signed-rank test, t tests, and Spearman correlation were performed with Prism GraphPad5.0. Results are expressed as medians, with a level of significance of p<0.05, two tailed. Single (n=28) or replicate (n=26) paired cell/plasma sequences were obtained from 54 patients. Although the clonalfalse-positive rate (FPR) value from both compartments strongly correlated (r=0.86 p<0.0001), discordance in tropism prediction was observed in both singles and replicates using geno2pheno or PSSM. Applying clonalFPR(10%) 46% (25/54) were X4 tropic, with a plasma/cell discordance of 11% in singles and 23% in replicates. Genetic distance (p<0.0001) and clonalFPR value dispersion (p=0.003) were significantly higher among replicate sequences from cells. Discordance of viral tropism prediction is not uncommon and the use of replicates does not decrease its occurrence, but improves X4 sensitivity. Sequences from provirus had greater genetic distance and dispersion of clonalFPR values. This may suggest that DNA replicate assays may better represent the diversity of HIV-1 variants, but the clinical significance of these findings needs further evaluation.

Performance of a clonal-based HIV-1 tropism phenotypic assay

Adequate determination of HIV-1 tropism is important in clinical and research settings. Genotypic and phenotypic approaches to evaluate tropism have been described. Phenotypic assays are widely used to determine HIV-1 tropism because of their sensitivity to detect minor CXCR4-using variants (X4). However they cannot differentiate mixed quasi-species of R5 and X4 viruses from dual-tropic viruses. We describe here a clonal-based HIV-1 tropism phenotypic assay. Env-pseudo-typed viruses were produced by co-transfection of the env expression plasmid pcDNA3.1/V5HisTOPO and a backbone vector pNL4-3.Luc.E-R- that expresses the entire HIV-1 genome except for env and vpr in 293T cell cultures. Co-receptor use was tested by infecting U87.CD4.CCR5+ and U87.CD4.CXCR4+ cells in the presence or absence of co-receptor inhibitors, using 10 clones from each sample. The ability of the assay to detect minor variants in a viral population was assessed by mixing X4 and R5 clones using different ratios. Both R5 and X4 minority variants were detected when present at greater than 0.4% in a mixture of envelope populations. This assay can be useful in both clinical and research laboratories.

Reply to “Issues on Results of the External Quality Assessment for Proviral DNA Testing of HIV-1 Tropism in the Maraviroc Switch Collaborative Study”

We appreciate the comments by Berg et al. regarding our article. First, the aim of the quality assessment program reported in this study was not to standardize the methodology across the network of laboratories involved but to assess the outcome of laboratories testing clinical material using their laboratory’s in-house standard protocol for determination of HIV-1 tropism using proviral DNA. As MARCH is an international study, the different HIV subtype distribution necessitated having nonstandardized methodologies and, hence, use of different primers required to amplify different clades. We agree that samples had a high degree of variability, with some samples having a very wide range in their false-positive rates (FPRs), and these were solid findings. Our external quality assessment (EQA) samples were derived from real patient samples and deliberately selected to be similar to those recruited to MARCH, i.e., long-term aviremic patients. The use of only clones or other homogeneous samples would therefore not be a true representation of the genetic diversity of real patient samples and would have led to a misrepresentation of the performance of laboratories testing such samples for diagnostics. One could have separately evaluated the two different issues, diversity of samples and diversity of laboratory performance. However, we presented the data in one single examination to assess the overall impact of such material as an international quality assessment program. Furthermore, it was interesting that Berg et al. compared our study to that of Svicher et al. This study used samples from viremic patients (10,000 copies/ml) and tested HIV RNA, which is more likely to deliver uniform results because the circulating virus is likely to reflect the fittest clone/quasispecies circulating in that patient at that time. The DNA in long-term aviremic patients is likely to be far more heterogeneous, reflecting multiple quasispecies of archived virus of varying fitness. We felt very strongly that a clonal panel would not be helpful for the purposes of our program. The observed variability validates the importance of performing this EQA, and these data will be critical for interpreting the results of the MARCH study itself. We acknowledge that triplicate testing is costly and not feasible in all laboratory settings. Nevertheless, we felt triplicate testing was essential, considering both the inherent variability of proviral DNA and the lack of information from rigorously conducted randomized clinical trials, to the clinical relevance of this test. Therefore, we consider it far more important to detect X4 viruses and be conservative in terms of patient safety. Overall, and for the reasons described both in the article and in this response, we do not believe that singlicate testing of a clonal standard would reliably ensure that a patient’s virus would likely respond to the protocol drug, maraviroc. In summary, we feel that Berg et al. have misinterpreted the intent of the EQA program established specifically for MARCH. Proviral DNA, obtained from real patient samples, was associated with a high degree of variability, and the most suitable approach for quality assessment for determination of viral tropism was developed for this study.

Correction: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG

Correction: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG

The Genotypic False Positive Rate Determined by V3 Population Sequencing Can Predict the Burden of HIV-1 CXCR4-using Species Detected by Pyrosequencing

ObjectiveThe 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).Methods54 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.ResultsBy 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%).ConclusionsFPR 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.

Phenotyping methods for determining HIV tropism and applications in clinical settings

HIV-1 enters CD4-expressing cells via one or both of the chemokine receptors CCR5 and CXCR4. Specific CCR5 antagonists are now in clinical use, but only for CCR5-tropic viruses. Hence, several methods have been developed for assessing HIV-1 tropism in patients who are candidates for CCR5 antagonists. This article reviews current data on phenotypic assays of tropism. Phenotypic assays are still used as reference, although genotypic methods have improved. The main advantages of phenotypic assays are their great sensitivity for detecting minor CXCR4-using variants and their capacity to assess non-B subtypes of HIV-1. Clinical trials of maraviroc have, thus, relied on the phenotypic determination of HIV-1 tropism. However, new genotypic approaches that are more sensitive for minor CXCR4-using variants, notably ultra-deep pyrosequencing, are now challenging phenotypic assays. Nevertheless, phenotypic assays are essential for improving genotypic algorithms for determining HIV-1 tropism as well as for assessing the resistance of R5-tropic viruses to CCR5 antagonists. HIV-1 tropism should be determined before using CCR5 antagonists. Phenotypic recombinant assays are still the benchmark tests for characterizing HIV-1 tropism as their great sensitivity enables them to detect minor CXCR4-using variants of both B and non-B HIV-1 subtypes.

Tropismo del VIH. Técnicas disponibles y utilidad

Determination of HIV-1 tropism is mandatory before using CCR5 antagonists in clinical practice. One drug of this class, maraviroc, has been approved for the treatment of HIV infection. The phenotypic assay, TrofileTM, was clinically validated in the clinical development program of maraviroc and has been widely used to select candidates for maraviroc therapy. Phenotypic tests, however, have the disadvantage of being complex, are costly and time-consuming, and their accessibility is limited, which hampers their routine use in clinical diagnosis. Genotypic assays, based on sequencing the third hypervariable (V3 loop) of the viral gene env, interpreted according to various genotypic bioinformatic tools, such as geno2pheno and PSSM, are faster and cheaper than phenotypic assays, and are also more accessible. In retrospective analyses of the maraviroc pivotal trials, genotypic methods using either conventional ("bulk") or deep-sequencing technology predicted virologic response to maraviroc similarly to phenotypic assays and are now included within several European recommendations to guide the clinical use of CCR5 antagonists.

Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism

To determine whether genotyping of HIV-1 by duplicate PCR amplification of the region encoding the V3 loop is more sensitive than single PCR for detecting CXCR4-using viruses. The V3 genotypes of the HIV-1 infecting 152 patients enrolled in the multicentre GenoTropism ANRS study were determined by all the participating laboratories using a single PCR and V3 bulk sequencing. In parallel, one laboratory determined the V3 genotype using duplicate PCR and bulk sequencing of pooled amplicons. HIV tropism was predicted with the geno2pheno10 algorithm. The phenotypes of all samples were determined with the Trofile assay and the Toulouse tropism test (TTT) recombinant virus assay. Geno2pheno10 was 56.8% sensitive and 75.9% specific when compared with the Trofile assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and bulk sequencing of the pooled PCR amplicons increased the sensitivity to 68.2% and specificity to 79.6%. Geno2pheno10 was 64.1% sensitive and 77.0% specific when compared with the TTT assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and sequencing of the pooled PCR amplicons increased sensitivity to 76.9% and specificity to 80.5%. The genotypic determination of HIV-1 tropism can be improved by duplicate amplifications and sequencing the pooled PCR products. This is a good compromise between improved sensitivity and reasonable cost for the genotype-based determination of tropism.

Genotypic Prediction of Human Immunodeficiency Virus Type 1 Tropism by Use of Plasma and Peripheral Blood Mononuclear Cells in the Routine Clinical Laboratory

We developed a sequencing assay for genotypic HIV-1 tropism determination. The assay allows examination of HIV RNA from plasma and HIV DNA from peripheral blood mononuclear cells (PBMC), including PBMC samples from patients with undetectable viral loads. Assessment of 100 pairs of plasma and PBMC samples showed a high concordance of 90%. With the limitations of population-based sequencing, the assay was found to be robust and suitable for the routine clinical laboratory.

A Highly Sensitive and Specific Model for Predicting HIV-1 Tropism in Treatment-Experienced Patients Combining Interpretation of V3 Loop Sequences and Clinical Parameters

Phenotypic assays are considered the gold standard for HIV-1 tropism assessment. However, they are expensive and not widely available. Genotypic assays may provide an easier alternative, but their sensitivity remains low. We hypothesize that combining clinical data with V3 sequences may improve the diagnostic accuracy of genotypic tools. We analyzed clinical and biological data from 159 HIV-1-infected adults, 88 (56%) of whom were treatment experienced. Coreceptor phenotype was performed with Trofile and ES Trofile assay. V3 loop sequences were interpreted according to genotypic algorithms available at website. Multivariate logistic regression analyses were used to identify variables predicting HIV-1 tropism. Cut-off values for the prediction of CXCR4-using virus were defined. A total of 170 samples with phenotypic and genotypic determination of HIV-1 tropism were included. When only treatment-experienced patients were selected, a predictive model of HIV-1 tropism had an area under the receiver operating characteristic curve of 0.966 (95% confidence interval: 0.930 to 1.000, P < 0.001). The equation of the model included 2 bioinformatic tools (Geno2pheno-clinical model and net charge rule), the false positive rate score of Geno2pheno, and the following clinical data: exposure to more than 3 antiretroviral classes, years since HIV infection diagnosis and log10 HIV-1 RNA. A cut-off value ≥ 5.75 showed the highest accuracy to predict CXCR4 usage (96.6% sensitivity and 92.3% specificity). A genotypic-clinical model is highly accurate in predicting phenotypic tropism of HIV-1 in treatment-experienced patients. This may provide a cheap and rapid tool to select candidates for treatment with CCR5 antagonists in a routine clinical setting.

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment

BackgroundDetermination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care.MethodsForty-nine consecutive plasma samples for which an original TrofileTM assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call.ResultsAccording to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with TrofileTM population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value.ConclusionsIn line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.

Genotypic Determination of Hiv-1 Tropism in the Clinical Setting

HIV enters cells via the CD4 receptor and a coreceptor, generally CCR5 or CXCR4. The specific coreceptor used by a patient’s virus is referred to as its tropism. Tropism testing is necessary prior to treatment with CCR5 antagonist medication to rule out the presence of CXCR4-using (X4) virus, with the phenotypic Trofile™ assay being the most commonly used test for HIV coreceptor usage. Genotypic tropism testing may offer some practical advantages to phenotypic tropism testing and Trofile. Genotypic tropism assays are typically based on sequencing the V3 loop of HIV env and analysis using bioinformatic algorithms to infer the likely coreceptor usage of the virus. Genotypic methods have been refined and improved over the years and have recently been used as retrospective (and occasionally prospective) screening tools for treatment with CCR5 antagonist medication, such as maraviroc. Alternative approaches to genotypic tropism testing include heteroduplex tracking assays, ‘deep’ V3 sequencing and testing of cell-associated HIV DNA. Genotyping for HIV tropism is a promising tool for determining whether patients will respond to a CCR5 antagonist.

Correlation between genotypic predictions based on V3 sequences and phenotypic determination of HIV-1 tropism

Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. The 103 patients had a median CD4+ T lymphocyte count of 268 x 10(6)cells/l and nadirs of 98 x 10(6)cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.