Comparative determination of HIV-1 co-receptor tropism by Enhanced Sensitivity Trofile, gp120 V3-loop RNA and DNA genotyping

Prosperi, Mattia; Bracciale, Laura; Fabbiani, Massimiliano; Giambenedetto, Simona Di; Razzolini, Francesca; Meini, Genny; Colafigli, Manuela; Marzocchetti, Angela; Cauda, Roberto; Zazzi, Maurizio; Luca, Andrea De

doi:10.1186/1742-4690-7-56

Cited by 52 publications

(40 citation statements)

References 35 publications

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“…In parallel, the same patients were analyzed with the TrofileES test. Previous studies have confirmed the good agreement between the phenotypic format of TrofileES and the genotyping tool Geno2Pheno on a retrospective comparison of a large study population in clinical studies (47,48). As further confirmation, we also performed a parallel analysis with TrofileES on a limited set of 20 clinical samples.…”

Section: Resultssupporting

confidence: 73%

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

et al. 2015

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dKey clinical studies for HIV coreceptor antagonists have used the phenotyping-based Trofile test. Meanwhile various simplerto-do genotypic tests have become available that are compatible with standard laboratory equipment and Web-based interpretation tools. However, these systems typically analyze only the most prominent virus sequence in a specimen. We present a new diagnostic HIV tropism test not needing DNA sequencing. The system, XTrack, uses physical properties of DNA duplexes after hybridization of single-stranded HIV-1 env V3 loop probes to the clinical specimen. Resulting "heteroduplexes" possess unique properties driven by sequence relatedness to the reference and resulting in a discrete electrophoretic mobility. A detailed optimization process identified diagnostic probe candidates relating best to a large number of HIV-1 sequences with known tropism. From over 500 V3 sequences representing all main HIV-1 subtypes (Los Alamos database), we obtained a small set of probes to determine the tropism in clinical samples. We found a high concordance with the commercial TrofileES test (84.9%) and the Web-based tool Geno2Pheno (83.0%). Moreover, the new system reveals mixed virus populations, and it was successful on specimens with low virus loads or on provirus from leukocytes. A replicative phenotyping system was used for validation. Our data show that the XTrack test is favorably suitable for routine diagnostics. It detects and dissects mixed virus populations and viral minorities; samples with viral loads (VL) of <200 copies/ml are successfully analyzed. We further expect that the principles of the platform can be adapted also to other sequence-divergent pathogens, such as hepatitis B and C viruses. The predominant virus variant in early stages of the clinical manifestation of disease, CCR5-tropic HIV, is found in approximately 80% of treatment-naive patients (1, 2). Although this number can vary for the different virus subtypes, the percentage of CXCR4-tropic HIV isolates is generally low and tends to rise with disease progression (3-6). Nevertheless, the fraction of CCR5-tropic viruses in clinical specimens continues to stay at Ͼ50% throughout the course of infection (7,8). As such, the molecular interactions between the viral envelope and the cellular chemokine receptor CCR5 were recognized as potentially attractive targets for drug development and have yielded compounds and drugs able to specifically block CCR5-tropic HIV (9-11). It is this selectivity of the inhibition of one (CCR5) and not the other (CXCR4) viral coreceptor that necessitates tropism testing prior to prescribing drugs of this particular class. Although the chemokine receptor binding site in the HIV envelope is constituted mainly by the V3 loop, the V1/V2 regions, and the C4 conserved region in the HIV protein gp120, coreceptor tropism is dictated predominantly by amino acid sequences of the V3 region (12, 13). But also sequences of other variable env regions can contribute as secondary sites to the viral tropism (14-17).Initially, all...

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Section: Resultssupporting

confidence: 73%

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

et al. 2015

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“…In this context, proviral DNA may be considered as a source of viral genetic material for GTT. Although evidence for a close correlation of GTT results obtained with plasma RNA and proviral DNA has previously been reported, those studies included a small number of Table 2 Correlation between phenotypic tropism prediction (PTT) and genotypic tropism prediction (GTT), using the results of PTT as the standard, for PTT carried out using (a) the MT2 assay, (b) the original Trofilet assay, (c) the enhanced Trofilet assay and (d) any of these assays, for an overall comparison patients [19][20][21]. The aim of the present study was to explore the possibility of using proviral DNA for GTT, by comparing large series of both simultaneous plasma RNA and proviral DNA samples from patients with a viral load of 4500 copies/ mL, and current proviral DNA samples and stored plasma RNA samples collected from treated patients with a current viral load of o500 copies/mL.…”

Section: Discussionmentioning

confidence: 99%

Concordance between HIV-1 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA

et al. 2011

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ObjectiveThe aim of the study was to evaluate the use of proviral DNA as a source of viral genetic material for genotypic coreceptor tropism testing (GTT). MethodsGTT consisted of bulk V3 sequencing followed by geno2pheno interpretation with the interpretative cut-off [false positive rate (FPR)] set at 5 and 10%. GTT was performed for 165 patients with a viral load of 4500 HIV-1 RNA copies/mL on simultaneously collected plasma RNA and proviral DNA, and for 126 patients with a viral load of o500 copies/mL on current proviral DNA and pretreatment plasma RNA. Phenotypic tropism testing (PTT) results were available for 142 samples. ResultsIn the simultaneous RNA/DNA comparison, concordance in prediction was 95.2% (at FPR 10%) and 96.4% (at FPR 5%). Six RNA-R5/DNA-X4 and two RNA-X4/DNA-R5 discordances were observed at an FPR of 10%, and six RNA-R5/DNA-X4 discordances were observed at an FPR of 5%. In the longitudinal RNA/DNA comparison, concordance was 88.1% (at FPR 10%) and 90.5% (at FPR 5%). Eight RNA-X4/DNA-R5 and seven RNA-R5/DNA-X4 discordances were seen at an FPR of 10%, and 10 RNA-R5/DNA-X4 and two RNA-X4/DNA-R5 discordances at an FPR of 5%. The overall concordance of RNA GTT with PTT was 82% (at FPR 10%) and 83% (at FPR 5%). The overall concordance of DNA GTT with PTT was 85% (at both 10 and 5% FPRs). ConclusionsGTT produced highly concordant tropism predictions for proviral DNA and plasma RNA. GTT on proviral DNA offers a promising approach for tropism prediction in clinical practice, particularly for the assessment of treated patients with low or suppressed viraemia.Keywords: chemokine (C-C motif) receptor 5 (CCR5) inhibitors, genotypic tropism testing, HIV-1 coreceptor, HIV-1 proviral DNA Accepted 25 January 2011Introduction Chemokine (C-C motif) receptor 5 (CCR5) antagonists, members of the class of HIV-1 entry inhibitors, selectively inhibit the replication of CCR5-using (R5) viral strains. Before introducing a CCR5 antagonist as a component of antiretroviral therapy (ART), coreceptor usage, or viral tropism, must be determined to exclude the possibility of the presence of chemokine (C-X-C motif) receptor 4 (CXCR4)-using (X4) strains, as these are associated with poor virological response to the drug [1]. The output of the earliest HIV-1 phenotypic tropism testing (PTT) assay was the formation of syncytia in cultured MT2 cells after virus inoculation. This assay is less well suited for use in routine clinical practice because of inherent difficulties with Correspondence: Prof. Chris Verhofstede, AIDS Reference Laboratory, University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. Tel: 1 32 9 332 51 61; fax: 1 32 9 332 38 41; e-mail: Chris.verhofstede@ugent.be DOI: 10.1111/j.1468-1293.2011.00922.x HIV Medicine (2011 GTT is based on analysis of the V3-loop sequence of the HIV-1 envelope (env) gene using bioinformatic prediction models to deduce coreceptor usage. GTT has the advantage of being less technically demanding, more rapid and less expensive than PTT, thereby meeting today's need for a fast and...

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“…These results, however, must be interpreted with caution given the fact that tropism was determined only in part of the maraviroc failures, which cannot exclude selection bias, and that different assays for tropism determination were employed at baseline and during follow-up, although concordance between genotyping, and Trofile or ESTA is expected to be high (80% or higher). 25,31 In conclusion, we observed significant virological and immunological responses with maraviroc-based regimens in antiretroviral-experienced patients. Any of the approved methods for determining R5 tropism is suitable for predicting virological and immunological responses in a real world setting.…”

Section: Rossetti Et Almentioning

confidence: 62%

Virological and Immunological Response to Antiretroviral Regimens Containing Maraviroc in HIV Type 1-Infected Patients in Clinical Practice: Role of Different Tropism Testing Results and of Concomitant Treatments

Rossetti¹,

Bianco

Bellazzi

et al. 2014

AIDS Research and Human Retroviruses

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We assessed the immunovirological response to antiretroviral regimens containing maraviroc in HIV-infected viremic patients with viral tropism predicted by different assays. We selected antiretroviral treatmentexperienced HIV-1-infected patients initiating regimens containing maraviroc after different phenotypic or genotypic viral tropism assays, with at least one HIV-1 RNA determination during follow-up. Survival analysis was employed to assess the virological response as time to HIV-1 RNA < 50 copies/ml and immunological response as time to a CD4 cell count increase of ‡ 100/ll from baseline. Predictors of these outcomes were analyzed by multivariate Cox regression models. In 191 treatments with maraviroc, virological response was achieved in 65.4% and the response was modestly influenced by the baseline viral load and concomitant drug activity but not influenced by the type of tropism assay employed. Immunological response was achieved in 58.1%; independent predictors were baseline HIV-1 RNA (per log 10 higher: HR 1.29, 95% CI 1.05-1.60) and concomitant therapy with enfuvirtide (HR 2.05, 0.96-4.39) but not tropism assay results. Of 17 patients with baseline R5-tropic virus and available tropism results while viremic during follow-up on maraviroc, seven (41%) showed a tropism switch to non-R5 virus. A significant proportion of experienced patients treated with regimens containing maraviroc achieved virological response. The tropism test type used was not associated with immunovirological response and concomitant treatment with enfuvirtide increased the chance of immunological response. More than half of virological failures with maraviroc were not accompanied by tropism switch.

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Comparative determination of HIV-1 co-receptor tropism by Enhanced Sensitivity Trofile, gp120 V3-loop RNA and DNA genotyping

Cited by 52 publications

References 35 publications

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

Concordance between HIV-1 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA

Virological and Immunological Response to Antiretroviral Regimens Containing Maraviroc in HIV Type 1-Infected Patients in Clinical Practice: Role of Different Tropism Testing Results and of Concomitant Treatments

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