TROCAI (Tropism Coreceptor Assay Information): a New Phenotypic Tropism Test and Its Correlation with Trofile Enhanced Sensitivity and Genotypic Approaches

Abstract

The only clinically validated assay available to determine HIV tropism is Trofile, an assay that possesses some limitations. Our first aim was to develop a new phenotypic tropism test (TROCAI [tropism coreceptor assay information]) and to categorize results generated by this test according to the virological response to a short-term exposure to the CCR5 receptor antagonist maraviroc (maraviroc clinical test). Our second aim was to compare TROCAI results to those obtained by Trofile enhanced sensitivity (ES) and to different genotypic algorithms. TROCAI assayed HIV tropism in 33 HIV-infected patient viral isolates obtained from a modified coculture, followed by multiple infection cycles of indicator cells. TROCAI obtained a reportable result in all patients with viral loads of >500 HIV RNA copies/ml and in 3/6 patients with <500 HIV RNA copies/ml (30/33 patients, 91.9%). Patients who responded to maraviroc had an X4-using virus proportion in indicator cell supernatant of 0 to 0.41%. Hence, we used the threshold of 0.5% to categorize TROCAI results as R5 (<0.5%) or dual/mixed (>0.5%). The concordance between TROCAI and Trofile (ES) was 22/24 (91.6%), and with genotypic approaches it was 22/26 (84.6%). TROCAI results, which were categorized in this study by the maraviroc clinical test, could be used as a test in addition to those currently used to select patients for treatment with CCR5 antagonists.

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

“…Plasma samples from 114 HIV-infected individuals screened to be treated with a maraviroc-containing regimen, a subset of samples from the same Seville and Madrid cohorts of patients, were analyzed using the novel deep-sequencing-based HIV-1 coreceptor tropism assay. These results were compared with those from a series of genotypic (population sequencing) and phenotypic (ESTA [42], TROCAI [73], and VeriTrop (Fig. 5B).…”

“…Patients provided written informed consent, and the ethics committee of the hospital approved the study (72). HIV-1 coreceptor tropism in these samples was determined at baseline using two different phenotypic assays, i.e., the enhanced-sensitivity Trofile assay (ESTA) (42) and TROCAI (73), and by population sequencing analyzed with Geno2Pheno (27) with an FPR of 10%, according to the recommenda-tions from the European Consensus Group on the clinical management of HIV-1 tropism testing, as described on the Geno2Pheno website (http: //coreceptor.bioinf.mpi-inf.mpg.de/index.php).…”

Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

“…Moreover, the subtype B backbone (HIV-1 NL4-3 ) used to clone the patient-derived gp120/gp41 region was compatible not only with env fragments from subtype B wild-type and multidrugresistant strains but also with that from all non-B HIV-1 group M subtypes analyzed (35). Furthermore, the VERITROP assay is efficient and reproducible, with a turnaround time (11 days) comparable to other phenotypic HIV-1 tropism tests, such as the enhanced-sensitivity Trofile assay (16 days) (42), the RVA Toulouse-TTT (9 days) (57), and TROCAI (21 to 42 days) (53).…”

“…A problem with these methods is that prolonged culturing during virus isolation usually leads to changes in the original HIV-1 quasispecies population (52). Thus, a viable alternative has been the use of env recombinant viruses and reporter cell lines expressing HIV-1 receptor and coreceptors to evaluate HIV-1 coreceptor usage (16,18,53), including the only commercially available assay (Trofile; Monogram Biosciences, South San Francisco, CA) (17,42). VERITROP is based on the introduction of patient-derived env fragments into a noninfectious vector using yeast-based cloning (34,35) and a modified version of the ␣-complementation assay for env-mediated cell-to-cell fusion (19).…”

Correlation of the Virological Response to Short-Term Maraviroc Monotherapy with Standard and Deep-Sequencing-Based Genotypic Tropism Prediction Methods

“…However, this phenotypic assay has some limitations, including an approximately 20% rate of nonreportable results, and specimens must be shipped to the unique reference laboratory in the United States. For these reasons, other clinical (8), phenotypic (10,11,23), or genotypic (12) alternatives for determining viral tropism have been examined.…”