Background: Antiretroviral therapeutic regimens that contain CCR5 antagonists can lead to various shifts in HIV co-receptor usage within HIV patients. Therefore, a diagnostic method that can simultaneously detect and quantify the amount of both CXCR4-tropic and CCR5-tropic HIV will be useful to identify candidates for specific therapies (e.g., CCR5 antagonist treatment); to monitor changes in HIV viral co-receptor tropism in response to antiretroviral treatments; and to further define the role of CXCR4-tropic HIV in disease progression.

Methods: The qualitative form of the SensiTrop assay uses heteroduplex tracking technology to detect CXCR4-tropic HIV with numerous mutations in the V3 loop by forming heteroduplexes with CCR5 V3 probes. We modified this technique by generating fluorescently labeled single-stranded CCR5 V3 probes, which allow the simultaneous separation, detection, and quantitation of both CCR5/CCR5 homoduplex DNA hybrids and CXCR4/CCR5 heteroduplex DNA hybrids.

Results: Using the SensiTrop QT assay format, we determined the imprecision and the limit of quantification of this assay using different X4/R5 DNA mixtures with ratio ranges from 0% to 100% CXCR4/CCR5. The data show SensiTrop QT assay can reproducibly quantify X4/R5 ratios down to 5% with acceptable inter-assay coefficient of variant values ranging from 1.8% to 16.9%. We have also tested the SensiTrop QT assay using spiked plasma containing X4/R5 viral mixtures with ratio ranges from 1% to 50%. This data clearly demonstrates that the SensiTrop QT assay can also similarly quantify X4/R5 ratios at the level of viral RNA when the HIV viral load is >1000 HIV copies/mL. Lastly, in random HIV patient specimens, we have used the quantitative form of the SensiTrop QT assay to confirm that the qualitative form of the SensiTrop assay can accurately detect CXCR4-tropic HIV when it is present at only 1% of the amount of CCR5-tropic HIV.

Conclusions: We believe that the ability to rapidly and sensitively quantify co-receptor tropism usage in HIV patients will be a valuable new diagnostic tool to help physicians, researchers, and pharmaceutical companies monitor how changes in ART cause positive or negative shifts in the HIV co-receptor status.