The dual-target approach in viral HIV-1 viremia testing: An added value to virological monitoring?

Abstract

New methods of HIV-1 RNA quantification based on dual-target detection are increasingly used in HIV viral load monitoring, but clinical implications and impact of dual-target detection on HIV-1 infection management are not established. Aptima HIV-1 Quant Dx assay is a last generation HIV viral load method, that uses pol and LTR as simultaneous target, providing quantitative results based mainly on pol target, while LTR target is used to report the results when pol signal is absent. In our laboratory, about 6% of results of all HIV-1 viral load tests performed with this platform in one year period resulted from LTR signal. Interestingly, LTR-based viremia (sometimes exceeding 1,000 copies/mL) was observed in a small proportion (up to 1%) of patients under ART, considered for long time virologically suppressed on the basis of a single target (pol-based) assay. Male gender, >700 vs <200 CD4 cell/mL and dual therapy including NRTI plus either NNRTI, or PI/b or INSTI were independently associated with increased risk of LTR-based HIV-1 viral load detection by multivariable logistic regression. A significant linear correlation was observed between LTR-based HIV-1 RNA levels and PBMC-associated proviral DNA. Moreover, in a small group of patients with HIV-1 RNA levels >200 copies/mL, longitudinal assessments showed parallel kinetics between plasma viremia and proviral DNA. Sequencing of pol region for drug resistance assessment in patients with LTR-based viremia failed on plasma HIV-1 RNA, while it was successful on proviral DNA. The detection/quantification of HIV-1 viremia based only on LTR signal with a dual target assay in samples resulting undetectable with the more conventional target pol needs accurate evaluation; unravelling the biological basis of this phenomenon, here described for the first time, is mandatory to establish relevance and implication by both pathogenetic (i.e. infectivity of LTR-detected viruses, reservoir turnover, immune activation, etc.) and clinical standpoint.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Distribution of samples (n = 148) that were quantified with the LTR signal according to HIV-1 RNA ranges (<50 copies/mL; 50–500 copies/mL; >500 copies/mL) by Aptima.

Fig 2. Correlation between LTR-detected HIV-1 RNA and PBMC-associated total HIV-1 DNA levels.

(A) Correlation between VL levels detected by LTR region and total HIV-1 DNA content measured in PBMC in 33 samples (p = 0.0063; 95% confidence interval of the regression, shown with dashed lines). (B) Kinetics of plasma HIV-1 RNA (blue square symbols) and PBMC-associated total HIV-1 DNA (red triangle symbols) in one representative patient, showing viremia repeatedly detected with LTR target.

Fig 3. Comparison of HIV-1 VL obtained with Aptima, RealTime and or CAP/CTM in longitudinal samples from two patients with HIV-RNA detected only with LTR signal.

Residual plasma samples quantified with LTR region by Aptima were re-tested with RealTime (n = 4 for patient A and n = 5 for patient B) and CAP/CTM (n = 2 for both patient A and B). Symbols: red square, RealTime result; green triangle, CAP/CTM result; blue diamond, Amtima LTR-based result. Light blue dot line indicates the conventional suppressed viremia, i.e. 50 copies/mL HIV-1 RNA.

Fig 4. Distribution of HIV-1 subtypes in patients whose VL result was based on the LTR or pol signal.

Dark red columns represent the HIV-1 subtype distribution (%) in patients whose viremia was detected with LTR region (n = 92); light grey columns refer to patients whose VL was calculated on the basis of pol signal (n = 1,749).