Detection of very low levels of cell-associated HIV-1 RNA in well suppressed children (CARMA study)

Authors: K. Gärtner, T. Gkouleli, J. Heaney, P. Grant, S. Domínguez-Rodríguez, E. Busby, D.M. O’Sullivan, M. Spyer, J.F. Huggett, E. Nastouli on behalf of the EPIICAL consortium

2019/09/16

The following poster was presented by Kathleen Gärtner on behalf of the EPIICAL consortium at the HIV Cure and Reservoir Symposium held at the University of Ghent, Belgium on 16 – 17 September 2019. The Symposium comprehensively summarized the state of the art in HIV cure strategies and diagnostic tools to monitor therapeutic interventions.

Background: Two methods (qPCR and dPCR) were used to quantify cell-associated HIV-1 RNA (CA-RNA) in a well-suppressed, early treated (ET) cohort of 40 perinatally infected children (CARMA). Total and unspliced (US) CA-RNA was measured to complement studies for ongoing HIV replication and/or immune activation in HIV-1 infected individuals.

Total and US CA-RNA were measured in PBMC extracts from 40 individuals using qPCR and dPCR. Copy numbers per reaction were very low for qPCR (mean 7.4) and dPCR (mean 1.3). Total and US CA-RNA was not detected in 9 and 17 of the 40 samples, respectively, by either method. The Bland-Altman concordance analysis showed 97.4% agreement for both, total and US CA-RNA, between qPCR and dPCR, suggesting comparability of the methods for detecting low copy numbers of CA-RNA in this cohort.

Copy numbers of US CA-RNA were generally lower than total CA-RNA, indicating expression of different forms of HIV RNA in general and very low expression of full-length RNA in early treated well-suppressed children. In addition, we measured viral load in plasma by an ultrasensitive method (VL). Values of VL were lower than CA-RNA and there was no correlation between them.

Taken together, our data show that expression of HIV-1 CA-RNA in ET well-suppressed children can be detected by qPCR and dPCR at very low levels. Further work including full-length single genome sequencing is ongoing to investigate whether presence of CA-RNA is correlated with immune activation, and/or if expression of full-length HIV RNA is derived from replication competent proviruses that contribute to the cell-free plasma VL.