Fig. 5.

Comparison of single cell HIV-1 usRNA results by scdPCR with traditional quantitative PCR measures of us RNA from participants on ART. usRNA copies/10⁶ CD4 + T cells by conventional assay on bulk lysates increased after 24 h of TCR stimulation by αCD3/αCD28 antibodies in all participants with undetectable plasma HIV load measurements at the time of blood collection (A, B, C, D). Three of these four individual (A, B, and D) also experienced an increase in usRNA copies ~ 18 h after romidepsin pulse. Despite these expected increases in bulk usRNA levels, changes in the number of cells producing usRNA as measured by scdPCR after stimulation were variable, and at times, discordant with results from traditional RNA measures. For example, despite a > 1 log10 increase in bulk RNA following HDAC inhibition for participant A, the number of cells expressing usRNA decreased. Conventional qPCR results were more variable in participants with low-level detectable viremia on ART at the time of sample collection (E, F, G). Assays were repeated two to four times with the exception of participant G (limited sample), and error bars represent standard error of the mean between independent experiments. Reproducibility between independent experiments was observed, and all scdPCR values were greater than the both the detection limit of the assay (1 log₁₀ RNA expressing CD4 + T cells) and limit of linearity (2 log₁₀ cells).