Limited Evolution of Inferred HIV-1 Tropism while Viremia Is Undetectable during Standard HAART Therapy

<div><p>Background</p><p>HIV patients on suppressive antiretroviral therapy have undetectable viremia making it impossible to screen plasma HIV tropism if regimen change is required during suppression. We investigated the prevalence and predictors of tropism switch from CCR5-using (“R5”) to non-CCR5-using (“non-R5”) before and after viral suppression in the initially therapy-naïve HOMER cohort from British Columbia, Canada.</p><p>Methods</p><p>We compared pre-therapy and post-suppression viral genotypic tropism in patients who initiated on PI/NNRTI-based antiretroviral regimens between 1996-1999 (n = 462). Virologic suppression was defined as having two consecutive viral loads of <500 copies/mL, which was the sensitivity limit of most viral load assays at the time. Viral tropism was inferred by V3-loop-population-sequencing and geno2pheno_[coreceptor] with cutoff at 5.75% false positive rate (FPR).</p><p>Results</p><p>When virologic suppression was defined as two-consecutive viral loads <500 copies/mL, 34 (9%) of the 397 patients with pre-therapy R5-virus switched to non-R5 at viral load rebound after a median of 19 months (IQR 8–41 months) of undetectable viremia. Duration of viral load suppression was not a predictor of switch, but lower CD4 count during suppression (median 400 versus 250 cells/mL) and an increased prevalence of pre-therapy non-R5 HIV by “deep” sequencing (median 0.2% versus 3.2%) were independently associated with switch (p = 0.03 and p<0.0001, respectively).</p><p>Conclusion</p><p>R5-to-non-R5 tropism switches in plasma virus after undetectable viremia were relatively rare events especially among patients with higher CD4 counts during virologic suppression. Our study supports the use of pre-suppression tropism results if maraviroc is being considered during virologic suppression in this subgroup of patients.</p></div

Study flow chart of our primary analysis: Virologic suppression definition: <500 copies/mL; geno2pheno_[coreceptor] FPR cutoff 5.75%.

<p>Study flow chart of our primary analysis: Virologic suppression definition: <500 copies/mL; geno2pheno_[coreceptor] FPR cutoff 5.75%.</p

454 “deep” sequencing results of pre-therapy “R5” samples by population sequencing.

<p>In patients with pre-therapy baseline R5-viruses (n = 156), “deep” sequencing reveals that the prevalence of non-R5 viruses before starting HAART was a significant predictor of R5-to-non-R5 change (p<0.0001, Mann-Whitney test). Median non-R5 prevalence by “deep” sequencing among subjects who were tested as having R5 virus that remained R5 by population sequencing was 0.2% in comparison to 3.2% among those who had switched from R5 to non-R5. Horizontal bars indicate median values. For graphing and visualization purposes, values less than or equal to 1 were given randomized numbers between 0.01 and 0.8 such that samples with <1% non-R5 prevalence would randomly disperse across the plot from −2 to −0.1 log copies/mL. The dotted line at 2% non-R5 represents our group's optimized cutoff value (>2% non-R5 sequences) used for dichotomizing samples into non-R5 or R5. The dashed line at 20% represents the approximate sensitivity limit of population sequencing; five samples in this figure had %non-R5 above this sensitivity limit indicating 454 and population sequencing discordance. Detailed examination of these five samples suggested the high %non-R5 observed was a summation effect from multiple less prevalent non-R5 sequences in four, and was due to random sampling bias in one sample (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099000#pone.0099000.s002" target="_blank">Table S1</a>).</p

Baseline and post-therapy characteristics of all subjects (column 1 “All Subjects n = 462”) followed by the same dataset stratified by tropism switch categories determined by population-sequencing (columns 2–5).

a<p>Age was categorized as follows: under 30, 30–39, 40–49, and 50 or more.</p>b<p>Duration (in months) between HAART-initiation and virologic suppression defined as 500 copies/mL.</p>c<p>Adherence ≥95% was defined as ≥95% compliance of prescription refills over first 12 months of therapy initiation.^d PI-containing therapy: drug category of a patient's first HAART therapy.</p>e<p>NNRTI-containing therapy: drug category of a patient's first HAART therapy</p>f<p>p-values were calculated based on comparisons between groups “Remained R5” and “R5-to-non-R5”.</p>g<p>Fisher's Exact test comparing PI and NNRTI-containing therapy against Remained R5 and R5-to-non-R5 switch.</p