Prolonged and Substantial Discordance in Prevalence of Raltegravir-Resistant HIV-1 in Plasma versus PBMC Samples Revealed by 454 “Deep” Sequencing

<div><p>The evolution of drug resistance mutations in plasma samples is relatively well-characterized. However, the viral population and diversity in other body compartments such as peripheral blood mononuclear cells (PBMC) remains poorly understood. Previous studies have mostly focused on protease and reverse transcriptase drug resistance mutations (DRMs). In this study, we used 454 “deep” sequencing technology to observe and quantify longitudinally the prevalence of resistance mutations associated with the integrase inhibitor, raltegravir, in plasma versus PBMC samples from a San Francisco-based cohort. Four heavily treatment-experienced subjects were monitored in this study over a median of 1.2 years since the initiation of raltegravir-containing regimens. We observed a consistent discordance in the prevalence of DRMs, but not resistance pathway(s), in the plasma versus PBMC viral populations. In the final paired samples that were tested while the subjects were on a raltegravir-containing regimen, DRM prevalence reached 100% in plasma but remained 1% in PBMC on day 177 post-therapy in Subject 3180 (Q148H/G140S), 100% in plasma and 36% in PBMC on day 224 in Subject 3242 (N155H), 78% in plasma and 11–12% in PBMC on day 338 in Subject 3501 (Q148H/G140S), and 100% in plasma and 0% in PBMC on day 197 in Subject 3508 (Y143R). Furthermore, absolute sequence homology comparison between the two compartments revealed that 21% - 99% of PBMC sequences had no match in plasma, whereas 14% - 100% of plasma sequences had no match in PBMC. Overall, our observations suggested that plasma and PBMC hosted drastically different HIV-1 populations even after a prolonged exposure to raltegravir selection pressure.</p> </div

Absolute sequence homology comparison revealed 21% to 99% of sequences derived from PBMC samples had no identical match in their plasma counterparts.

<p>An absolute sequence homology comparison revealed prolonged and substantial differences between plasma and PBMC viral populations. (A) Subject 3180, (B) Subject 3242, (C) Subject 3501, and (D) Subject 3508. 454 “deep” sequencing sampled an average of 3345 sequences from each sample. The forward 454 primer covered HIV-1 integrase (<i>int</i>) amino acids at approximately position 83–152 (210 bases), and the reverse primer covered approximately position 117–192 (228 bases). Within each sample, identical sequences were grouped, and the prevalence of each group of unique sequences was calculated. Next, a pair of plasma and PBMC collected from the same subject at the same time point was compared. Each group of identical sequences within a PBMC sample was compared with all the sequences derived from the plasma. The result was graphed on individual horizontal bars in this figure, in which each bar represents 100% of the total viral population sampled. When a match was found (defined by 100% nucleotide similarity and a minimum overlap of 73 bases and a maximum overlap of 228 bases), the corresponding sections in the horizontal bars were shaded white and a vertical/slanted line was drawn to link the two identical groups from the two compartments. Solid grey shading represents sequences that found no match. Note that the white sections represent groups of identical sequences, but in contrast the grey sections represent all sequences that found no match and thus represent a heterogeneous mixture of sequences.</p

Technical and Regulatory Shortcomings of the TaqMan Version 1 HIV Viral Load Assay

<div><h3>Background</h3><p>The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay.</p> <h3>Methods</h3><p>Between October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40–250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy (N = 279; median 3.2 months follow-up).</p> <h3>Results</h3><p>TaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40–250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36–283] copies/mL). In addition, in ∼2.4% of samples pVL was dramatically <em>underestimated</em> by TaqMan due to poor binding of the proprietary TaqMan primers.</p> <h3>Conclusions</h3><p>The replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure.</p> </div

Patients characteristics (N = 4).

<p>“Baseline” is defined as the initiation of a raltegravir-containing regimen. ABC (abacavir), 3TC (lamivudine), ETV (etravirine), RGV (raltegravir), FTC (emtricitabine), TDF (tenofovir), RTV (ritonavir), DRV (darunavir), MVR (maraviroc), TPV (tipranavir), HGH (human growth hormone).</p

Plasma viral load testing and reporting protocol in British Columbia between October 2009 and April 2010.

<p>All plasma samples were initially tested with the Roche COBAS AmpliPrep/COBAS TaqMan v1 HIV-1 Test (“TaqMan v1”) assay. TaqMan v1 pVL results <40 or ≥250 copies/mL were reported to physicians. Samples with TaqMan v1 pVL ≥40 and <250 copies/mL were re-tested by the Roche COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR UltraSensitive Test, version 1.5 (“Amplicor v1.5”). The Amplicor v1.5 test results were reported to physicians.</p

Simulated trajectories of genotype frequencies (solid and dashed lines) and population-level coreceptor usage phenotype (shaded regions) under the fitness valley and gradual models of HIV coreceptor usage evolution.

<p>Simulations were generated under a five-allele Moran model with mortality selection <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002753#pcbi.1002753-Muirhead1" target="_blank">[42]</a>, effective population size , forward mutation rate of per replication, and fitness vectors of (1, 1.025, 1.05, 1.075, 1.1) and (1, 0.999, 0.999, 0.999, 1.1) corresponding to gradual and valley landscapes, respectively. Note that the relatively rapid and complete fixation of the fifth variant is partly due to the model assumption of no back mutation, and is not consistent with the observation that CXCR4-using variants tend to remain a minority species in HIV infections.</p

Poor concordance between TaqMan v1 and Amplicor v1.5 at low plasma viral load levels (40

<p>–<b>250 HIV RNA copies/mL by TaqMan v1).</b> Between October 2009 and April 2010 plasma samples from British Columbia with low-level viremia (40–250 copies/mL) by the Roche TaqMan v1 assay (N = 1198) were systematically re-tested by the Amplicor v1.5 assay. Poor concordance was observed between TaqMan v1 and Amplicor v1.5 results, with 82% of values falling below the line of identity (red solid line). Points above the blue dashed line are samples in which TaqMan v1 <i>underestimated</i> pVL by ≥0.5 log₁₀ copies/mL relative to the Amplicor v1.5 assay.</p

Low-level viremia by TaqMan v1 does not predict short-term virological failure.

<p>A subset of patients (N = 279) with low-level viremia (40–250 copies/mL) by TaqMan v1 were followed longitudinally for a median of 3.2 months (IQR: 2.0–4.2 months). Patients initiated HAART at least 6 months prior, and treatment regimens remained unchanged over the course of follow-up. Samples from patients' latest follow-up visit were re-tested with the TaqMan v1 and Amplicor v1.5 assays. Overall 17% of patients had a detectable viral load by Amplicor v1.5 at their latest follow-up visit, while 38% were detectable by TaqMan v1. When patients were grouped according to their baseline TaqMan v1 pVL into 50 copies/mL strata we observed a stepwise increase in the proportion of patients with detectable pVL at their latest follow-up visit by both Amplicor v1.5 (red bars) and TaqMan v1 (blue bars). Consistent with previous results, more patients had detectable pVL by TaqMan v1 than by Amplicor v1.5 at follow-up in all strata.</p

“Deep” Sequencing Accuracy and Reproducibility Using Roche/454 Technology for Inferring Co-Receptor Usage in HIV-1

<div><p>Next generation, “deep”, sequencing has increasing applications both clinically and in disparate fields of research. This study investigates the accuracy and reproducibility of “deep” sequencing as applied to co-receptor prediction using the V3 loop of Human Immunodeficiency Virus-1. Despite increasing use in HIV co-receptor prediction, the accuracy and reproducibility of deep sequencing technology, and the factors which can affect it, have received only a limited level of investigation. To accomplish this, repeated deep sequencing results were generated using the Roche GS-FLX (454) from a number of sources including a non-homogeneous clinical sample (N = 47 replicates over 18 deep sequencing runs), and a large clinical cohort from the MOTIVATE and A400129 studies (N = 1521). For repeated measurements of a non-homogeneous clinical sample, increasing input copy number both decreased variance in the measured proportion of non-R5 using virus (p<<0.001 and 0.02 for single replicates and triplicates respectively) and increased measured viral diversity (p<0.001; multiple measures). Detection of sequences with a mean abundance less than 1% abundance showed a 2 fold increase in median coefficient of variation (CV) in repeated measurements of a non-homogeneous clinical sample, and a 2.7 fold increase in CV in the MOTIVATE/A400129 dataset compared to sequences with ≥1% abundance. An unexpected source of error included read position, with low accuracy reads occurring more frequently towards the edge of sequencing regions (p<<0.001). Overall, the primary source of variability was sampling error caused by low input copy number/minority species prevalence, though other sources of error including sequence intrinsic, temporal, and read-position related errors were detected.</p></div

Prolonged and substantial discordance in the prevalence of drug resistance mutations in plasma versus PBMC. Percentages represent the prevalence of the indicated resistance mutations revealed by 454 “deep” sequencing.

<p>Asterisks (*) indicate unavailable samples. <b>Bolded</b> font indicates time points at which subjects were prescribed raltegravir-containing regimens. Superscript ^a indicates the termination of a raltegravir-containing regimen. Blank cells indicate a wildtype genotype.</p