Abstract
BackgroundMaraviroc is an HIV-1 coreceptor antagonist that has shown good efficacy and tolerability in treatment-naive and treatment-experienced patients harboring CCR5-tropic virus. The use of Maraviroc in treatment simplification in patients with suppressed plasma HIV-1 RNA requires analysis of HIV-1 DNA. Coreceptor tropism testing is often performed remotely at reference laboratories. In this study paired whole blood stored at + 4°C and at−20°C were compared as a source for genotypic coreceptor tropism testing.MethodsTwo hundred paired whole blood samples from different patients were analysed. Each sample was stored in two different conditions: one aliquot was stored at−20°C until spin column DNA extraction (WB20) and one aliquot was stored at +4°C for two weeks and then placed at room temperature (22-24°C) for two days before DNA extraction (WB4). Subsequently, a fragment encompassing the HIV-1 gp120 V3 domain was amplified by a singlicate nested PCR followed by triplicate nested PCR in the negative samples. A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor].ResultsWB20 yielded a higher amount of DNA than WB4 (median [IQR] values 332.5 ng/μl [117.5-401] and 107 ng/μl [56.6-318], respectively; P < 0.001). However, the DNA purity was higher for WB4 than for WB20 (median distance from the optimal OD260/280 ratio, 0.14 [0.07-0.79] and 0.96 [0.36-1.10], respectively; P < 0.0001). The number of samples successfully amplified was 152 (76.0%) for WB20 and 155 (77.5%) for WB4 with the first PCR and 179 (89.5%) for WB20 and 181 (90.5%) for WB4 (P = ns) following subsequent triplicate analysis. The inferred coreceptor tropism was concordant in 18 out of 20 paired WB4 and WB20 samples. Two samples yielded discordant results, consistent with the discordance rate within duplicates from the same sample source (2/20 with WB4 and 1/20 with WB20) due to the inherent gp120 V3 variability.ConclusionsStoring whole blood at +4°C for up to two weeks and shipping at room temperature is a convenient method for obtaining HIV-1 gp120 V3 sequence information via testing at a remote laboratory in patients with suppressed viremia.
Highlights
Maraviroc is an Human Immunodeficiency Virus type 1 (HIV-1) coreceptor antagonist that has shown good efficacy and tolerability in treatment-naive and treatment-experienced patients harboring C-C chemokine receptor type 5 (CCR5)-tropic virus
For each sample, (i) one 500-microliter whole blood aliquot was frozen within 4 hours after drawing and stored at−20°C until Deoxyribonucleic acid (DNA) extraction (WB20) and (ii) one 500microliter whole blood aliquot was stored at +4°C for two weeks within 4 hours after drawing, placed at room temperature (22-24°C) for two days (WB4) and subjected to the same DNA extraction procedure
There was no significant difference in the rate of positive samples with Whole blood stored at + 4°C (WB4) and Whole blood stored at−20°C (WB20), either in the first singlicate test or in the subsequent triplicate analysis performed on negative samples
Summary
Maraviroc is an HIV-1 coreceptor antagonist that has shown good efficacy and tolerability in treatment-naive and treatment-experienced patients harboring CCR5-tropic virus. Maraviroc containing regimens are used in patients with suppressed viremia [4] This strategy is supported by the safety profile of this drug, decreasing treatment toxicity [5]. In such patients, HIV coreceptor tropism cannot be determined on plasma RNA as recommended but proviral DNA can be considered as an alternative source of viral genetic material [6]. Previous studies have shown a good correlation between genotype based tropism results obtained from paired HIV-1 DNA and RNA [7,8] and preliminary evidence of the clinical relevance of proviral HIV-1 DNA tropism testing in the context of suppressed viremia has been provided [9,10]
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