A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor]

A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor]. Results WB20 yielded a higher amount of DNA than WB4 (median [IQR] values 332.5?ng/l [117.5-401] and 107?ng/l [56.6-318], respectively; P? ?0.001). singlicate nested PCR followed by triplicate nested PCR in the unfavorable samples. A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor]. Results WB20 yielded a higher amount of DNA than WB4 (median [IQR] values 332.5?ng/l [117.5-401] and 107?ng/l [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 your same sample source (2/20 with WB4 and 1/20 with WB20) due to the inherent gp120 V3 variability. Conclusions Storing whole blood at +4C for up to two weeks and shipping at room heat is a convenient method for obtaining HIV-1 gp120 V3 sequence information via DprE1-IN-2 screening at a remote laboratory in patients with suppressed viremia. region sequence and coreceptor usage [3]. In Europe, Rabbit polyclonal to WNK1.WNK1 a serine-threonine protein kinase that controls sodium and chloride ion transport.May regulate the activity of the thiazide-sensitive Na-Cl cotransporter SLC12A3 by phosphorylation.May also play a role in actin cytoskeletal reorganization. Maraviroc is licensed for therapy-experienced patients but not yet for first-line therapy. Maraviroc made up of regimens are also used in patients with suppressed viremia [4]. This strategy is usually supported by the security profile of this drug, decreasing treatment toxicity [5]. In such patients, however, HIV coreceptor tropism cannot be decided on plasma RNA as recommended but proviral DNA can be considered as an alternative source of viral genetic material [6]. Previous studies have indeed 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 screening in DprE1-IN-2 the context of suppressed viremia has been provided [9,10]. While genotypic coreceptor tropism screening is gaining wide acceptance, this process may not usually be available in all clinical settings. Standard sample handling for remote screening requires storage of frozen specimens and shipment in dry ice, adding complexity to routine analysis. In this study, whole blood storage at +4C and shipment at room heat was evaluated as a more convenient handling method for remote HIV-1 DNA coreceptor tropism screening. To test this strategy, 200 paired whole blood samples were analysed. Methods A total of 200 whole blood samples were collected from 200 patients with suppressed viremia as defined as HIV-1 RNA 40 copies/ml by the Abbott RealTime assay. Patients signed an informed consent allowing anonymous use of samples for research purposes and the study was approved by the Ethical Committee of the Siena University or college Hospital. Of these, 43 experienced HIV-1 RNA target detected and 157 experienced HIV-1 RNA target not detected. For each sample, (i) one 500-microliter whole blood aliquot was frozen within 4?hours after drawing and stored at?20C until DNA extraction (WB20) and (ii) one 500-microliter whole blood aliquot was stored at +4C for two weeks within 4?hours after drawing, then placed at room heat (22-24C) for two days (WB4) and subjected to the same DNA extraction process. Whole blood DNA was extracted by using the High Pure Viral Nucleic Acid Kit (Roche Applied Science, catalogue number 11858874001) following the DprE1-IN-2 manufacturer instructions. The choice of this system was based on previous comparisons showing that DNA yield is increased with respect to the QIAamp DNA Blood Mini Kit (Qiagen) (data not shown). To make the process as straightforward as you possibly can, DprE1-IN-2 the DNA extracted was not measured and 5 of the total 50 microliters obtained from the extraction process were directly utilized for PCR. However, DNA concentration and purity were subsequently measured spectrophotometrically (NanoPhotometer P360, Implen) as a post hoc analysis to compare the yield of the two extraction procedures. A 421-bp fragment encompassing the HIV-1 gp120 V3 domain name was amplified by nested PCR. Blood DNA extracted from WB4 or WB20 was amplified by a nested PCR protocol using primer P150 (5-AATGTCAGCACAGTACAATGYACACAT-3,.