Field Study of Dried Blood Spot Specimens for HIV-1 Drug Resistance Genotyping

Journal of Clinical Microbiology (Impact Factor: 3.99). 05/2014; 52(8). DOI: 10.1128/JCM.00544-14
Source: PubMed


Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared genotyping efficiency and resistance profiles of DBS stored and shipped at different temperatures to plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood, and a 5th card from finger-prick blood were prepared from 103 HIV-patients with a median VL of 56,795 copies/ml (range 1,081 - 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at -80°C, and shipped from Uganda to the United States, at ambient temperature or frozen on dry ice, for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiency. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiency. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P=0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P<0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This study delineates the optimal DBS collection, storage and shipping conditions and opens a new avenue for cost-saving ambient temperature DBS specimen shipments for HIVDR surveillances in resource-limited settings.

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    ABSTRACT: World Health Organization-recommended surveys of acquired HIV-1 drug resistance include assessment of HIV-1 viral load suppression to levels below 1,000 copies/ml and drug resistance-associated mutation patterns in subjects on antiretroviral therapy. Surveys are being conducted in regions of the world that cannot support the collection, storage, and shipping of frozen plasma. Therefore, dried blood spots are often the specimen type of choice for both genotyping and viral load measurement. Furthermore, viral load testing for individual patient management in these regions is being scaled-up in accordance with WHO 2013 Guidelines for Antiretroviral Treatment. Technical issues related to the adaptation of viral load assays to dried blood spots, especially with respect to sensitivity (limit of detection), specificity (cell-free RNA vs. cell-associated DNA or RNA), and assay method, affect the interpretation of a viral load result from dried blood spots. Amongst published studies of commercial assay performance with dried blood spots, the bioMérieux EasyQ® and Abbott RealTime assays tended to show high (> 90%) specificity and sensitivity; the Biocentric Generic or Roche TaqMan® assays tended to show high sensitivity but lower specificity, using a 1,000 copies/ml threshold. The relative contribution of cell-associated DNA or RNA to a viral load measurement is likely to vary between patients, depending on clinical parameters and treatment status. A model was developed that predicts that in patients on antiretroviral therapy with low plasma viral load, cellular DNA is the predominant source of non-plasma virus-derived nucleic acid in dried blood spots. The extent of viral load overestimation from dried blood spots becomes less important when plasma viral load is over about 5,000 copies/ml. To avoid misclassifying subjects with plasma viral load suppression, the World Health Organization-recommended threshold of 1,000 copies/ml can be applied only when an assay that can distinguish between DNA and RNA is used (e.g. bioMérieux EasyQ® or Abbott RealTime). There is a need for additional affordable technologies with the ability to discriminate between cell-free (plasma) and cell-associated nucleic acids.
    No preview · Article · Jul 2014 · AIDS reviews
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    ABSTRACT: Objectives: We evaluated an In-house assay for HIV-1 drug resistance genotyping by using DBS samples in China. Methods: The amplification sensitivity was assessed using 79 DBS specimens with plasma viral load ranging from 1,000 to 6,000 copies/ml. Precision was assessed using 5 DBS specimens with 5 replicates tested in one test run. Reproducibility was evaluated using other 5 DBS specimens with 5 replicates genotyped in 5 test runs. Nucleotide sequence identity and the degree of concordance in detecting drug resistance mutations were assessed within and between test runs. In addition, nucleotide sequence and drug resistance mutations were compared between 64 matched plasma and DBS specimens. Results: The amplification rate of DBS specimens with plasma viral load of 1,000-6,000 copies/ml was 96.2% (76/79). The nucleotide sequence identity was 99.7±0.34% and 99.6±0.25% within and between test runs, respectively. Moreover, there was a near perfect agreement of detecting drug resistance mutations intra- and inter- test runs with kappa value of 0.972 and 0.963, respectively. Between 64 pairs of plasma and DBS specimens, the nucleotide identity was excellent with 99.5±0.34%. As compared to the results of plasma specimens, the sensitivity and specificity for detecting drug resistance mutations in DBS specimens were 99.4 % (95% CI, 97.4-99.8%) and 99.8% (95% CI, 99.7-99.9%), respectively. Totally 15 discordant drug resistance mutations were found. Among them, 53.3 % (8/15) were caused by mixture base. Conclusion: The In-house HIVDR genotyping assay could be used for testing DBS samples with viral load above 1,000 copies/ml in China and had a low intra- and inter- assay variability. DBS is an excellent alternative to plasma for HIV-1 drug resistance genotyping at population levels in China.
    No preview · Article · Dec 2014 · Current HIV Research
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    ABSTRACT: Access to antiretroviral treatment (ART) becomes more and more effective in resource-limited settings (RLS). However, this global effort would be even more profitable if the access to laboratory services especially in decentralized settings was strengthened. We report the virological outcome and HIV-1 drug resistance in three West African countries using dried blood spots (DBS) samples. We included HIV-1-infected adults on ART ≥6 months and followed up in capital cities and decentralized sites in Senegal, Mali and Guinea-Conakry. Patients were consecutively enrolled and DBS were collected in field conditions and kept at ambient temperature before transfer to the reference laboratory. Viral load (VL) was quantified using the NucliSENS EasyQ HIV-1 v1.2. Genotyping of HIV-1 pol gene was performed using in-house protocol. Of the 407 participants, 119, 152 and 136 were from Senegal, Mali and Guinea-Conakry, respectively. The median treatment duration was 36 months [IQR: 6-136]. Virological failure (VF) (VL≥3log10 copies/mL) was observed in 26% (95% confidence interval (CI), 18-35; n=31), 11% (95% CI, 6-17; n=16) and 24% (95% CI, 17-32; n=33) of patients in Senegal, Mali and Guinea-Conakry, respectively (p=0.001). Of samples presenting VL≥3log10 copies/mL (n=80), 70 were successfully genotyped. At least one drug resistance mutation (DRM) was detected in the following proportions: 70% (95% CI, 50-86; n=19), 93% (95% CI, 68-100; n=14) and 68% (95% CI, 48-84; n=19) in Senegal, Mali and Guinea-Conakry, respectively (p=0.22). Twenty-six per cent (26%; 95% CI, 16-38; n=18) of patients in VF harboured wild-type viruses, which is likely indicative of weak adherence. Phylogenetic analysis showed the predominance of CRF02_AG subtype (73%; 95% CI, 61-83; n=51). We describe the ART outcome in capital and rural settings of Senegal, Mali and Guinea-Conakry. Our results in all of the three countries highlight the need to reinforce the ART adherence in order to minimize the occurrence of drug resistance. In addition, these findings provide additional evidence that the use of DBS as a sampling support could assist virological monitoring of patients on ART in remote areas.
    Full-text · Article · Dec 2014 · Journal of the International AIDS Society