Article

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

ABSTRACT

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.
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    Full-text · Article · Dec 2014 · Journal of the International AIDS Society