Dried Blood Spot Specimens Are a Suitable Alternative Sample Type for HIV-1 Viral Load Measurement and Drug Resistance Genotyping in Patients Receiving First-Line Antiretroviral Therapy

Rottinghaus, Erin; Ugbena, Richard; Diallo, Karidia; Bassey, Orji; Azeez, Aderemi; DeVos, Joshua; Zhang, Guoqing; Aberle‐Grasse, John; Nkengasong, John N.; Yang, Chunfu

doi:10.1093/cid/cis015

Cited by 53 publications

(52 citation statements)

References 28 publications

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“…A recent study performed in a controlled laboratory environment reported no significant impact of ambient-temperature shipment on genotyping efficiency in comparison to frozen shipment on dry ice (22). The aim of this study was to determine the optimal ambient-temperature storage and shipment conditions of DBS specimens for HIVDR genotyping using a relatively low-cost and broadly sensitive in-house genotyping assay (25). (These data were presented in part at the International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative Strategies, 5 to 9 June 2012, Sitges, Spain.…”

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confidence: 99%

“…The contribution of cellular RNA or proviral DNA in DBS specimens has also been investigated, with one study documenting DNA contribution in over half of patients with undetectable VL and poor sequence reproducibility from replicate PCRs (23). In another study, proviral DNA contributed to 24 of the 37 sequences produced from DBS specimens; nonetheless, the nucleotide sequence identity of paired DBS and plasma specimens was high (98.1% to 98.8%) (24,25). Based on these and other data, DBS specimens are recommended as a suitable substitute for plasma in HIVDR surveillance in resource-limited settings (14-16, 18, 21, 25-28).…”

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

Parry

Parkin

Diallo³

et al. 2014

J Clin Microbiol

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h 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 the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 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 efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. 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 report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings.

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

Parry

Parkin

Diallo³

et al. 2014

J Clin Microbiol

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“…Second, in order to implement the WHO recommendations of task shifting and decentralization of ART to remote settings, the consequence would be that complex procedures, including drawing blood, isolation and storage of plasma samples, and cold chain shipments to qualified labs for VL testing, should be avoided. Rather, VF should be detectable on dried blood spots (DBS), a sampling alternative that is inexpensive and easy to collect and transport and has proven application for VL testing (13,14). Third, given the fact that for accurate detection of VF, a nucleic acid amplification step remains necessary and taking into consideration the realities of contamination risks in remote labs, it was decided to concentrate on a real-time PCR approach.…”

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Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

et al. 2013

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h Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n ‫؍‬ 191) and Tanzania (n ‫؍‬ 42). Field evaluation was performed in Uganda using local clinical plasma samples (n ‫؍‬ 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E؉03 copies/ml for plasma samples and 5.00E؉03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for "open platform" applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.

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“…Such small discordances, which may be unavoidable, might be related to PCR, storage time and RNA degradation, VL, and exclusive existence of proviral DNA in filter-based analytes (11,13).…”

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HemaSpot, a Novel Blood Storage Device for HIV-1 Drug Resistance Testing

et al. 2016

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dHemaSpot, a novel dried-blood storage filter device, was used for HIV-1 pol resistance testing in 30 fresh United States blood samples and 54 previously frozen Kenyan blood samples. Genotyping succeeded in 79% and 58% of samples, respectively, improved with shorter storage and higher viral load, and had good (86%) resistance mutation concordance to plasma. D rug resistance is a major challenge to sustained treatment success, particularly in resource-limited settings (RLS) with few antiretroviral therapy (ART) regimens and limited access to virologic monitoring and drug resistance testing (DRT) (1, 2). Availability of these tests is restricted mostly due to cost and expertise.HemaSpot is a novel dried-blood storage device that combines an absorbent paper to hold samples and a desiccant to maintain dryness, within a plastic cartridge (Spot On Sciences, Austin, TX). To date, HemaSpots had only been applied to Leishmania diagnosis via antibody detection in dogs, showing high sensitivity and specificity (3). We examine the potential of HemaSpot use for HIV-1 DRT.HemaSpot DRT was evaluated using fresh blood in the United States study (30 samples from two patients with various storage times and viral load [VL] dilutions) and previously frozen blood in the Kenya study (54 samples from patients failing first-line ART). Sequences were compared to those derived from plasma. Further study design and laboratory and data analysis methods are given in the supplemental material.In the U.S. study, genotyping was successful in 67% (20/30) of HemaSpots at all tested time points, with detectable VL (range, 1,000 to 100,000 copies/ml) (Table 1), 79% with a VL of Ͼ1,000 copies/ml, and 83% with a VL of Ͼ5,000 copies/ml (see Fig. S1 in the supplemental material). Odds of successful genotyping were 23.1 times higher for each 1-log-unit-higher VL (confidence interval [CI], 1.98 to 270.1; P ϭ 0.01). Genotyping success was significantly lower at 2 weeks (50%) of storage compared to 24 h (90%; odds ratio [OR], 0.01; CI, 0.00 to 0.78; P ϭ 0.04) and marginally significantly lower at 4 weeks (60%; OR, 0.03; CI, 0.00 to 1.40; P ϭ 0.075), with very small odds ratios.In the Kenya study (Table 2), genotyping was successful in 35% (19/54) of HemaSpots with detectable VL (range, 110 to 1,175,462 copies/ml) and in 65% (35/54) from paired plasma samples (VL range, 41 to 1,175,462 copies/ml), including all 19 for which HemaSpot genotyping was successful. Using a cutoff VL of Ͼ1,000 copies/ml, genotyping was successful in 58% of HemaSpots (89% in plasma), and for a VL of Ͼ5,000 copies/ml, genotyping was successful in 68% (94% in plasma).Plasma samples had 7.29 times the odds of successful amplification (95% CI, 2.68 to 19.83; P Ͻ 0.001) compared to HemaSpots. Additionally, successful amplification was related to higher VL (OR, 4.50 per 1-log-unit-higher log 10 VL; CI, 2.32 to 8.70; P Ͻ 0.001) and shorter storage time (OR, 0.11 for time of Ͼ8 months versus Յ8 months; 95% CI, 0.03 to 0.41; P ϭ 0.001). The interaction between analyte type and VL was insig...

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Dried Blood Spot Specimens Are a Suitable Alternative Sample Type for HIV-1 Viral Load Measurement and Drug Resistance Genotyping in Patients Receiving First-Line Antiretroviral Therapy

Abstract: Our results indicate that DBS specimens could be used for surveys to monitor HIVDR prevention failure in resource-limited settings.

Cited by 53 publications

References 28 publications

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

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

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

HemaSpot, a Novel Blood Storage Device for HIV-1 Drug Resistance Testing

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