Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance

Buckton, Andrew J.; Bissett, Sara L.; Beddows, Simon; Edwards, Simon; Cane, Patricia A.; Pillay, Deenan

doi:10.1093/jac/dkn412

Cited by 39 publications

(47 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Amplification of the protease and reverse transcriptase (RT) regions using a variety of testing methods has been reported. The sensitivities of PCR amplification from DBS differ significantly, with a few in-house assays reporting success from DBS specimens with VL as low as 1,000 to 2,000 copies/ml (14)(15)(16). All reports indicate lower genotyping sensitivities from DBS than from plasma specimens (17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

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

Parry

Parkin

Diallo³

et al. 2014

J Clin Microbiol

View full text Add to dashboard Cite

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.

show abstract

mentioning

confidence: 99%

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

Parry

Parkin

Diallo³

et al. 2014

J Clin Microbiol

View full text Add to dashboard Cite

show abstract

“…In order to mitigate the high cost associated with commercial genotyping tests and streamline the sample collection process, there have been systematic efforts to develop and evaluate inhouse assays and adopt the assays for use with the DBS sample type, and several assays have shown promising results (24,(28)(29)(30). With the publication of the WHO manual for HIV drug resistance testing using dried blood spot specimens in 2010 and the promising results obtained from the in-house genotyping assays (31), we initiated a study to adopt and validate a broadly sensitive in-house assay (32,33) against the FDA-approved ViroSeq HIV-1 drug resistance genotyping system using both plasma and DBS specimens.…”

mentioning

confidence: 99%

Field Evaluation of a Broadly Sensitive HIV-1 In-House Genotyping Assay for Use with both Plasma and Dried Blood Spot Specimens in a Resource-Limited Country

et al. 2013

View full text Add to dashboard Cite

c HIV-1 drug resistance (HIVDR) assays are important tools in clinical management of HIV-infected patients on antiretroviral therapy (ART) and surveillance of drug-resistant variants at population levels. The high cost associated with commercial assays hinders their use in resource-limited settings. We adopted and validated a low-cost in-house assay using 68 matched plasma and dried blood spot (DBS) samples with a median viral load (VL) of 58,187 copies/ml, ranging from 253 to 3,264,850 against the commercial assay ViroSeq. Results indicated that the in-house assay not only had a higher plasma genotyping rate than did ViroSeq (94% versus 78%) but also was able to genotype 89.5% (51/57) of the matched DBS samples with VLs of >1,000 copies/ml. The sensitivity in detecting DR mutations by the in-house assay was 98.29% (95% confidence interval [CI], 97.86 to 98.72) on plasma and 96.54 (95% CI, 95.93 to 97.15) on DBS, and the specificity was 99.97% (95% CI, 99.91 to 100.00) for both sample types compared to ViroSeq. The minor DR mutation differences detected by the in-house assay against ViroSeq did not result in clinical significance. In addition, cost analysis showed that the in-house assay could reduce the genotyping cost by about 60% for both plasma and DBS compared to ViroSeq. This field condition evaluation highlights the potential utility of a cost-effective, subtype-independent, in-house genotyping assay using both plasma and DBS specimens for HIVDR clinical monitoring and population-based surveillance in resource-limited settings.

show abstract

“…The size of the amplicon is roughly half the length of those generated using commercial and in-house HIV-1 drug resistance genotyping assays (11,15,16). By amplifying as little as possible of the RT using highly fine-tuned primer combinations, focusing on the region encompassing all of the relevant HIV-1 drug resistance mutations, it was possible to achieve a genotyping success rate of 94.8% for clinical plasma samples with Ն1.00Eϩ3 RNA copies/ml in a single-round RT-PCR.…”

Section: Discussionmentioning

confidence: 99%

“…By amplifying as little as possible of the RT using highly fine-tuned primer combinations, focusing on the region encompassing all of the relevant HIV-1 drug resistance mutations, it was possible to achieve a genotyping success rate of 94.8% for clinical plasma samples with Ն1.00Eϩ3 RNA copies/ml in a single-round RT-PCR. Subsequent sequencing requiring only a single forward and a single reverse primer, compared to the four to six primers needed for commercial and in-house assays (11,(14)(15)(16), increases the throughput for processing and decreases the analysis time per sample. In turn, decreasing the number of reactions required decreases the overall cost of the assay and minimizes the hands-on time, contamination risk, and turnaround time.…”

Section: Discussionmentioning

confidence: 99%

A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

et al. 2013

View full text Add to dashboard Cite

Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance

Abstract: Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy.

Cited by 39 publications

References 26 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

Field Evaluation of a Broadly Sensitive HIV-1 In-House Genotyping Assay for Use with both Plasma and Dried Blood Spot Specimens in a Resource-Limited Country

A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

Contact Info

Product

Resources

About