HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 C

Youngpairoj, Ae S.; Masciotra, Silvina; Garrido, Carolina; Zahonero, Natalia; Mendoza, Carmen de; Garcı́a-Lerma, J. Gerardo

doi:10.1093/jac/dkn100

Cited by 57 publications

(86 citation statements)

References 11 publications

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“…The facility to prepare, transport, and store dried spots is the major advantage of this sample support. Different studies have shown that dried spots can be kept for long periods when refrigerated or frozen in hermetic bags with desiccant, such as 1 year at 4°C (25) and 4 years at Ϫ20°C (17) for genotypic drug resistance testing and at least 15 days at 4°C (1, 11) and 1 year at Ϫ70°C (10) for RNA quantification. These data are important but are only applicable to reference laboratories where the infrastructure for long-term storage is avail- able.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

et al. 2009

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The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays-a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)-were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n ‫؍‬ 47; median VL, 4.13 log 10 copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log 10 copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log 10 copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log 10 copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20°C but for only 1 month at 37°C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37°C and for 1 month at 20°C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.Due to the efforts of national programs and the support of a wide range of international partners, the number of people receiving antiretroviral therapy (ART) in resource-limited countries has increased significantly over recent years, most notably in sub-Saharan Africa (24). In order to limit the emergence of resistance to antiretroviral drugs, human immunodeficiency virus (HIV) treatment should ideally be accompanied by periodic virological monitoring, such as viral load and resistance testing. However, in resource-limited countries, viral load monitoring and drug resistance tests are not yet available at an affordable cost, and infrastructure requirements limit the scale-up in access to these tests. Treatment initiation and modifications are thus guided mainly by clinical disease progression and by CD4 cell counts, when possible (13, 24). As a consequence, many people with virological failure stay on inadequate drug regimens for long periods. Rapid or uncontrolled emergence of HIV drug resistance is thus feared as a potential consequence of ART scale-up in resource-limited countries. Therefore, ART resistance monitoring is not recommended at the individual patient level in low-income countries, and the WHO has established a sys...

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Section: Discussionmentioning

confidence: 99%

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

et al. 2009

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“…The study was reviewed by the ethical review committee of Bridge Consultants Foundation and Vanderbilt University Institutional Review Board. DBS were prepared according to a protocol described by Youngpairoj et al 8 Fifty microliters of whole blood was spotted on 903 filter paper (Schleicher & Schuell, Keene, NH) and dried at room temperature for 3 h. The DBS were then sent to the China Center for Disease Control and Prevention for further genotypic testing at room temperature.…”

Section: Study Participants and Specimen Collectionmentioning

confidence: 99%

Antiretroviral Drug Resistance Mutations Among Treated and Treatment-Naive Patients in Pakistan: Diversity of the HIV Type 1 pol Gene in Pakistan

Shah

Xing

Altaf

et al. 2011

AIDS Research and Human Retroviruses

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Pakistan is experiencing a growing HIV epidemic. Antiretroviral drugs (ARV) have been smuggled into the country and available without prescription since the early 1990s, but are now provided free of cost by the government. We assessed the prevalence of HIV-1, drug resistance, and subtype distributions. Blood specimens were collected from HIV-1-infected participants registered in Sindh Province on dry blood spot (DBS) cards in 2008. Pol, protease, and partial reverse transcriptase regions were sequenced after reverse transcriptase PCR (RT-PCR). HIV-1 subtype was assigned by phylogenetic analysis. Primary drug resistance was analyzed by the Calibrated Population Resistance (CPR) tool using the Stanford Surveillance Drug Resistance Mutation (SDRM) major mutation list. Out of 100 blood samples collected, 42 were suitable for testing. Out of 42, 11 were ARVreceiving and 31 ARV-naive patients. Among them, 24 were injection drug users (IDUs), four immigrants, two hijras (male transvestites), two men who have sex with men (MSM), four prisoners, one female sex workers, two spouses of HIV-infected persons, and four from the general population. ARV resistance among naive patients was 2/31 (6.5%) and 36.4% (4/11) among ARV-experienced patients making an overall resistance of 14.2%. HIV-1 subtype A1 was the predominant subtype found in 35/42 (83.3%) followed by CRF35_AD and C, 6.5% each. Subtype D and G were found in one (2.4%) each. A significant proportion of Pakistani HIV patients has ARV drug resistance. Physicians treating patients should consider the magnitude of drug resistance while selecting regimens, and address drug adherence aggressively.

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“…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). Notably, genotyping sensitivity of 1,000 copies/ml was achieved by only two laboratories in a recent WHO proficiency testing program (22).…”

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

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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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HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 C

Cited by 57 publications

References 11 publications

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

Antiretroviral Drug Resistance Mutations Among Treated and Treatment-Naive Patients in Pakistan: Diversity of the HIV Type 1 pol Gene in Pakistan

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

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