Evaluation of Dried Blood Spots for Human Immunodeficiency Virus Type 1 Drug Resistance Testing

Abstract

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at ؊20°C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at ؊20°C or at ؊70°C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that ؊20°C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.The introduction of highly active antiretroviral therapy and the demonstration of dramatic improvements in human immunodeficiency virus (HIV)-and AIDS-related mortality and morbidity in North America and Europe have fueled international efforts to expand access to care and treatment in lessdeveloped countries. Several major initiatives to provide treatment in resource-limited settings, including the U.S. President's Emergency Plan for AIDS Relief and the Global Fund against AIDS, TB and Malaria, are now in progress (16). The implementation of these programs requires the development of appropriate and effective patient-monitoring systems, including surveillance for antiretroviral drug resistance. Sentinel drug resistance surveillance systems are important public health tools that can provide information on trends in the prevalence of resistance at the population level and can be used to modify treatment guidelines.Plasma and serum are considered the preferred specimen types for HIV type 1 (HIV-1) drug resistance testing. However, these types of specimens are not optimal in resource-limited settings where the equipment necessary for PCR amplification and sequencing may not be available at collection sites and resistance testing requires transportation of the samples to a refer...

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

“…Few studies examined the possibilities of performing drug resistance testing on dried blood, plasma, or serum on filter paper (24,33,43). Because of the ease to collect, store, and transport DBS, use of DBS is the ideal method for blood sampling in resource-poor rural settings.…”

“…Results of published reports in this regard are very inconsistent (6,10,14,21,24,34). The present study aims to investigate both the possibility of DNA sequencing from whole blood cells or from DBS and the usefulness of DNA sequencing for determination of drug resistance in patients from a resource-poor setting with a high level of HIV-1 subtype diversity.…”

“…The fact that a reverse transcription step is no longer needed for the amplification of the genes of interest also reduces the overall cost of the procedure. Blood spots collected on filter paper have been used for DNA amplification purposes, but reports on the feasibility of performing genotypic analysis for HIV-1 drug resistance remain scarce (24,33,43). In resource-poor settings, dried blood spots (DBS) are the preferred method of sample collection in the…”

Human Immunodeficiency Virus

“…One study showed that high-quality sequencing data could be obtained from DBS stored at −20˚C for 5 to 6 years but not from DBS stored at room temperature (53). There was also good agreement of sequencing data obtained from stored DBS compared to the stored plasma samples (54). The size of the amplicon also influences the quality of sequencing; only 58% of DBS stored at 4˚C for one year could be amplified using the ViroSeq test, which generates an 1800-base-pair amplicon, compared to a 95% success rate using a laboratorydeveloped assay that amplifies a 1000-base-pair fragment (55).…”

“…The size of the amplicon also influences the quality of sequencing; only 58% of DBS stored at 4˚C for one year could be amplified using the ViroSeq test, which generates an 1800-base-pair amplicon, compared to a 95% success rate using a laboratorydeveloped assay that amplifies a 1000-base-pair fragment (55). DBS may be a good alternative for resistance testing, although proviral DNA can contribute significantly to the viral sequences (54), and storage conditions are critical (56).…”