With an estimated 38.6 million individuals worldwide living with HIV at the end of 2005, HIV/AIDS represents a major global health challenge. Large-scale antiretroviral treatment is considered fundamental for an effective global response to HIV [1,2]. Several initiatives, including the US President's Emergency Plan for AIDS Relief and the Global Fund against AIDS, TB and Malaria, have demonstrated the feasibility of delivering HIV treatment in less-developed countries [3–5]. The introduction of antiretroviral drugs in these settings requires the development of appropriate and effective patient monitoring systems, including surveillance for antiretroviral drug resistance. Sentinel drug resistance surveillance systems are an important public health tool that can provide information on trends in the prevalence of resistance at the population level and can be used to develop better treatment guidelines. Resistance testing may also complement basic diagnostic and clinical tests as part of individual patient management .
Drug resistance genotyping is usually performed using plasma or serum specimens. These types of specimens are, however, not always optimal in resource-limited countries where testing usually requires the transportation of specimens to a centralized location. In these areas, alternative specimen types with simplified processing and less stringent storage and transport requirements would simplify drug resistance testing. Dried blood spots (DBS) have been extensively used for HIV-1 antibody testing [7,8], molecular diagnostic [9,10], and viral load quantification [11–15], and are considered an attractive alternative to plasma or sera for resistance testing. DBS require minimal laboratory manipulation, are less expensive to collect, store, and transport than plasma, and can be easily prepared by trained nonspecialists.
We recently showed that HIV-1 pol can be efficiently genotyped from DBS prepared from untreated patients having a wide range of plasma viral loads . We also found that proviral DNA was amplifiable in a significant number of the DBS specimens. The frequent amplification of HIV DNA raised questions regarding a potential interference of proviral/archived DNA in the genotypic profiles generated from DBS as a result of the different dynamics of emergence and persistence of mutations in proviral DNA and plasma RNA [17–21]. These observations highlighted the need for a wider evaluation of the concordance between resistance genotypes from plasma and DBS in treated populations.
In this study, we evaluated the concordance between plasma and DBS genotypes generated from treated individuals with detectable plasma viremia. We showed that despite the presence of proviral DNA, resistance genotypes from DBS were comparable with those obtained from plasma. We also showed a high efficiency of genotyping from DBS using an existing commercial genotypic assay. These findings highlight the promise of DBS as an alternative to plasma for drug resistance testing in treated patients.
Material and methods
A total of 60 DBS were prepared from residual diagnostic specimens collected from HIV subtype B-infected individuals receiving care in Hospital Carlos III, Madrid, Spain. Of these patients, 58 were antiretroviral experienced and two were antiretroviral naive. The average age was 42 years (range 14–57) and 78% were men. Information on plasma viral loads, CD4 cell counts, and antiretroviral drug treatment is shown in Table 1. Plasma viral loads were quantified using the Versant HIV-1 RNA 3.0 Assay (branched DNA; Bayer HealthCare Diagnostic Division, Tarrytown, New York, USA). DBS specimens were anonymized before they were sent to our laboratory at the Centers for Disease Control and Prevention (CDC) for testing.
Preparation of dried blood spots
DBS were prepared by pipetting 50 μl whole blood onto premarked circles on 903 filter paper cards (Schleicher and Schuell, Keene, New Hampshire, USA). Cards were dried overnight at room temperature, covered with glossy paper, and placed in a gas-sealed plastic bag (Fisher Scientific Company, Pittsburgh, Pennsylvania, USA) containing a silica gel desiccant (Mini Pax Sorbent; Multisorb Technologies, Buffalo, New York, USA). All DBS were stored at −20°C before they were shipped to CDC for testing. To prepare the DBS for transport, bagged samples were removed from the freezer and kept at room temperature for 30 min before opening the bag. Ziplock bags were then opened and fresh desiccants were added before shipment. Specimens were shipped in dry ice and stored at −20°C upon arrival at CDC. The average time of storage at −20°C was 21 weeks (range 18–26 weeks).
Nucleic acid extraction from dried blood spots
Total HIV-1 nucleic acids were extracted from one or two spots by using the silica-based Boom method with some modifications for DBS processing . Briefly, a whole spot containing 50 μl blood was cut with scissors and added into 9 ml Nuclisens lysis buffer (bioMerieux, Inc., Durham, North Carolina, USA). Special care was taken to avoid contact of the scissors with the blood spots; scissors and forceps were sprayed with 70% ethanol after each use. After a 2-h incubation with lysis buffer at room temperature under gentle rotation, the supernatant was clarified by centrifugation at 250g for 5 min and then transferred to a clean 15 ml conical tube. Nucleic acids were then extracted following the manufacturer's instructions, resuspended in 45 μl elution buffer, and stored at −80°C until use.
Amplification of proviral HIV-1 DNA from dried blood spots
The frequency of amplification of proviral DNA from DBS was investigated using an in-house reverse transcriptase (RT)-nested polymerase chain reaction (PCR) method. This assay amplifies a 1023 base pair fragment of HIV-1 pol (amino acids 15–99 of the protease and 1–256 of the RT) and has previously been validated for its use in DBS . We performed parallel RT–PCR reactions in the presence of MuLV RT (amplification of RNA and DNA) and in the absence of MuLV RT to amplify DNA  selectively. All amplifications were performed in a Biometra T3 thermocycler (Biometra GmbH, Goettingen, Germany).
HIV-1 drug-resistance testing from dried blood spots and plasma specimens
Genotypic testing from DBS was performed using the ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plains, Illinois, USA) and 10 μl of the extracted HIV-1 nucleic acids. The ViroSeq assay amplifies a 1.8 kb fragment and provides amino acid sequences for the entire protease and codons 1–335 of the RT. Genotypic testing from plasma samples was also performed using the ViroSeq HIV-1 assay with the exception of seven specimens that were genotyped using the TrueGene HIV-1 genotyping system (Bayer HealthCare LLC, Tarrytown, New York, USA). Protease and RT sequences from plasma and DBS were interpreted using the Stanford Genotypic Resistance interpretation algorithm (version 4.2.6) available at http://hivdb.stanford.edu/pages/algs/HIVdb.html . Nucleotide sequences from 66 matched plasma and DBS specimens have been deposited in the GenBank database (accession numbers EF397436–EF397501).
Analysis of the relationship between nucleotide sequences from matched plasma and DBS specimens was performed by using the Clustal multiple sequence alignment program. Sequences from a subtype A (AB098330) isolate from Uganda and a subtype B isolate (HIVHXB2R) from France were used as references. Phylogenetic analysis was performed by the neighbor-joining method, with the nucleotide distance calculated by Kimura's two-parameter method included in the Phylip package version 3.5c with bootstrapping (1000 replicates) . The tree was derived from a nucleotide alignment of the pol region totaling 837 bp after eliminating columns containing gaps. Sequences were aligned using the Clustal W 1.82 multiple sequence alignment program with the default gap-penalty values.
The comparison between HIV-1 plasma viral loads or CD4 cell counts among samples with and without amplifiable DNA was performed using the Mann–Whitney test. Statistical significance was defined by a two-tailed P value less than 0.05. All statistical analyses were performed using GraphPad Prism 4 software for Windows (version 4.03, 2005).
Characteristics of the study population
We evaluated 60 matched plasma/DBS samples from antiretroviral-experienced (n = 58) and naive (n = 2) patients. The median CD4 cell count was 371 cells/μl (range 32–1320 cells/μl), and the median plasma viral load was 9135 copies/ml (range 78–676 694 copies/ml). Of the 60 samples, 29 had plasma viral loads higher than 10 000 copies/ml (median 25 678), nine had viral loads between 10 000 and 2000 copies/ml (median 4156) and 22 had viral loads of less than 2000 copies/ml (median 347). Table 1 shows the characteristics of the study population including antiretroviral treatment, plasma viral loads, and absolute CD4 cell counts.
HIV pol genotyping from dried blood spots
Using the ViroSeq assay, HIV-1 pol was successfully genotyped in 50 of the 60 (83.3%) DBS specimens, including all 38 DBS specimens obtained from patients with plasma viral loads higher than 2000 RNA copies/ml and 12 of the 22 DBS specimens from patients with plasma viral loads lower than 2000 copies/ml. Of the 12 DBS specimens with plasma viral loads lower than 2000 copies/ml, eight were only amplifiable using RNA extracted from two spots (Table 1). Overall, these findings demonstrate the efficient genotyping of HIV-1 pol from DBS using the ViroSeq assay.
Correlation between genotypic results from plasma and dried blood spots
We next compared the genotypic profiles obtained from DBS with those generated from plasma. Genotypes from plasma were only available in 40 specimens and were generated using the ViroSeq assay (33 samples) or the TrueGene assay (seven samples; Table 1). Our comparison between plasma and DBS sequences was therefore restricted to these 40 specimens with available genotypes in both plasma and DBS. Resistance genotypes from DBS were edited before the plasma genotypes were available for the comparison.
Table 2 shows all the major and minor protease and RT mutations seen in the 40 matched plasma/DBS samples. Overall, protease genotypes from plasma and DBS were highly concordant. Of the 121 protease mutations found in plasma sequences, 115 (95%) were also found in DBS sequences. Of the six discordant mutations, five were mixtures at minor positions (G73GS in two samples, L35ED, and L33LI,) and only one (M46L in patient 36) was a major mutation found as a mixture in plasma but not in DBS (Table 2). We also evaluated if additional mutations could be seen in DBS and not in plasma. Table 2 shows that of the 121 mutations found in DBS sequences, 116 (95.9%) were also detected in plasma sequences; all the five additional mutations found in DBS were minor (A71I, T74TP, and L10AIT).
Similar findings were observed when RT mutations from plasma and DBS were compared (Table 2). Of the 195 RT mutations found in plasma sequences, 191 (97.9%) were also found in DBS, and only mutations M41L and a T215S revertant in sample 28, mutation K103N in sample 38, and mutation T69S in sample 42 were not detected in DBS sequences. Conversely, of the 200 mutations found in DBS sequences, only 11 (5.5%) were absent in plasma sequences. Of these mutations, D67A, D67Y, K103D, K103I, L210G, T69I, and Q151E are unusual mutations with unknown effects on nucleoside reverse transcriptase inhibitor/non-nucleoside reverse transcriptase inhibitor susceptibility, G333D (two samples) is a polymorphism that occurs slightly more frequently in individuals receiving nucleoside reverse transcriptase inhibitors than in untreated individuals , and P236L causes delavirdine resistance and increased susceptibility to nevirapine . Overall, our results indicate that in this population of treated patients, plasma and DBS genotypes are highly correlated.
We also compared the overall nucleotide similarity between plasma and DBS sequences from the 33 patients that had genotypes generated by the ViroSeq assay. The seven remaining pairs were left out of the analysis because they had segmented protease and RT sequences. The overall nucleotide similarity between paired plasma and DBS sequences was 98.8 ± 0.6% and ranged between 97.5 and 99.6%. A high amino acid similarity between plasma and DBS sequences was also noted (mean 98.9 ± 0.8%, range 97–100%). Figure 1 shows the phylogenetic tree derived with all these specimens (837 bp after gap stripping). Bootstrap values obtained for plasma/DBS pairs were higher than 99% in all specimens. Figure 1 also shows two clear clusters with a bootstrap value of 100. These clusters correspond to samples taken from the same two patients at different timepoints (SpDBS/PL2 and SpDSB/PL7, and SpDBS/PL23 and SpDBS/PL30).
Frequency of amplification of proviral DNA from dried blood spots
We previously showed that proviral DNA is amplifiable in a significant proportion of DBS . We therefore evaluated the extent of DNA amplification in these DBS specimens by using an in-house nested PCR assay. We compared the rate of amplification among RT–PCR reactions performed with and without reverse transcription. In the presence of reverse transcription, pol sequences were amplifiable in 45 of the 60 DBS specimens (75%) including 37 of the 43 samples (86%) with plasma viral loads higher than 1000 RNA copies/ml. In the absence of reverse transcription, 20 of the 45 RT–PCR-positive samples (44.4%) were also amplified, confirming the presence of HIV-1 DNA in a substantial number of DBS specimens. The amplification of HIV-1 DNA was more frequent in DBS prepared from patients with higher plasma viral loads. Of the 26 RT–PCR-positive DBS specimens with viral loads higher than 10 000 RNA copies/ml, 15 (57.7%) were amplified in the absence of reverse transcription. In contrast, proviral DNA was amplified in only five of the 19 samples (26.3%) with viral loads lower than 10 000.
We also compared median viral loads and CD4 cell counts among samples that were positive and negative for proviral DNA amplification. Figure 2 shows that the median plasma viral load in specimens with detectable DNA was higher than in DNA-negative specimens (median 19 128 copies/ml and 8316, respectively; P = 0.038). Median CD4 cell counts in DNA-positive specimens were also higher than in DNA negative specimens (415 and 330 cell/μl, respectively) although the difference was not statistically significant (P = 0.25; Fig. 2).
We have investigated the concordance between HIV drug resistance genotypes generated from plasma and DBS in individuals failing antiretroviral treatment. We wanted to compare the genotypic profiles between these two types of specimens because proviral DNA can be amplified in a substantial number of DBS and may potentially interfere with the genotypic results obtained from DBS . We show that drug resistance genotypes from plasma and DBS were comparable, and that discordances were generally caused by minor mutations or unusual amino acid changes at resistance-related positions. The high concordance between plasma and DBS genotypes was especially significant because plasma and DBS sequences were edited by different personnel at two separate locations. Our results indicate that DBS represent a valid alternative to plasma for resistance testing in treated individuals, and expand previous findings showing the feasibility of using DBS for resistance testing in untreated populations .
The amplification of proviral DNA sequences in a substantial number of DBS specimens raised questions regarding potential interferences of proviral pol sequences in the genotypic results generated from DBS. Our results showing a high concordance between plasma and DBS genotypes suggest that such interference may be minimal. The extent to which DNA was contributing to the genotypes obtained from DBS could not be inferred from our analysis because amplifications of proviral sequences from DBS might be masked by a higher RNA content. Such a possibility cannot be excluded because DNA amplifications were more common among samples with high plasma viremia and were less frequent in patients with low viral loads.
The degree of interference of proviral DNA sequences in the genotypic profiles generated from DBS might also differ according to the treatment characteristics of the population as a result of the different dynamics of emergence and persistence of resistance mutations in plasma and peripheral blood mononuclear cells (PBMC). For example, patients who fail treatment tend to have more detectable mutations in plasma sequences than in PBMC, particularly at lower viral loads. In those patients, mutations are generally detected first in plasma then in PBMC, with delays of up to one year . The amplification of proviral wild-type sequences from PBMC in those patients might potentially interfere with the detection of drug resistance mutations present in plasma RNA. The opposite usually occurs in patients undergoing treatment interruptions or modifications who typically have more detectable mutations in PBMC [17–21]. A more detailed analysis of resistance genotypes originating from selective amplifications of RNA and DNA from DBS prepared from different populations would help to understand the degree of interferences from proviral DNA sequences.
Our study was primarily designed to evaluate the concordance between drug resistance genotypes generated from plasma and DBS, and sought to maximize the generation of resistance information from DBS. We included patients infected with subtype B viruses because the ViroSeq assay has been extensively validated for HIV-1 subtype B, although recent reports indicate that this assay performs well for other subtypes [25–29]. We prepared, transported, and stored the DBS under optimal conditions to minimize the degradation of HIV nucleic acids and maximize the generation of meaningful resistance information. The DBS specimens were stored at −20°C because this temperature was found to be suitable for the long-term storage of DBS for resistance testing . Desiccant packs were added to remove moisture given the extreme sensitivity of HIV nucleic acids to degradation in the presence of humidity. Spots were transported in dry ice and were allowed to equilibrate thoroughly to room temperature for 30 min before opening the bag. Under these controlled conditions, we found that the efficiency of genotyping from DBS was comparable to that seen in plasma. Such a high efficiency has also been noted using in-house nested PCR methods that amplify smaller (663–1023 bp) pol fragments [16,30]. The great success of HIV genotyping by the ViroSeq assay was especially significant because this assay amplifies a much larger (1.8 kb) pol fragment and does not include a nested PCR step.
Appropriate storage conditions are thought to be critical for the efficient genotyping from DBS. The high rate of amplification from our DBS stored at −20°C confirms our recent observations indicating that −20°C may be a suitable temperature for the long-term storage of DBS . The storage of DBS specimens at −20°C may not be possible at DBS collection sites in less-developed areas. In these settings, short-term storage at 4°C or room temperature may represent a more feasible alternative. However, neither the impact of 4°C or room temperature on the ability to amplify large pol fragments from DBS nor how different levels of humidity will affect the stability of nucleic acids on DBS have been evaluated. Such analysis is essential to define appropriate guidelines for the correct storage of DBS at collection sites.
The effective implementation of first-line antiretroviral regimens in resource-limited settings is considered particularly critical because such regimens usually involve simple co-formulated, generic, fixed-dose drug combinations . The suboptimal delivery of these regimens could lead to an increase in therapeutic failures, the transmission of drug-resistant virus, and reductions in program effectiveness. Surveillance for antiretroviral drug resistance may assist the effective implementation of these programs by assessing the frequency and patterns of circulating drug-resistant viruses. The availability of inexpensive and simple methods for sample collection, such as DBS, represents an advantage in areas that lack the appropriate infrastructure for plasma processing and transport, and may help to simplify resistance surveillance in these settings.
In summary, we show that despite the presence of proviral DNA in DBS, resistance genotypes from DBS and plasma were highly comparable when DBS were stored under optimal conditions. We also show that resistance testing from DBS can achieve sensitive levels similar to those seen using plasma specimens. Our results indicate that DBS may represent a feasible alternative to plasma for drug resistance testing in treated individuals. This specimen type may be particularly useful for resistance surveillance in resource-limited settings in which the use of antiretroviral drugs continues to increase.
Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
The use of trade names is for identification only and does not constitute endorsement by the United States Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
antiretroviral therapy; dried blood spots; HIV drug resistance; protease inhibitors; reverse transcriptase inhibitors; surveillance