The availability of clinical specimens that can be easily collected, stored and transported is advantageous in areas that lack appropriate infrastructure for blood processing. Whole blood samples collected by finger stick and dried onto a filter paper [dried blood spots (DBSs)] represent an attractive alternative to the conventional collection of blood in tubes. DBSs have been extensively used for HIV-1 antibody testing,¹ molecular diagnostics² and virus load (VL) quantification^3–5 and are now considered a convenient alternative to plasma for HIV-1 drug resistance testing. We recently found that drug resistance genotypes generated from DBSs were similar to those derived from plasma in antiretroviral-naive and -experienced patients.^6,7 We also found that resistance testing from DBSs can be as sensitive as with plasma when using a genotypic assay that amplifies a large (1.8 kb) pol fragment.⁶ The high success of amplification was noted using DBSs stored at −20°C in the presence of desiccant, suggesting that −20°C may represent a feasible storage temperature for DBSs. Although these findings were encouraging, storage at −20°C is not always possible in resource-limited settings where 4°C or room temperature may represent a more realistic alternative. Studies on HIV-1 VL determinations using DBSs have reported the efficient amplification of short HIV-1 RNA sequences from filter papers stored at 4°C or room temperature.^3,8,9 However, genotypic assays usually rely on the amplification of large pol fragments and are thought to be particularly sensitive to HIV-1 RNA degradation that may occur under suboptimal storage conditions. Here, we report on the efficiency of amplification and drug resistance genotyping from DBSs stored at 4°C for 1 year.

When the ViroSeq assay was used, only 23 of the 40 (57.5%) DBS specimens stored at 4°C were successfully genotyped; 22 of the successful amplifications were from specimens that had plasma viraemia >10 000 RNA copies/mL (Table 1). An additional five specimens produced amplicons that did not generate full-length RT and protease sequences. Visual inspection of the desiccant bags after 6 months of storage at 4°C showed evidence of humidity in 21 of the 40 specimens. Of these 21 specimens, only those with plasma VL higher than 14 000 RNA copies/mL (12 specimens) were successfully genotyped by the ViroSeq assay at 12 months (data not shown).

As we previously noted using parallel DBSs stored at −20°C, resistance genotypes generated from DBSs and matched plasma specimens were highly concordant. Of the 163 drug resistance mutations identified in these 23 plasma sequences, 158 were also found in sequences generated from DBSs. Of the five mutations absent in DBSs, three were protease mutations, two were major (V82A and I54M) and one minor (L10IT), and two were polymorphisms at codon 333 of the RT (G333E and G333D) (data not shown). We also compared the efficiency of amplification seen in these DBSs with that previously seen in DBSs prepared in parallel from the same patients and stored at −20°C for 6 months.⁶ Under storage at −20°C, all of the 40 DBS specimens were successfully amplified and genotyped (Table 1).

The ViroSeq assay amplifies a large (1.8 kb) pol fragment in a single round of PCR and may be particularly sensitive to the degradation of HIV-1 nucleic acids that may occur during long-term storage. We, therefore, assessed if the rate of amplification from DBSs stored at 4°C could be improved by using an in-house assay that amplifies a smaller (1023 bp) fragment and uses a nested PCR step.⁷ Using this method, viruses from 38 of the 40 DBSs (95%) were successfully genotyped (Table 1). Despite the use of a different assay to genotype viruses from these 38 DBS specimens, resistance genotypes were highly concordant with those generated from plasma using the ViroSeq assay; 275 of the 291 mutations found in plasma viruses were also found in viruses from DBSs (data not shown).

To investigate if the decreased amplification success rate observed with the ViroSeq assay was due to insufficient or degraded HIV nucleic acids, we performed serial dilutions of RNA extracts from high VL specimens and tested them by both the in-house and the ViroSeq assays (Figure 1). Although both assays have comparable sensitivity, amplification signals were only maintained when the shorter pol fragment was amplified by the in-house method. Figure 1 shows that such signals were consistently lost when the larger pol fragment was amplified using the ViroSeq assay, suggesting the possibility of nucleic acid degradation during storage at 4°C. Such a possibility was further supported by parallel testing of serial dilutions of RNA extracts prepared from specimens stored at −20°C. Figure 1(c) shows that amplification signals generated from these specimens using the in-house assay were generally stronger than those obtained from the same DBS specimens stored at 4°C (Figure 1b).

Our study on a small number of specimens suggests that storage at 4°C may represent a feasible alternative to −20°C for long-term storage of DBSs. However, we noted that the efficient genotyping from these DBS specimens required the use of an in-house nested PCR assay and that the ViroSeq assay failed to genotype a substantial proportion of specimens. The results with the ViroSeq assay differed from our previous findings with this assay showing a high success of genotyping from DBSs stored at −20°C and suggest that some degradation of RNA may have occurred during long-term storage at 4°C possibly due to suboptimal storage temperature, humidity or both. Such a possibility was suggested by the weaker amplification signals observed in RNA extracts from specimens stored at 4°C compared with those stored at −20°C upon serial dilutions. Although different efficiencies of RNA extraction could partially explain these differences, we found that a high proportion of the specimens stored at 4°C had evidence of humidity at 6 months, whereas none of the specimens stored at −20°C did. High humidity conditions are thought to be detrimental to resistance testing from DBSs given the extreme sensitivity of HIV nucleic acids to degradation in the presence of humidity.¹⁰ Nonetheless, we showed that it is possible to overcome potential losses in HIV-1 RNA integrity and efficiently genotype from DBSs stored at 4°C by using an in-house nested PCR protocol that amplifies a smaller fragment using stringent, quality-controlled reagents. These results are encouraging and expand on our earlier findings showing the efficient genotyping of HIV-1 pol from DBSs stored at −20°C for up to 4 years.^6,7 A high success of genotyping has also been noted from DBSs stored for 3 months at 37°C and 85% humidity in the presence of desiccant.¹⁰ Under these extreme conditions, viruses from ∼85% of the DBS specimens could be genotyped by using in-house nested PCR assays that amplified small (700 bp), overlapping pol sequences.¹⁰ The use of nested PCR protocols and/or the amplification of small fragments may likely result in higher amplification success rates from DBSs stored under less optimal conditions. Such a possibility was supported by our findings, showing a better maintenance of amplification efficiencies using the in-house method upon serial dilution of RNA extracts. Despite having comparable sensitivity, amplifications were rapidly lost when a larger fragment was amplified using the ViroSeq assay.

The main objective of our study was to evaluate the amplification success rate from DBSs stored at 4°C for a prolonged period of time. We used an in-house assay validated for HIV-1 subtype B since our study population included patients infected with subtype B viruses. It is important to note, however, that information on the performance of our assay with non-B subtypes is limited to a small number of specimens.⁷ Therefore, we caution that resistance testing from DBSs collected in areas with prevalent non-B subtypes will require in-house assays appropriately validated for other HIV subtypes.

The ability to collect blood samples on filter paper represents an advantage for HIV drug resistance surveillance and monitoring, particularly in areas that lack the appropriate infrastructure for plasma processing and transport. Much effort is currently underway to define the best conditions that will facilitate resistance testing from DBSs including the most appropriate storage temperature and time. The findings reported here suggest that 4°C may represent a feasible storage temperature for long-term storage of DBSs and add to the promise of DBSs as a convenient specimen type for HIV-1 drug resistance testing.

Work at the CDC was done with intramural funding. Work at Hospital Carlos III was supported by grants from the Fondo de Investigación Sanitaria (FIS, CP06/284 and PI06/1826).

None to declare.

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.

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