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Dried blood spots (DBS) and dried plasma spots (DPS) are considered convenient alternatives to serum and plasma for HIV drug resistance testing in resource-limited settings. We sought to investigate how extreme conditions could affect the short-term ability to amplify and genotype HIV from DBS.
A panel of six matched DPS/DBS was generated using blood collected from HIV-infected donors. Replicate cards were prepared in 903 filter paper using 50 µL of blood and stored at either −20°C or at 37°C/100% humidity. Nucleic acids were extracted at baseline and after 1, 2, 8 and 16 weeks of storage and were amplified and sequenced using an in-house RT-nested PCR method or the ViroSeq assay.
HIV-1 pol was successfully amplified in all DBS/DPS at baseline and in those stored for up to 16 weeks at −20°C by the in-house assay. In contrast, amplification was rapidly lost during storage at 37°C/100% humidity with only 6/6 and 4/6 DBS specimens amplifiable by the in-house assay at weeks 1 and 2, respectively. Similarly, only two DPS stored at 37°C/100% humidity were amplified by the in-house assay at week 1.
We show that resistance testing from DBS and DPS is severely compromised after 2 and 1 weeks of storage at 37°C/100% humidity with desiccant, respectively. These findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport specimens from collection sites to locations that have appropriate storage conditions such as −20°C.
With an estimated 33.2 million people worldwide living with HIV at the end of 2007 and an estimated 2.5 million new infections in 2007, HIV/AIDS remains a major global health challenge.1 Rapid scale-up of antiretroviral treatment in resource-limited settings is an international priority and is fundamental for an effective global response to HIV. Several initiatives including the US President’s Emergency Plan for AIDS Relief and the Global Fund to Fight AIDS, Tuberculosis and Malaria have now demonstrated the feasibility of delivering HIV treatment in less-developed countries.2–4 In these areas, first-line regimens are considered particularly critical since such regimens usually involve simple co-formulated, generic, fixed-dose drug combinations. Suboptimal delivery programmes could lead to an increase in therapeutic failures, reductions in programme effectiveness, and emergence and transmission of drug-resistant HIV-1. Laboratory surveillance of drug resistance can provide essential information on trends in the prevalence of resistance at the population level and can be used to improve antiretroviral programme effectiveness.5
Plasma or serum are the preferred specimen types for HIV-1 drug resistance testing. However, these specimens are not practical in areas that lack the appropriate infrastructure for blood processing, shipping and storage. In these areas, alternative specimen types with simplified processing and less stringent storage and transport requirements can simplify resistance testing. Dried blood spots (DBS) are easy to prepare and store and have been extensively used for HIV-1 antibody testing, molecular diagnostics, virus load quantification and, more recently, drug resistance testing.6 We recently showed that HIV-1 pol can be efficiently genotyped from DBS stored for prolonged periods at −20°C or 4°C, and that resistance genotypes from DBS and plasma were generally concordant.7,8 However, we noted that the efficient genotyping from DBS stored at 4°C for 1 year required the amplification of small pol fragments in a nested PCR protocol, and was compromised using assays that amplified a larger fragment.8 Such assay requirements were associated with RNA degradation that occurred during long-term storage at 4°C, and illustrated how suboptimal storage conditions can influence the efficiency of genotyping from DBS.8 These observations emphasized the need to better define the impact of humidity and temperature on the ability to genotype from DBS.
Resistance testing from DBS in resource-limited countries usually involves transportation of specimens from DBS collection sites to a centralized location that has appropriate storage conditions. In this scenario, different humidities and temperatures are possible and may potentially affect the efficiency of HIV genotyping from DBS. In this study, we sought to investigate how extreme conditions that may be encountered at DBS collection sites and during transport could affect the short-term ability to amplify and genotype HIV from DBS.
We prepared a panel of DBS and DPS using blood collected from six subtype B HIV-infected donors enrolled in the Virology Quality Assurance (VQA) Program. Samples were collected by protocols approved by the Institutional Review Board at Rush University Medical Center. Plasma RNA virus loads in these patients ranged from 570 240 to 38 520 RNA copies/mL (Roche Amplicor HIV-1 Monitor, Roche Molecular Systems, Branchburg, NJ, USA). DBS and DPS were prepared by pipetting 50 µL of venous whole blood or plasma onto pre-marked circles on 903 filter paper cards (Schleicher & Schuell, Keene, NH, USA), respectively. A total of 18 replicate cards were prepared per patient; cards were packaged individually with desiccant (Mini Pax Sorbent, Multisorb technologies, Buffalo, NY, USA), grouped and bagged by panel (zip-lock bags, Whatman) and stored at the indicated temperature and time (Figure 1). Two group panels (A and C) were stored at 37°C and 100% humidity and tested using an in-house RT-nested PCR assay (panel A) or the ViroSeq assay (Abbott Molecular, Des Plains, IL, USA) (panel C, Figure 1). Two other control group panels (panels B and D) were stored at −20°C and tested in parallel using the same two assays (Figure 1).
Group panels A and B were shipped overnight at room temperature to the Centers for Disease Control and Prevention (CDC) for storage and group panels C and D were stored on-site (VQA). Upon arrival at CDC, an additional bag of desiccant with humidity indicator was added to each individually packaged bag and to each group panels A and B prior to storage; these extra desiccant bags were not added to the specimens stored on-site. DPS were prepared in parallel from the same patients (18 cards per patient) using 50 µL of plasma and were stored under the same conditions and for the same periods of time.
Total HIV-1 nucleic acids were extracted from one spot at baseline and after 1, 2, 8 and 16 weeks of storage by using the silica-based Boom manual extraction method (bioMerieux, Inc., Durham, NC, USA) with some modifications for DBS processing as previously described.9 We extracted nucleic acids from a total of 216 samples (108 DBS and 108 DPS). Genotypic testing at the CDC was done in duplicate using an in-house RT-nested PCR method validated only for HIV-1 subtype B viruses.9 This method amplifies a 1023 bp fragment of HIV-1 pol comprising amino acids 15–99 of the protease and 1–256 of the RT and has a sensitivity of detection of 1000 HIV-1 RNA copies/mL. Genotypic testing at the VQA was done using the ViroSeq HIV-1 Genotyping System. The ViroSeq assay amplifies a 1.8 kb pol fragment and has a sensitivity of detection of 2000 RNA copies/mL of plasma. Input sample for each amplification reaction was one-third of the extracted nucleic acid material (10 µL).
Using the in-house assay, HIV-1 pol was successfully amplified and genotyped in all DBS and DPS specimens at baseline and in those stored for up to 16 weeks at −20°C (Table 1). In contrast, amplification was substantially reduced during storage at 37°C/100% humidity. Table 1 shows that while all six DBS specimens were amplified at week 1 using this assay, only four of the specimens were amplified at week 2 and none was amplifiable at week 8 or 16. Visual inspection of humidity indicator bags in specimens stored at 37°C/100% humidity showed evidence of moisture at week 6, as indicated by changes in the colour of the desiccant packs from blue to pink. Table 1 also shows that only two of the DPS prepared and stored in parallel were amplifiable after 1 week at 37°C/100% humidity. A comparison of RT and protease genotypes obtained from DPS or DBS at baseline and after 16 weeks at −20°C showed full genotypic concordance at positions associated with drug resistance in all six specimens; two specimens had RT resistance mutations (K103N; specimens 2 and 5) and the six specimens had common protease polymorphisms (L63P, V77I, I93L, M36I, D60E). The overall nucleotide similarity between paired plasma and DBS sequences was 99.4% (range, 98%–100%) at baseline and 99.7% (range, 99%–100%) after 16 weeks of storage at −20°C (data not shown).
Using the ViroSeq assay, all the DBS specimens stored at −20°C were amplified through the 16 weeks with the only exception of one specimen that was negative at week 1 (Table (Table1).1). However, none of the specimens stored at 37°C/100% humidity was amplifiable at any timepoint (Table (Table1);1); a visual examination of these specimens showed evidence of mould.
Our results on a small number of DBS and DPS specimens suggest the existence of a short window of opportunity to genotype specimens exposed to 37°C and high humidity conditions. We noted a rapid decline in amplification efficiencies by a validated in-house assay that has been successfully used to genotype specimens stored for 1 year at 4°C or 2–3 years at −20°C, suggesting that high temperature, humidity or both may have degraded HIV-1 nucleic acids in our DBS specimens.8,9 The inability of the ViroSeq assay to genotype specimens stored under suboptimal conditions is not unique to this study and was recently noted with specimens maintained at 4°C for prolonged periods of time.8 Such low amplification efficiencies were explained by a higher sensitivity of the ViroSeq assay to RNA degradations since this assay amplifies a larger pol fragment in a single PCR amplification step.8 However, the differences in amplification efficiencies between the two assays observed in the present study might also be due to variations in the initial processing of the two panels. While extra desiccant bags were added to the group panels tested by the in-house method upon arrival at CDC, such extra desiccants were not added in the panels evaluated using the ViroSeq assay, resulting in higher humidity and likely explaining the presence of mould in these specimens.
Humidity is thought to be detrimental for the successful amplification of HIV from DBS and DPS specimens as RNases present in blood may easily degrade HIV RNA in the presence of moisture. Indeed, different amplification efficiencies have been noted in specimens stored at 4°C in the presence or absence of desiccant using similar assays.8,10 Higher amplification efficiencies than those reported here have also been noted upon storage at 37°C but lower (85%) humidity conditions for up to 3 months although the study amplified smaller 700 bp pol fragments.11 Our study shows that even in the presence of desiccant, the window of opportunity to genotype specimens exposed to extreme conditions is short. However, our design did not contemplate changes in desiccant, which may be advisable upon evidence of moisture.12 The addition of extra desiccants might better control humidity and expand the window of opportunity to genotype specimens exposed to high humidities.
Our experimental design contemplated specimens with medium to high plasma virus loads and nucleic acid extractions from only one 50 µL spot. However, extractions from two spots have generally resulted in improved amplification efficiencies from specimens with low plasma virus loads and are currently recommended by the WHO for resistance testing.7,13 The use of two spots may be particularly important for the efficient genotyping of HIV from DBS specimens stored under suboptimal conditions.
In resource-limited settings where antiretroviral treatment is being scaled up, the WHO recommends periodic surveillance of transmitted HIV drug resistance to assess and prevent drug resistance emergence and improve programme effectiveness.5 The standardization of procedures for collection, handling, shipment and storage of DBS specimens is a key component of the current WHO efforts.12 Recent guidelines recommend that short-term storage of DBS at room temperature (15°C–30°C) should not exceed a maximum period of 2 weeks and that transport from DBS collection sites to the processing laboratory for genotypic testing should be done within 7–14 days. Our findings support these recommendations. While our conditions do not necessarily represent all possible field conditions, the findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport and store DBS and DPS from collection sites to locations that have appropriate and more controlled storage conditions such as −20°C.
Work at the CDC was done with intramural funding. Work at Rush Medical College was supported by NIAID contract HHSN266200500044C/NO1-AI-50044.
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. Use of trade names is for identification only and does not constitute endorsement by the US Department of Health and Human Services, the Public Health Service or the Centers for Disease Control and Prevention.