We demonstrate under field conditions in Tanzania that an HIV-1 RNA dried blood spot program performs well against liquid plasma HIV-1 RNA for viral load monitoring and against quantitative liquid plasma HIV-1 RNA and qualitative dried blood spot DNA for early infant diagnosis. Performance was best for samples from patients with plasma RNA levels above 5,000 copies/mL. DBS RNA levels appear to be stable after approximately 10 weeks of storage compared with a baseline sample tested at a median of 21 days following collection. Furthermore, we show that a DBS program performs well using existing staff at remote health care facilities, a robust central laboratory, and that using the local mail service for DBS transport is reliable and is less expensive than ground transportation of frozen plasma on dry ice.
For early infant diagnosis, our study showed agreement between DBS RNA and both plasma RNA and DBS DNA using an RNA threshold of ≥1,000 copies/mL. Although we were able to test only one sample rather than the two recommended by the American Academy of Pediatrics (AAP), several participant samples positive by DBS DNA would have been classified as HIV-uninfected using the AAP threshold of ≥10,000 copies/mL [11
]. Since untreated HIV-infected infants usually have high HIV RNA levels, it is unclear why four patients positive by DBS DNA had low HIV RNA levels, between 1,000 and 10,000 copies/mL. Low plasma HIV RNA levels have been seen in other studies [16
]. Possible explanations include that these patients are able to control HIV RNA levels [18
], they had early acute HIV infection with low HIV RNA levels prior to viral load ramp up [20
] either following breastfeeding [21
] or peripartum transmission, or that HIV-1 subtype variation resulted in underquantitation. Additionally, cross-contamination of DBS samples at sites could cause false positive results. The training provided to staff for this study would make cross-contamination unlikely but difficult to rule out. Other groups have shown that most infants with initial low levels of plasma RNA followed over time subsequently are confirmed to be HIV-infected [14
], but a proportion are found to be HIV-uninfected [16
Our findings on the use of DBS RNA for early infant diagnosis are consistent with those of others. A study in Thailand yielded sensitivity of 97–100% and specificity of 100% using DBS RNA with the ROCHE Amplicor, NucliSens, and an in-house assay [7
]. A South African study found sensitivity and specificity of 99.7% and100% using the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test [13
Compared with plasma RNA, DBS RNA was highly sensitive and specific for diagnosis of virologic failure at a threshold of ≥5,000 copies/mL and retained high sensitivity but lower specificity for virologic failure at a threshold of ≥400 copies/mL. Across a range of HIV-1 RNA levels, there was excellent agreement ≥5,000 copies/mL and fair agreement <5,000 copies/mL between plasma and DBS RNA ( and ). HIV-1 RNA levels from DBS tended to be higher than from plasma <5,000 copies/mL and we identified 25 samples with HIV RNA not detected on DBS but with low levels of plasma RNA detected. Entrapment of RNA in filter paper and amplification of proviral DNA may contribute to these findings. Our study was consistent with others showing disagreement and the frequency of lack of detection of DBS RNA increasing below 4,000–6,000 copies/mL [6
Our data suggests that DBS are stable over an 80 day period under laboratory conditions. DBS RNA samples for early infant diagnosis using a qualitative assay have been shown to remain 99.2% sensitive and 100% specific on retesting 4 years later [26
]. Using a quantitative HIV-1 RNA assay, DBS RNA concentration was shown stable 9 months when stored at a range of temperatures [7
] and in another study stable over 1 year if stored at room temperature or −70°C [6
]. Thus DBS RNA samples appear to be stable over a duration that exceeds the period of clinical value of the result.
We demonstrate that a DBS RNA program performs well under field conditions in Tanzania. The local mail service rapidly and reliably transported DBS samples to the central laboratory and plasma RNA results to the clinical sites. The program achieved a median turnaround time from sample collection to receipt of the result at the remote site of 23 days. Since many follow-up visits in HIV Care and Treatment programs in Tanzania are scheduled monthly, most results were available to the clinician at the next follow-up visit. Some results took longer, leading to inconvenience for clinicians and for patients. The main contributor to total turnaround time was the interval from sample receipt to testing at the central laboratory. Laboratory turnaround time could be shortened by improved maintenance of back-up electricity infrastructure, increased technical support, and greater experience of laboratory staff with the instrument and with DBS. Total turnaround time could be further reduced by using electronic or telephone transmission of results. Despite the total turnaround time, all RNA results were deemed to be clinically useful for patient management.
A DBS RNA service with centralized testing could reach a very large population requiring or receiving ART. In 2007 it was estimated that 136,000 people were receiving ART in Tanzania [27
]. The World Health Organization (WHO) recommends that, where available, HIV-1 RNA testing be offered to patients receiving ART. With annual testing, given that the assay evaluated in this study can process 93 patient samples per run, and assuming that an instrument at a central laboratory completes one run per day 365 days a year then 26,242 DBS samples could be processed per year. If two runs were done per day then 52,484 DBS RNA tests could be done per year. These test volumes would cover 39% of Tanzanians receiving ART in 2007.
Our study had a number of limitations. The remote healthcare facilities in this study had active research programs and capacities which may not be found in all rural and remote areas. The sites were served by a reliable and rapid mail service which may not be available everywhere. Our stability testing data are limited by a small sample size and would need to be verified with large numbers of samples. A number of samples were lost with assay errors and could not be included in the final analysis, possibly resulting in bias. Finally, because the study used patient samples the range of HIV-1 RNA levels may not have thorough evaluated the entire dynamic range of the assay.
We demonstrate that a DBS HIV RNA program serving rural and remote healthcare settings is feasible in Tanzania both in terms of the technical quality of the results and the operation of the program. We suggest DBS are a viable alternative to a plasma sample type within RNA programs and DBS RNA services could be scaled up to a national level. Having established the feasibility of such a program technically and operationally, we suggest that detailed cost-effectiveness analyses be conducted to allow Ministries of Health to determine costs and benefits of such a program. Larger studies that evaluate DBS program performance at non-research sites and that incorporate the impact of HIV-1 RNA results on patient management are needed.