Lofgren SM, Morrissey AB, Chevallier CC. Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote healthcare facilities. 2009 Nov 27;23(18):2459-66.
Efforts to expand early HIV treatment to perinatally infected infants is hampered by the difficulties associated with diagnosis. Nucleic acid amplification testing (NAT) is an effective way to diagnose HIV in this group(1) and also may offer a way to bypass the inaccuracy of using clinical and immunological markers to gauge response to treatment in infants.(2) In resource-constrained areas, NAT is not easily accomplished because the storage and transport requirements for the plasma are not routinely available in these areas. Dried blood spots (DBSs) are used in developing areas for other tests and may offer an alternative to the collection, storage, and transport of plasma. Dried blood spots have been used successfully for DNA and RNA polymerase chain reaction (PCR) to diagnose infant HIV(3) and to determine HIV RNA concentration(4) under laboratory conditions.
To assess the technical and operational performance of DBSs to diagnose HIV by RNA testing (Part A) and to monitor HIV in rural/remote clinics (Part B)
Two rural and remote healthcare facilities in northeastern Tanzania
Cross-sectional comparison of laboratory methods
Infants aged less than 18 months who were HIV-exposed or clinically suspected to have HIV infection (Part A) and HIV-infected persons aged 18 months or older (part B)
Sensitivity and specificity of DBS RNA compared to plasma RNA and compared to DBS DNA for infant HIV diagnosis and DBS RNA compared to plasma for detection of virologic failure
Blood specimens of 5-10 mL were collected from subjects and placed into EDTA tubes. Fifty µL whole blood aliquots were spotted onto Guthrie 903 filter paper cards and air dried for at least four hours in the laboratory. Up to 10 DBSs were prepared for each participant. After drying, the cards were placed into gas-permeable zip-locked bags with desiccant and stored at ambient temperature. The samples were mailed weekly to the central laboratory (Kilimanjaro Christian Medical Centre (KCMC)) using a mail service of the Tanzania Posts Corporation. Remaining blood was separated by centrifugation and the plasma frozen at -80°C and transported on dry ice to KCMC weekly via courier.
At KCMC, DBSs were cut from the cards and transferred to a 50 mL tube containing lysis buffer and incubated at room temperature for two hours with intermittent mixing; 1 ml of the solution was tested for HIV RNA using the DBS protocol.
Dried blood spots from Part A of the study also were sent to the University of Witwatersrand, South Africa for HIV DNA testing using standard procedures. Dried blood spots from 32 patients enrolled in Part B were stored for 10 weeks before testing. The baseline RNA level was the first DBS RNA measurement at 40 days or less and the follow-up level was the second DBS measurement taken between 41 and 80 days.
Assessment of the performance of DBSs under field conditions, the duration of transit times and proportion of sample damage using mail transport were monitored. Turn around times were calculated using the time from sample collection to shipping, time in transit, time in KCMC before testing, and time from testing to sending the result.
There were 375 participants enrolled from October 29, 2008 to March 6, 2009. Of these, 313 (84%) had samples available; 176 samples for Part A and 137 for Part B.
In the early infant diagnosis group, 39 (22%) participants had detectable plasma RNA compared with 35 (20%) by DBS RNA. At the threshold of at least 1000 copies/mL, 34 (19%) infants were classified as infected by plasma RNA compared with 35 (20%) by DBS RNA. At the threshold of at least 10,000 copies/mL, 31 (18%) infants were classified as infected by plasma RNA compared with 31 (18%) for DBS RNA. With the exception of one specimen (with RNA in plasma and DNA but not RNA on DBS), there was complete concordance between DBS RNA and DBS DNA at the DBS RNA threshold of at least 1000 copies/mL. Of 36 samples that tested positive by DBS DNA, four (11.1%) had DBS RNA levels of 1000-10,000 copies/mL from patients aged 0, 1, 7, and 15 months. There were also three patients aged 10, 13, and 14 months, negative by DBS DNA, with DBS RNA not detected and plasma RNA levels of 70, less than 40, and 237 copies/mL, respectively. None of the patients was on antiretroviral therapy (ART).
Comparing plasma RNA with DBS RNA at the threshold of at least 1000 copies/mL, estimated sensitivity and specificity (95% confidence interval (CI)) were 1.00 (0.90-1.00) and 0.99 (0.96-1.00), and were 1.00 (0.89-1.00) and 1.00 (0.97-1.00) at the threshold of at least 10,000 copies/mL. Comparing DBS RNA with DBS DNA at the threshold of at least 1000 copies/mL, estimated sensitivity and specificity (95% CI) was 0.97 (0.86-1.00) and 1.00 (0.97-1.00), respectively, and was 0.86 (0.71-0.94) and 1.00 (0.97-1.00), respectively, at the threshold of at least 10,000 copies/mL.
Among participants in Part B, the median age was 34 years (range 21 months to 77 years) and the median CD4 cell count was 253 cells/µL. Seventy-three patients were receiving ART. At the threshold of at least 400 copies/mL, 82 (60%) participants were classified as having virologic failure by plasma RNA compared with 88 (64%) by DBS RNA. At the threshold of at least 5000 copies/mL, 74 (54%) participants were classified as having virologic failure by plasma RNA compared with 76 (55%) by DBS RNA. Compared with plasma RNA, estimated sensitivity and specificity (95% CI) for classifying patients with virologic failure of DBS RNA at the threshold of at least 400 copies/mL was 0.99 (0.93-1.00) and 0.87 (0.76-0.94), respectively, and at the threshold of at least 5000 copies/mL was 1.00 (0.95-1.00) and 0.97 (0.89-0.99), respectively.
Using data from both parts A and B of the study, the r value produced was 0.9709; r value was 0.9675 for at least 5000 copies/mL but was 0.7301 for less than 5000 copies/mL.
Of the 32 samples re-tested to determine stability, the log RNA levels were essentially the same between baseline and follow-up.
The Tanzania mail service successfully transmitted all DBSs and results between sites and the central laboratory and none were damaged. The median time from collection to shipping was 1.5 days. Median time for testing was 13 days and the median time from testing to receipt of results was 1.5 days.
These results support the use of DBSs for RNA testing in remote areas.
This is a nicely done study. Although the participants may not be representative of the larger population, they do represent clinic attendees in rural Tanzania. The measures of laboratory performance and selection of a "gold standard" were appropriate.
This study demonstrated agreement between DBS RNA and plasma RNA and DNA for infant diagnosis. These findings are consistent with those from other studies(5, 6) and support the use of DBSs for infant diagnosis in remote areas. The high sensitivity and specificity DBS RNA and DNA when compared with plasma at a threshold of 5000 copies/mL and the high specificity of DBS at a threshold of 400 copies/mL also support using DBSs for detecting virologic failure. Finally, the success of transport of specimens and return of results from the central laboratory demonstrate that using DBSs is feasible and can produce valid results. These findings hold promise for better diagnosis of HIV in infants and for determining virologic failure in remote areas.
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