Reverse Transcription–Polymerase Chain Reaction Testing on Filter Paper–Dried Serum for Laboratory-Based Dengue Surveillance—American Samoa, 2018

Abstract

Laboratory-based surveillance for arboviral diseases is challenging in resource-limited settings. We evaluated the use of filter paper-dried sera for detection of dengue virus (DENV) RNA during an outbreak in American Samoa. Matched liquid and filter paper-dried sera were collected from patients with suspected dengue and shipped to a reference laboratory for diagnostic testing. RNA was extracted from each sample and tested for DENV RNA by real-time reverse transcriptionpolymerase chain reaction (RT-PCR). Of 18 RT-PCR-positive liquid specimens, 14 matched filter paper-dried specimens were positive for a sensitivity of 78% (95% CI, 55-91%). Of 82 RT-PCR-negative liquid specimens, all filter paper-dried specimens were negative for a specificity of 100% (95% CI, 96-100%). Shipping of filter paper-dried specimens was similarly timely but less expensive than shipping liquid sera. Using filter paper-dried serum or blood can be a cost-effective and sustainable approach to surveillance of dengue and other arboviral diseases in resource-limited settings.

Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics

“…Cards incubated at high humidity showed no difference in the time limit for detection of positive C t values when compared to dry cards incubated at low humidity ( S2 Table ). Despite this, the negative effect of humidity on the ability of FTA cards to stabilize RNA has been frequently documented in regions endemic to YFV where high humidity is common [ 28 ]. To assay if the reported loss in RNA yield was due to the card having absorbed moisture from the air prior to inoculation, cards were pre-incubated in the humidity chamber for one, two or three days before inoculation with YFV (illustrated in S3 Fig ).…”

“…In a study of the 2016–2018 Brazilian YFV outbreak viremia at time of death ranged from over 10 5 pfu/mL to less than 1 pfu/mL [ 39 ]. In many cases, the sensitivity of molecular assays was shown to decrease with the use of FTA cards [ 23 , 28 , 31 ]. It is hypothesized that this is due to many factors, including the volume of inoculum used, the humidity of endemic regions and incomplete drying of FTA card post-inoculation with sample [ 23 , 28 , 40 ].…”

“…In many cases, the sensitivity of molecular assays was shown to decrease with the use of FTA cards [ 23 , 28 , 31 ]. It is hypothesized that this is due to many factors, including the volume of inoculum used, the humidity of endemic regions and incomplete drying of FTA card post-inoculation with sample [ 23 , 28 , 40 ]. Although the LOD of YFV 17D RNA on FTA punches was shown to be less than 10 pfu/mL in both PBS and flavivirus-negative human sera, the LOD of FTA cards was significantly different than the gold standard of direct RNA extraction.…”

“…The high humidity of transmission season is also proposed to limit RNA stabilization by FTA cards [ 28 ]. One study showed that incomplete drying of FTA cards caused the most significant loss of RNA yield [ 23 ].…”

“…Cards that were inoculated dry and then placed at high humidity showed no significant loss in yield; however, cards exposed to a highly humid environment for days prior to inoculation ( Table 2 ), displayed a significant loss in RNA detection. This suggests that high humidity decreases the ability of FTA cards absorb inoculum as water is absorbed from the environment by the FTA card, limiting sample capacity as others have noted [ 22 , 23 , 28 , 32 ]. To counteract absorption of environmental humidity, ‘pre-humidified’ FTA cards were desiccated prior to, and after inoculation.…”