Evaluation of PCR Testing of Ethanol-Fixed Nasal Swab Specimens as an Augmented Surveillance Strategy for Influenza Virus and Adenovirus Identification

Abstract

Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at ؊70°C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

Effect of Preservative on Recoverable Rt-PCR Amplicon Length From Influenza a Virus in Bird Feces

“…This was measured by real-time PCR with a conserved 93-bp region of the matrix gene, as described (3). Although the data were not quantitative, we found that samples preserved with either commercial or guanidine buffers had approximately 10-to 100-fold less amplifiable viral RNA than observed for cloacal swabs on the basis of C T values (data not shown), whereas alcohols had approximately 1000-to 10,000-fold less (data not shown).…”

“…Our experience with fixed specimens in virus surveillance previously employed ethanol as a preservative (3,9). Although ethanol has been useful in the identification of influenza Apositive samples, it was insufficient to efficiently identify subtypes by RT-PCR (data not shown).…”

“…We became interested in preserved samples as an alternative surveillance method because they are expected to be cheaper, faster, less labor intensive, and more practical in remote areas, which would provide the capability of a world-wide influenza A virus surveillance network (3). However, the data that can be obtained from live virus cultures are currently more robust than those from fixed samples.…”

“…However, the data that can be obtained from live virus cultures are currently more robust than those from fixed samples. Preserved samples are not renewable, and their genomic analysis is limited by the quality of the RNA recovered followed by molecular methods such as reverse transcriptase-polymerase chain reaction (RT-PCR) (3). With short segments of recovered RNA, hemagglutinin (HA) and neuraminidase (NA) classifications become more difficult and inefficient.…”

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“…Newer methods that avoid this growth step, however, will facilitate direct high-throughput analysis of native samples from surveillance. This will include active samples preserved by cold chain as well as inactive samples preserved by chemical inactivation (Krafft 2005, Ilyushina 2005, Runstadler 2007). Since very little sequence information exists for may influenza subtypes, PCR primers will be updated and improved in an iterative fashion (see paper by Song et al in Appendices).…”