Operational evaluation of the rapid viability PCR method for post-decontamination clearance sampling.

Abstract

The Rapid Viability Polymerase Chain Reaction (RV-PCR) method was evaluated during the Bio-Response Operational Testing and Evaluation (BOTE), an interagency project to evaluate field-level facility biological remediation, using leading decontamination technologies. The tests were performed using an intentional release (aerosolization) of spores of Bacillus atrophaeus subspecies globigii (BG), as a surrogate for Bacillus anthracis, the etiologic agent for anthrax. Three decontamination methods were assessed including fumigation with vaporized hydrogen peroxide (VHP), fumigation with chlorine dioxide (CD), and a surface treatment process using pH-adjusted bleach.The RV-PCR method was developed to rapidly detect live B. anthracis spores during a bioterrorism event. The method uses a change in realtime PCR response before and after a nine hour incubation step, to determine the presence of viable bacterial spores in the sample; the method was recently verified for air filter, wipe and water samples at the 10-spore level for B. anthracis Ames spores, and was also developed for swab, sponge-stick, and vacuum sock/filter samples. In the method, high throughput sample processing is combined with PCR-based analysis before and after a rapid culture step to speed viability determination, especially for complex surface and environmental samples that present challenges to current culture-based methods. In the BOTE project, a total of 159 surface wipe samples from post-decontamination events were analyzed by splitting the suspension after spore recovery into two equal parts, with one part analyzed by RV-PCR and the other part by culture after concentrating to the same volume. In the BOTE project, the RV-PCR method provided rapid results for post-decontamination samples that were 98% (156/159 samples) consistent with results from culture analysis. The percentage agreement was noteworthy, given the large number of samples containing low spore levels. For the Post-VHP, Post-Bleach, and Post-CD event samples, the percentage agreement was 93% (41/44 samples), 100% (47/47 samples), and 100% (68/68 samples), respectively. The RV-PCR method performed well for the surrogate BG spores exposed to decontaminants at real-world application levels, and with wipe samples containing background debris and indigenous microbial populations.