Background: Microbiologic agents such as variola virus (VAR) are very attractive for terrorism. As a result of international collaboration under the WHO eradication campaign, smallpox was declared eradicated in 1980. Therefore, the immunization programs were discontinued worldwide. Because most people are now immunologically naive, VAR is considered to be a potential threat agent or bioterrorist weapon. Real-time polymerase chain reaction (PCR) followed by melting analysis was developed for fast and safe analysis and allows differentiation of VAR from other orthopoxviruses (OPVs) like vaccinia or camelpox virus.
Study design and methods: A RealArt Orthopox LC PCR kit (Artus GmbH) was used to amplify OPV sequences from blood donor samples. A total of 31,500 blood donor samples were tested in minipools of up to 96 samples. To evaluate the sensitivity of the assay, routine donor minipools (90 +/- 6 samples per pool) were spiked with vaccinia virus used as positive control.
Results: Specificity was 100 percent because none of 31,500 blood donors was positive for the presence OPV. The detection limit of the assay was 10.6 copies per PCR procedure. Therefore, a sensitivity of 1590 copies per mL was calculated. Overall, 0.28 percent of test results had to be considered invalid owing to negative internal controls.
Conclusion: The RealArt Orthopox LC PCR kit enables reliable detection of OPV DNA in viremic blood donor samples, even at the beginning of the disease when patients present minor clinical symptoms, and could be implemented in our routine screening procedure immediately. Thus, the assay could potentially help to prevent dispersion of viral agents by blood transfusion in case of bioterrorism.