A 10 Year Analysis of the Use of Multiplex Real-Time PCR Screening for Botulinum Neurotoxin-Producing Clostridium Species

Prompt testing of specimens suspected of containingbotulinum neurotoxins produced by Clostridium botulinum, C. butyricum, and C. baratiiisis essential due to the potential of these toxins to produce rapid morbidity and mortality in humans. The Standard Mouse Bioassay (SMB) is the gold standard for C. botulinum neurotoxin (BoNT) testing but has several limitations including a long, labor intensive testing process and difficult result interpretation. We have developed and evaluated a sensitive screening tool for the detection of Clostridium spp. neurotoxin genes, BoNT types A, B, E, F and C. baratii F, by real-time PCR using an automated DNA extraction. Clinical specimens and environmental samples were analyzed over a 10-year period by real-time PCR, SMB, and culture. A total of 61 cases, clinically compatible with foodborne or infant botulism were submitted to our laboratory between 2003-2013. PCR was positive for 31 cases, 81% of these were confirmed by culture and 45% were confirmed by SMB. We also found that screening specimens by PCR provides an early indication of botulism in approximately 4 hours on all specimen types. Comparatively, SMB testing requires at least four days, is not appropriate for all specimen types, and requires sufficient quantities of specimen for analysis which precluded its use in 28 cases in our study. This rapid, high-confidence, cost-effective assay that detects the major BoNT types is a great addition to the investigation of suspect cases of botulism.