Molecular-genetic diagnosis of Botulism using polymerase chain reaction in Georgia

Author: Mari Gavashelidze
Keywords: BoNT, q-PCR
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Clostridium botulinum bacteria is spore-forming, obligate anaerobic, gram-positive rod which produces a neurotoxin (BoNT) and thereby is the etiological agent of botulism. Botulism is an acute neuroparalytic disease caused by the BoNTs the most potent toxins known. Therefore, it might be misused as anbiological weapon. BoNTs can be divided in seven antigenic types (A-G), of which human cases are caused primarily by neurotoxin types A, B, E and F. Based on the entry route to the body foodborne-, cutaneous-, injection- or inhalation botulism are known. Georgia, having a tradition of home canning, is one of the most affected countries worldwide by foodborne botulism. For the last 2 years canned vegetables prepared at home have been the main cause of botulism. In 2019 11 cases were identified and in 2020 all 9 cases were related to canned vegetables. National Center for Disease Control and Public Health (NCDC) in Georgia regularly receives clinical samples for the diagnosis of botulism. So far, routine diagnostic is based on isolation, cultivation and confirmation of the BoNTs by mouse bioassay, which is still the gold standard. However, this method is expensive, labour-intensive and slow. Quantitative real-time PCR (qPCR), with its high sensitivity, specificity and short duration, is an essential method for molecular testing in clinical diagnostics. Therefore, our aim is to broaden the routine diagnostics of BoNTs at NCDC by quadruplex real-time PCR assay. For verification of the qPCR, specifically designed plasmids, containing four BoNTs gene fragments (A, B, E, F) are used as positive controls. The whole process is carried out in the laboratory of the Richard Lugar Public Health Research Center and after successful completion of the procedure, this qPCR will be included to the national laboratory diagnostic algorithm along with serological and bacteriological tests. Overall, this achievement is significant for early and accurate diagnosis, epidemiological surveillance and public health. This work is part of the German Biosecurity Programme: Establishment of a Western Asian Network for the Improvement of Biosecurity in the Caucasus Region.