Laboratory Diagnostics of Botulism

Abstract

SUMMARY Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…Despite enormous progress in the development of alternative in vitro methods for botulinum neurotoxin detection [23,24,25], the mouse lethality assay has remained the only accepted standard test to confirm active BoNTs [26]. This test is based on intraperitoneal injection of mice with dilutions of BoNT-suspected samples and subsequent observation of these mice for symptoms of botulism and, ultimately, death.…”

Infant botulism

“…39 An enzyme-linked immunosorbent assay (ELISA) has recently been developed for rapid detection of toxins A and B in IB. 40 This test allows detection within 24 h as compared to 4 days that are required for the mouse assay. The toxin can be identified in the stool of affected infants as long as 4 months after onset of symptoms, well into recovery.…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…Depending on the form of botulism, different sample materials are usually analyzed: in the case of food-borne botulism serum, feces, and suspected food; in wound botulism wound swabs, pus, tissue, and serum; and in infant botulism feces, serum, intestinal contents, suspected food, and environmental samples (Lindström and Korkeala 2006). With respect to diagnostics of suspicious botulism samples, attention has to be paid to the fact that the toxin occurs in different forms in different sample matrices.…”

“…By far, the most commonly employed methods are PCR-based techniques (Mullis et al 1986;Saiki et al 1988), many of which aim at detecting bont genes by conventional or quantitative amplification reactions (Szabo et al 1992(Szabo et al , 1993Franciosa et al 1994Franciosa et al , 1996Fach et al 1995Fach et al , 2009Takeshi et al 1996;Aranda et al 1997;Braconnier et al 2001;Kimura et al 2001;Craven et al 2002;Popoff and Walker 2003;Akbulut et al 2004;Takeda et al 2005;Yoon et al 2005;Lindström and Korkeala 2006;Artin et al 2007;Fenicia et al 2007;Heffron and Poxton 2007;Prévot et al 2007;Sánchez-Hernández et al 2008;Sakuma et al 2009;Hill et al 2010;Lindberg et al 2010;Takahashi et al 2010). Since conventional PCR is difficult to quantify and requires a post-PCR step to visualize and to verify the PCR product, many modern approaches use quantitative PCR (qPCR) formats.…”