An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler.

Rapid and specific detection of botulinum neurotoxin (BoNT) producing Clostridia is a priority for public health authorities, in case of both natural and intentional botulism outbreaks. This study reports on the evaluation of a detection system based on the GeneDisc Cycler designed for simultaneously testing the bont/A, bont/B, bont/E and bont/F genes encoding for the botulinum neurotoxins types A, B, E and F. BoNT-producing Clostridia (n = 102) and non-BoNT-producing bacteria (n = 52) isolated from clinical, food and environmental samples were tested using this macro-array and results were compared to the reference lethality test on mice. The bont genes were correctly detected in all C. botulinum type A, B, E and F strains available, as well as in toxigenic C. baratii type F and toxigenic C. butyricum type E. No cross reactivity was observed with non human-toxigenic bacteria, C. botulinum types C, D and G. The identification of the bont genotype using the macro-array was correlated to toxino-typing of the BoNTs as determined by the mouse bioassay. An "evaluation trial" of the GeneDisc array performed blind in four European laboratories with 77 BoNT-producing Clostridia as well as 10 food and clinical samples showed that the developed macro-array is specific and reliable for identifying BoNT/A-, BoNT/B-, BoNT/E- and BoNT/F-producing clostridial strains and for screening naturally contaminated food and fecal samples. The test is robust, has a low detection limit (c.a. 5 to 50 genome copies in the PCR reaction microwell) and is promising for monitoring BoNT-producing Clostridia in different kinds of samples including food and clinical samples.

Wound botulism in drug users: a still underestimated diagnosis.

Wound botulism is a rare infectious disease that is becoming a frequent complication of parental drug use. Diagnosis is often difficult and based on clinical suspicion. We report the first Italian case of wound botulism due to intramuscular heroin injection in a 48-year-old man with an acute onset of slurred speech and dysphagia. The most considerable finding of electrophysiological study was the reduction in amplitude of compound muscle action potential which should be considered a useful initial electrodiagnostic sign in the clinical context of botulism. Alerting clinicians to botulism is crucial for a rapid diagnosis and appropriate treatment and thus decreasing mortality and complications.

Intestinal toxemia botulism in Italy, 1984-2005.

Botulism in humans is caused by botulinum neurotoxins, produced in most cases by Clostridium botulinum, although other Clostridia species are implicated as well. Of the five forms of botulism in humans, three are referred to as "infective": wound botulism, infant botulism, and adult intestinal botulism; the latter two forms are also referred to as "intestinal toxemia botulism" because the organism colonizes the lumen of the intestinal tract and produces botulinum neurotoxin in vivo. Twenty-three cases of infant botulism and three cases of adult intestinal botulism occurred in Italy between 1984 and 2005. Microbiological analyses of clinical, environmental, and food samples and analysis of clinical and epidemiological data revealed two main characteristics of intestinal toxemia botulism in Italy that are not common in cases in other countries: the isolation of a strain of C. butyricum that produced botulinum neurotoxin type E in 6 of 26 cases, including two cases of adult intestinal toxemia botulism, and the onset of botulism in these cases with concomitant severe gastrointestinal symptomatology. This report summarizes the microbiological, clinical, and epidemiological data of all cases of intestinal toxemia botulism that have occurred in Italy in the period 1984-2005.

A case if infant botulism due to neurotoxigenic Clostridium butyricum type E associated with Clostridium difficile colitis.

Reported here is the sixth case of intestinal toxemia botulism caused by Clostridium butyricum type E in Italy since 1984. In this case, the patient was concomitantly affected with colitis due to Clostridium difficile toxin. A review of previously reported cases revealed that some of these patients may also have had intestinal toxemia botulism associated with Clostridium difficile colitis, based on the reported symptoms. Given that this association has been shown to exist not only in Italy but also in the USA, it is recommended that individuals with intestinal botulism and symptoms of colitis undergo testing for Clostridium difficile and its toxins in fecal samples.

Intestinal toxemia botulism in two young people, caused by Clostridium butyricum type E.

Two unconnected cases of type E botulism involving a 19-year-old woman and a 9-year-old child are described. The hospital courses of their illness were similar and included initial acute abdominal pain accompanied by progressive neurological impairment. Both patients were suspected of having appendicitis and underwent laparotomy, during which voluminous Meckel's diverticula were resected. Unusual neurotoxigenic Clostridium butyricum strains that produced botulinum-like toxin type E were isolated from the feces of the patients. These isolates were genotypically and phenotypically identical to other neurotoxigenic C. butyricum strains discovered in Italy in 1985-1986. No cytotoxic activity of the strains that might explain the associated gastrointestinal symptoms was demonstrated. The clinical picture of the illness and the persistence of neurotoxigenic clostridia in the feces of these patients suggested a colonization of the large intestine, with in vivo toxin production. The possibility that Meckel's diverticulum may predispose to intestinal toxemia botulism may warrant further investigation.

Neurophysiological assessment in the diagnosis of botulism: usefulness of single-fiber EMG.

We report the clinical, serological, and neurophysiological findings in seven patients with foodborne botulism caused by ingestion of black olives in water. The clinical picture was characterized by mild symptoms with a long latency of onset and by involvement of cranial and upper limb muscles; only one patient, a child, developed respiratory failure. Spores of Clostridium botulinum were found in stools in some but not all cases. Conventional neurophysiological tests had low sensitivity; abnormal findings were present only in the patient with severe clinical involvement, in whom compound muscle action potentials (CMAPs) appeared reduced. Repetitive nerve stimulation at a high rate showed pseudofacilitation and not true posttetanic facilitation, but single-fiber electromyography (SFEMG) showed abnormalities of neuromuscular transmission in every case. Neurophysiological evaluation, particularly SFEMG, is important because it allows rapid identification of abnormal neuromuscular transmission while bioassay studies are in progress.

Clostridium botulinum spores and toxin in mascarpone cheese and other milk products.

A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential). In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin. Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C. botulinum. Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A. The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C. botulinum growth and toxinogenesis. Of the other milk products, 2.7% were positive for C. botulinum spores. Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively.

Transient tonic pupils in botulism type B.

We report a 29-year-old woman who developed unilateral unreactive mydriasis and cycloplegia after 5 days of persistent constipation. During the next hours the patient complained of dry mouth and difficulties in swallowing food; iris and ciliary muscle palsies spread over the second eye. Ocular motility was normal and there were no clinical signs of neuromuscular involvement. Conventional electromyography and evoked muscle action potentials following repetitive nerve stimulation were normal; single-fiber electromyography showed normal jitter and absence of blocking. The diagnosis of botulism was considered as most likely, and the patient was given botulinum antitoxin. The post-treatment course was characterized by bilateral tonic pupillary reaction to near, sectoral iris contractions to light and pupillary constriction to 2 mm in 40 min following topical instillation of 0.1% pilocarpine. Ocular manifestations completely disappeared within 5 weeks. Botulism type B toxin was demonstrated in the pretreatment stool of the patient but not the serum.

Recovery of a strain of Clostridium botulinum producing both neurotoxin A and neurotoxin B from canned macrobiotic food.

A rare strain of Clostridium botulinum subtype Ab was isolated from a canned macrobiotic food suspected of being linked to a fatal case of food-borne botulism. The strain was recovered and identified by conventional methods modified by the inclusion of a PCR assay (G. Franciosa, J.L. Ferreira, and C.L. Hatheway, J. Clin. Microbiol. 32:1911-1917, 1994). The titers of neurotoxins produced by the strain were evaluated by a mouse bioassay.

PCR for detection of Clostridium botulinum type C in avian and environmental samples.

A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak.

A case of infant botulism associated with honey feeding in Italy.

A case of infant botulism in a 9 week-old female is described. A strain of C. botulinum type B was isolated from the feces of the baby. The epidemiologic study detected in a sample of home canned honey Clostridium botulinum spores of the same serotype that was isolated from the patient. The honey had been used only to sweeten the pacifier of the baby. This is the first case of infant botulism in Europe linked conclusively to honey.

Wound botulism: clinical and microbiological findings of an Italian case.

A case of wound botulism in a 41-year old man is reported. The patient had accidently been wounded when he fell on an iron bar. Some days later he developed typical clinical manifestations of botulism. Wound botulism was confirmed by detection and quantification of type B botulinal toxin in the serum. Ventilatory supportive care was necessary and botulinal antitoxin was not given. The patient was hospitalized for 30 days and recovery was complete.

Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy.

The first two confirmed cases of type E infant botulism occurred in two 16-week-old girls in Rome, Italy. The original diagnosis for the first patient was intestinal blockage due to an ileocecal invagination, which was treated surgically. Postoperatively, the patient became unresponsive and required ventilatory assistance. A diagnosis of infant botulism was then made. The second infant presented to the same hospital 7 1/2 months later with profound weakness, hypotonicity, mydriasis, and areflexia. This case was recognized as possible botulism at admission. Both cases were confirmed by detection and identification of type E botulinal toxin in stool specimens and in enrichment cultures of those specimens. The toxigenic organisms isolated were quite different from Clostridium botulinum type E. The apparent causative organism in each case resembles Clostridium butyricum but produces a neurotoxin that is indistinguishable from type E botulinal toxin by its effects on mice and by its neutralization with type E botulinal antitoxin.

Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism.

The apparent causative organism from the only reported case of type E infant botulism was isolated and characterized. Except for its ability to produce type E botulinal toxin, this organism (strain 5262) would be unquestionably identified as Clostridium butyricum. This is the second time an organism resembling a defined Clostridium species other than a member of the C. botulinum group has been implicated in infant botulism.

[Food poisoning caused by Clostridium botulinum type E]. / Intossicazione alimentare da Clostridium botulinum tipo E.

The results of a microbiological investigation carried out into a home-canned tuna fish are reported in relation to a suspected botulism case. Toxin of Cl. botulinum type E was detected by mouse toxicity and neutralization tests. The food specimen were also cultured for Cl. botulinum. The isolates was identified as Cl. botulinum type E by biochemical, gas chromatographic and immunological tests. The outbreak in which for the first time in Italy, the Cl. botulinum type E is involved, concerns one person who showed typical signs and symptoms consistent with botulism (abdominal cramps, dilatated pupils, diplopia, dysphagia, paralysis of lower upper limbs). The laboratory results are discussed with relation to environmental characteristics of the micro-organism and their resistance to same chemical and physical factors with are involved in the canning practice.