Botulism: Laboratory Methods and Epidemiology

Anaerobe (1999) 5, 165-168

Article No. anae.1999.0226

BOTULlSM (FACULTY PRESENTATION)

Botulism: Laboratory Methods and Epidemiology Rafael Alfredo Fernández* and Alberto Segundo Ciccarelli Cátedra de Microbiologia, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 33, (5500) Mendoza, Argentina

Although food botulism (FB) in Argentina was described by 1911, the first documented outbreak was recorded in 1922. In 1957, an outbreak of type A FB caused by red bell peppers was the first laboratory confirmation of botulism in Argentina. From 1922 to 1997, 70 FB outbreaks affecting 242 persons with 111 deaths (case fatality rate, 46%) were reported in Argentina. Infant botulism (lB) was recognized in 1976 and has been mostly diagnosed in the US.A. More than 146 lB cases have been reported in Argentina since 1982. Additional cases may go undiagnosed due to physician inexperience Key Words: botulism, Clostridium and limited access to diagnostic services. A single laboratory-confirmed botulínum, food botulism, case of wound botulism (WB) occurred in Argentina in 1995. The botulinal infant botulism neurotoxins (BoNTs) identified in Argentina have been types A, B, E, F and Af in FB, and exclusively type A in lB and WB. For the laboratory diagnosis of botulism, semm, gastrointestinal sample, food, and wounds should be tested for BoNT. Gastrointestinal, wound and food sample must also be cultured for toxigenic organisms. When higher volumes of semm were tested, BoNT was found in 61 % of lB patients in Mendoza compared with 13% in a previous series from the U.s.A. Reliable typing can only be achieved when the BoNT belongs to a known serotype and the toxin titer is aboye 4000 LDso/mL. When these criteria are not met, as in most clinical samples, bacterial isolation, purification and adequate toxin production in culture are required. Neutralization testing must be performed at not less than three lO-fold doses of toxin because of (1) the existEiQce of subtypes, where a second, minor serotype is present, (2) the sharing of epitopes between certain serotypes, and (3) the occurrence of serological variants. Three basic properties of working antitoxins, specifi­ city, protency and avidity, must be known for BoNT typing. The efficiency index (El), which expresses the avidity of antitoxins, is an important instmment for recognizing BoNT subtypes. (j')

1999 Academic Press

Epidemiology

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Although food botulism (FB) in Argentina was described by 1911, the first documented outbreak was recorded in 1922. Before 1957, the diagnosis of FB was made only on clinical grounds. In that year, a 1999 Acadernic Press

R.A. Fernández and A.S.. Ciccarelli

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botulism outbreak traced to commerciaHy processed sweet red beH peppers affected 21 persons and caused 12 deaths in La Plata, the capital of Buenos Aires Province. Type A botulinal toxin was detected in the pimentos by the mouse neutralization test, and Clostridium botulinum type A was isolated from a patient's feces [1]. Until 1981, the only botulism recognized in Argentina was FB due to type A neurotoxin. Subsequent outbreaks of FB have been caused by toxin types Af, B, E, and F toxins. eontemporary canning methods have decreased the global inciden ce of FB to low levels. From 1992, to 1997, 70 FB outbreaks affecting 242 persons with 111 deaths (case fatality rate, 46%) were reported in Argentina. Home-canned foods were incriminated in 66% of outbreaks and commercial foods in 13%. Preserved foods of vegetable origin accounted for 47% of outbreaks, mixtures of vegeta­ bIes and meat for 20%, and foods of purely animal origin for 11 %. A toxic food was not identified in the remaining 21 % of outbreaks. Infant botulism (lB), first recognized in 1976, occurs in infants between 1 week to 12 months of age. lB results from the absorption of botulinal neurotoxin produced in situ by clostridia colonizing the intestinal tracto The majority of published cases reside in the U.s.A. In Argentina, the first lB case was described in 1982. By 1997, 146 Argentine cases had been reported. For the diagnosis of lB, serum and fecal specimens were tested for botulinal toxin, and fecal samples were also cultured for the toxigenic organismo In aH Argentine patients, botulinal toxin and the organism were detected in feces. When higher volumes of serum were tested in Mendoza, toxin was found in 28 of 46 (61 %) lB patients [2] compared with nine of 67 (13%) of infants evaluated at the eDe in the U.s.A. [3]. The ages of the lB cases were between 1 "and 48 weeks with a mean of 13.7 weeks and a median of 12 Table 1. Cases of infant botulism in Argentina by province, 1982­ 1997 Province Mendoza Buenos Aires Neuquén San Luis Río Negro Chubut Córdoba La Pampa San Juan Misiones Salta Santa Fe Tierra del Fuego Tucumán Total

No. of cases

% of total cases

46 45 16 13 7 4 4 3 3 1 1 1 1 1

32% 31% 11% 9% 5% 3% 3% 2% 2% 1% 1% 1% 1% 1%

146

100%

weeks: 95% were 24 weeks old or younger. Of 93 (64%) patients whose sex was repoted, 55 (59%) were male and 38 (41 %) were female. Table 1 lists the geographic distribution of the cases; over 80% were diagnosed in the Mendoza, Buenos Aires, Neuquén or San Luis Provinces. Most of the patients lived in suburban or rural areas characterized by dryness, strong winds and dust. The date of illness was available for aH but two cases: of the remainder, 43 (29%) occurred in winter, 43 (29%) in spring, 36 (25%) in summer and 22 (15%) in autumn. AH strains isolated from lB patients belonged to C. botulinum type A, consistent with the predominance of this type in Argentine soils. It is possible that many cases are still unnoticed. Improvements in physician awareness of lB and in access to diagnostic labora­ tories may lead to better identification of cases. Wound botulism (WB) is arare disease. Between the first description of WB in 1943 and 1990, only 47 cases were reported in the U.s.A. [4,5]. Although WB in Argentina was described in 1992 [6], the first and only laboratory-confirmed case occurred in 1995 [7]. The patient was a girl whose right knee was punctured by a broken tree branch. She was admitted to an intensive care unit with somnolence, dysphagia and difficulty in walking. On examination, she had generalized hypotonia, sluggishly reactive pupils, nystagmus and diplopia. The diagnosis of WB was confirmed by the identification of type A C. botulinum neurotoxin and organism in the wound, and neuro­ toxin in her serum. She was treated with mechanical ventilation, antibiotics and botulinal antitoxin. After a period of stability, she suddenly died from hemody­ namic decompensation 20 days after admission.

Laboratory Methods The detection of circulating botulinal neurotoxin (BoNT) in serum is the most conclusive evidence of botulismo The direct detection of BoNT in suspected foods from outbreaks of FB is important because non­ toxic foods may contain toxigenic spores. BoNT's are the most potent bacterial toxins. Their potency derives from their enzymatic activity at the neuromuscular junction, resulting in a potentiaHy fatal, flaccid paralysis. The neurotoxins are divided into scvcn toxin types (A, B, el' D, E, F and G) and four subtypes (Af, Ab, Ba and Bn according to their antigenic properties but are closely related in structure and function. BoNT types A, B and E are mainly responsible for human botulism, and types el and D cause animal botulism [8]. In addition to the hetero­ genous anaerobic bacteria grouped as C. botulinum, at least three other clostridia, C. baratii (type F) [9],

Botulism: laboratory methods and epidemiology C. butyricum (type E) [10] and C. argantínense (type G) [11, 12] produce botulinum neurotoxin.

Currently, the mouse lethality test [13] is the only accepted method for the detection of BoNT. This is the most sensitive method available, with a detection limit around 10 pg/mL or < 5 mouse MLOso/mL [14, 15]. However, the test has a number of disadvantages. Mouse toxicity is not specific; toxin identification necessitates neutralization with botulinal antisera. This technique is tíme-consumíng, often taking at least 2 days, and requires laboratory animals. There is considerable need for rapid, specific and sensitive assays for BoNTs that do not use laboratory animals. Polymerase chain reaction (PCR) provides a very sensitive and specific method for identification of a known ONA sequence. Fach et al. [16] reported a PCR method for the rapid detection of type A BoNT from food samples. Franciosa et al. [17] reported detection of type A, B, and E BoNT genes in C. botulinum and other Clostridium species by PCR; however, not a11 BoNT genes were expressed. The type B toxin gene was detected in 43 type A toxin-producing strains, but only one (2%) of these strains produced functional type B toxin in culture. The type B toxin gene was also detected in two strains of C. subterminale, which were also non-toxigenic by bioassay. The potential for false­ positive results from unexpressed toxin genes sug­ gests that PCR alone is inadequate for establishing neurotoxigenicity [17]. Enzyme-linked immunosorbent assays (EUSAs) developed to date for BoNTs, while rapid and specific, are insufficiently sensitive to replace the mouse bioassay [14]. Over the past decade, amplified immunoassay systems with sensitivities similar to that of the mouse bioassay have been developed. Most of these are complicated, expensive and not yet widely available. Ooe11gast el al. [15] developed a sensitive modified EUSA for the detectíon o,",c. botulinum neurotoxins A, B, and E usíng signal amplification vía an enzyme-linked coagulation assay (ELCA). They demonstrated increases in sensitivity of 100- to 1000-fold compared with a non-amplified ELISA, and the method detected neurotoxin at less than 5 pg/mL. Another sensitive immunoassay for BoNT A, using a monoclonal antibody in conjunctíon with an enzyme amplíficatíon system, a110ws detec­ tíon of 5 to 10 MLOso mL of purified neurotoxín [14]. The average value for the mínimum level of detect­ able toxin in food extracts was 9 3.1/MLOso mL. Monoclonal antibodies may give false-negative results beca use of antigenic variations within botulinal toxins of the same type. In addition, immunoassay systems do not measure the biological activity of the neuro­ toxins [18]. The BoNTs act by cleaving proteins involved in exocytosis, vesicle-associated membrane protein

167

(VAMP), synaptosomal associated protein (SNAP-25) or syntaxin, thereby preventing neurotransmitter release at the neuromuscular junction. The neurotox­ ins appear to comprise a distinct class of endopro­ teases characterized by high specificity and strict substrate requirements. As an alternative to the mouse bioassay, EUSAs have been modified to measure the endopeptidase activity of the neurotox­ ins. These assays match the mouse assay for sensitiv­ ity, provide toxin type specificity and detect functional neurotoxin [19]. Hallis el al. [18] developed an assay for BoNT types A and B based on their endopeptidase activitíes with a sensitivity for BoNT B of 0.6 to 4.5 ng/mL, which could be increased to 0.1 to 0.2 ng/mL by using an amplification system based on catalysed reporter deposition. Until alternative assays are validated and accessible, the mouse lethality test remains the standard for the detection of BoNT in foods and pathological samples. Methods and techniques recommended for BoNT typing, including culture, metabolic characterization, purification procedures, toxin production and activa­ tíon, toxoiding and animal immunization, are dis­ cussed elsewhere [20-24]. Here we summarize relevant aspects of typing BoNT as developed and discussed by Giménez and Ciccarelli [22,24,25]. The typing of BoNT is usua11y performed from (1) samples where toxigenic clostridia have naturally grown (e.g. preserved foods, soil or silt), (2) clínical specimens, or (3) a culture medium growing toxigenic bacteria. Reliable toxin typing can only be achieved when BoNT of a known serotypes is produced at a level of at least 4000 LOso / mL, a titer higher than that found in clínical specimens. Sound culturíng techni­ ques are needed, therefore, for the isolation and purification of toxigenic strains and for obtaining a level of neurotoxin adequate for typing. The identification of strains producing subtypes, from which two toxin serotypes are present in varying proportions, is challenging. The minor serotype may account for only 1 to 10% of toxin produced by a subtype strain. In the routine typing of BoNT, it is common to perform a non-quantitative neutralization test using a single dose of 10 to 20 LOso and 1 international unit of antitoxin. The results of one dose neutralization testing can be misleading [25], as the multiple BoNTs of a subtype may not behave as separate antigens. Neutralization of the minor toxin can be achieved by increasing the dose.of antitoxin corresponding to the major toxin of the subtype [1t 21]. Furthermore, partial cross-neutralization may be expected within certain serotypes [20,26]. Accordingly, neutralization tests must be performed at no less than three doses of toxin starting from 20 LOso and increasing 10-fold (e.g. 20, 200, and 2000 LOso)·

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A delayed death of mice inQculated with toxin and antitoxin of the same serotype indicates incomplete neutralization. This may result from (a) an error in the estimation of the toxin or antitoxin dose, (b) the emergence of a second toxin not detected in a prior dilution or neutralization, or (c) a serologic variant, which requires a higher dose of antitoxin for its neutralization than that expected for a standard toxin. Three basic properties of working antitoxins, specificity, potency and avidity, must be characterized for typing a BoNTs. The efficiency index (El), which expresses the avidity of antitoxins [251, is an im­ portant instrument for recognizing BoNT subtypes. During neutralization tests with increasing doses of a BoNT, the downward shift in the trend of the El ímplies the appearance of an additional antigen not detected in the lower dose.

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