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Temperature abuse of tuna (Thunnus alalunga) was carried out in order to assess the histamine buildup in fish-processing facilities where fish can be exposed to high temperatures for short periods of time. Histamine production was studied in tuna loins under different storage and abuse conditions. Tuna was stored at 0-2 degrees C, 3-4 degrees C, and 6-7 degrees C, and abused for 2 h daily at 20 degrees C and 30 degrees C for 7-12 days. Loins abused at 30 degrees C for 2 h daily contained potentially toxic histamine concentrations (67-382 mg kg(-1)) when stored at a low refrigeration temperature (0-2 degrees C), whereas when stored at 6-7 degrees C, the loins contained highly toxic histamine concentrations (544.5-4156.6 mg kg(-1)). Lower histamine concentrations (23-48 mg kg(-1) in loins stored at 0-2 degrees C and 124.7-2435.8 mg kg(-1) in loins stored at 6-7 degrees C) were observed in temperature-abused loins that were initially frozen. An increase over time was observed in most microbial counts tested. Bacteria isolated from the temperature-abused loins showed a varied ability of histamine production, with Morganella morganii, Klebsiella oxytoca, Staphylococcus hominis, and Enterococcus hirae being the most active histamine-producing bacteria.

Clostridium perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused by C. perfringens plasmid cpe isolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14 cpe+ C. perfringens fecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified as cpe+ type A, and 12 of the 14 cpe+ isolates carry their cpe gene on the plasmid and 2 carry it on the chromosome. Interestingly, cpb2 is present in only 12 plasmid cpe isolates; 11 isolates carry cpe and cpb2 on different plasmids, but cpe and cpb2 are located on the same plasmid in one isolate. C. perfringens enterotoxin is produced by all 14 cpe+ SD isolates. However, only 10 of the 12 cpe+/cpb2+ SD isolates produced CPB2, with significant variation in amounts. The levels of cpb2 mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypical cpb2 was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression of cpb2 mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94 cpb2, which overexpressed cpb2 mRNA, restored CPB2 production in F4969 (a cpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin in C. perfringens-associated SD.

Infant botulism was confirmed in a 5-month-old female by both isolation of Clostridium botulinum type B and by detection of type B botulinum neurotoxin in rectal washout and faeces. DNA fingerprinting of nine isolates from faeces yielded two different amplified-fragment length polymorphism (AFLP) patterns. C. botulinum was isolated from two of 14 food and drink items from the patient's home: C. botulinum type A was recovered from an opened container of dried rice pudding and C. botulinum type B from opened infant formula milk powder. Ten C. botulinum type B isolates from the opened infant formula yielded four AFLP patterns, two of which were indistinguishable from the clinical isolates. Fifteen unopened foods were tested and C. botulinum type B of a unique AFLP pattern was recovered from one unopened infant formula of the same batch as the opened container. It is suggested that multiple C. botulinum were present in both food and the intestine during infant botulism.

There are three main, naturally occurring, epidemiological types of botulism: food-borne, intestinal colonization (infant botulism) and wound botulism. The neurological signs and symptoms are the same for all three epidemiological types and may include respiratory paralysis. Wound botulism is caused by growth of cells and release of toxin in vivo, is associated with traumatic wounds and abscesses and has been reported in drug users, such as those injecting heroin or sniffing cocaine. Up to the end of 1999 there were no confirmed cases of wound botulism in the UK. Between the beginning of 2000 and the end of December 2002, there were 33 clinically diagnosed cases of wound botulism in the UK and Ireland. All cases had injected heroin into muscle or by 'skin popping'. The clinical diagnosis was confirmed by laboratory tests in 20 of these cases. Eighteen cases were caused by type A toxin and two by type B toxin.

Purpose of Review: In recent times the number of blooms of algae that produce toxins has increased in frequency, intensity and geographical distribution. This review describes some of the illnesses caused by fish and shellfish contaminated with toxins produced by marine algae and by bacteria., Recent Findings: The increase in toxic algal blooms may be a result of increased awareness, aquaculture, eutrophication, or transport of cysts in ship ballast. Improved chemical methods for the detection of algal toxins are now being developed, and so the number of toxins recognized is increasing. Toxicological data on some of these algal toxins are lacking. Despite the increase in occurrence of algal toxins, scombrotoxic poisoning remains the most common cause of food poisoning associated with the consumption of fish and shellfish. This may be real or it may be a reflection of lack of suitable tests for algal toxins or under-recognition by workers in health care., Summary: The major problem worldwide in this field is the lack of pure toxins for use in developing and standardizing chemical methods for toxin detection. Such methods would permit increased testing of both food and clinical specimens, and hence would prevent the entry of toxic food into the food chain and increase laboratory confirmation of incidents of illness.

Faecal specimens from 843 cases of diarrhoea in the community were tested for the presence of Clostridium difficile cytotoxin and Clostridium perfringens enterotoxin. C. difficile cytotoxin was detected in faecal specimens from 0.6 % of cases aged at least 2 years by using a Vero cell assay. Factors associated with detection of C. difficile cytotoxin were antibiotic therapy, age over 60 years and living in a home with other elderly people. Three methods were used for the detection of C. perfringens enterotoxin: a Vero cell assay, a commercial (TechLab) enzyme immunoassay (EIA) and an in-house EIA. The lower level of detection of pure C. perfringens enterotoxin in buffer was 0.01 micro g ml(-1) by the TechLab EIA and 1.0 micro g ml(-1) by the Vero cell assay. C. perfringens enterotoxin was detected by using the TechLab EIA in faecal specimens from 2.5 % of cases. This commercial EIA was less sensitive than the in-house EIA, detecting only 31 % of positive cases, but was specific and could be used for outbreak investigation by routine diagnostic laboratories. Age over 60 years was a factor associated with C. perfringens enterotoxin detection; this age group may be targeted for testing.

Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease. C. novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly. The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification. Additional phenotypic tests that are useful in recognising the main pathogenic species are described. Differentiation of C. novyi type A from C. botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture.

As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

A large outbreak of suspected botulism occurred on a dairy farm. The affected animals were listless and showed signs ranging from hindlimb unsteadiness to lateral recumbency, although the most common presentation was sternal recumbency with an apparent hindlimb weakness when stimulated to rise. Postmortem examinations revealed no conclusive gross pathology or histopathology. The affected cattle were found to have neutrophilia and hyperglycaemia with no other consistent haematological or biochemical abnormalities. The combination of clinical signs, disease epidemiology and the ruling out of other differential diagnoses strongly supported a diagnosis of unconfirmed botulism; however, the source of toxin was not demonstrated. Botulism is a severe disease in human beings and there are uncertainties about the pharmacokinetics and pharmacodynamics of Clostridium botulinum toxins. In such circumstances, a precautionary approach to food safety is essential. Restrictions were placed on the movement of livestock and sale of milk from the farm premises until 14 days after the onset of the last clinical case.

Between 1992 and 1999 1425 foodborne general outbreaks of Infectious Intestinal Disease (IID) were reported to the PHLS Communicable Disease Surveillance Centre. Of these, 148 (10%) were associated with the consumption of fish and shellfish. Three main aetiologies were identified. Outbreaks associated with fish (47%) occurred more frequently in the summer months, and were linked with Scombrotoxic fish poisoning caused by the consumption of tuna that was improperly stored. Outbreaks associated with molluscs (36%) were associated with the consumption of oysters contaminated with viral pathogens, particularly in February. Outbreaks associated with the consumption of crustaceans (11%) often involved eating prawns that contained either salmonellas or viral pathogens. The maintenance of microbial quality from prior to capture/harvesting until the moment of consumption, based on a Hazard Analysis and Critical Control Point style approach, is essential if gastrointestinal illness associated with such produce is to be avoided.

Thirty-five Clostridium perfringens isolates from patients and foods implicated in seven outbreaks of suspected Cl. perfringens food poisoning together with five unrelated incidents were analysed by serotyping and amplified fragment length polymorphism (AFLP). Despite minor band differences, AFLP was found to be highly reproducible and 16 different profiles (each unique to the 12 incidents) were recognised. The results from both serotyping and AFLP analysis identified exactly the same groups of related cultures. It is concluded that AFLP can provide a rapid, sensitive and reproducible method for the typing of Cl. perfringens for outbreak investigation.

A study was undertaken to identify the microorganisms and toxins in stool specimens associated with infectious intestinal disease (IID) among cases in the community and presenting to general practitioners (GPs) and in asymptomatic controls. Population based cohorts were recruited from practice lists in 70 practices and followed for 26 weeks (cohort component). Seven hundred and sixty-one cases of IID identified from the cohorts, 2893 cases who presented to GPs in 34 of the practices (GP component), and age/sex matched control subjects (555 and 2264, respectively) submitted stool specimens by post for comprehensive microbiological examination. Campylobacter spp (12.2% of stools tested), rotavirus group A (7.7%), and small round structured virus (SRSV) (6.5%) were the organisms most commonly detected in the GP component. SRSV was identified in 7.0% of cases in the community cohort. No target microorganisms or toxins were identified in 45.1% and 63.1% of cases in the two components. Aeromonas spp, Yersinia spp, and some enterovirulent groups of Escherichia coli were detected as frequently in controls as in cases. The higher frequency of detection of campylobacter, salmonella, and rotavirus among cases who presented to GPs than among those in the community suggests that those pathogens cause more severe illness. No enteropathogens were detected from a large proportion of cases although comprehensive standard methods were used to seek them.

A male patient with acute myeloid leukaemia received a pooled platelet preparation prepared by Optipress system on the last day of its shelf life. The patient collapsed after two-thirds of the contents had been transfused. Clostridium perfringens was isolated from the platelet bag within 18 h of the acute event. Metronidazole, gentamicin and Clostridium antiserum were then administered in addition to the broad spectrum antibiotics started previously. However, the patient died 4 days after the platelets were transfused. The cause of death was given as cardiovascular shock, entirely compatible with an overwhelming bacteraemic and septic episode. A coroner's verdict of accidental death due to transfusion of a contaminated unit of platelets was recorded. On subsequent investigation Cl. perfringens type A serotype PS68,PS80 (identical to that found in the platelet bag) was cultured from the venepuncture site of the arm of one of the donors who contributed towards the platelet pool. The donor had two young children and frequently changed nappies. Faecal contamination of the venepuncture site was the suspected source for the transmission of Cl. perfringens, an organism commonly found in the soil and intestinal tract of humans. This case dramatically highlights the consequences of transfusing a bacterially contaminated unit. It is vital that such incidents are investigated and reported so that the extent of transfusion-associated bacterial transmission can be monitored and preventative measures taken if possible.

The neurotoxins produced by Clostridium botulinum are amongst the most potent known to man. Toxin production is detected by a mouse bioassay, which requires several days for a result and is not acceptable for routine use unless there is a high level of suspicion. The Rapid ID32 A kit produced by bioMerieux gives an identification of an isolate within 4 h. The aim of this study was to examine the efficiency of the identification of Cl. botulinum using the Rapid ID32 A. Forty-two strains of Cl. botulinum, one strain each of botulinum toxin-producing Cl. butyricum and Cl. baratii, and four strains of Cl. sporogenes, were tested. One strain of Group I Cl. botulinum gave a presumptive identification of Group II Cl. botulinum, six strains of Cl. botulinum were identified as 50-98% Cl. botulinum in some tests, and 17 strains of Cl. botulinum were identified as < 50% Cl. botulinum. Thirteen strains of Cl. botulinum were identified as > 99% Cl. sporogenes or 86% Cl. histolyticum, and five strains gave a combination of these results. All strains of Cl. sporogenes were correctly identified. These results show that some strains of Cl. botulinum may not be correctly identified using the Rapid ID32A.

The purpose of this study was to investigate the incidence of cases of sporadic diarrhoea associated with enterotoxigenic Clostridium perfringens. Cases were identified by detection of C. perfringens enterotoxin with the Oxoid RPLA kit, with confirmation by ELISA, in faecal specimens from isolated incidents of diarrhoea and from which no other enteropathogen had been isolated. In a 2-month study, 65 (18%) of 370 specimens were enterotoxin positive. There was no predominant age group or sex in the enterotoxin-positive group, but higher proportion (79%) was resident in the community than were enterotoxin-negative cases (34%). Only four of the 65 enterotoxin-positive patients had received antibiotic therapy. Spore counts in most enterotoxin-positive patients were < 10(5)/g, indicating that detection of high numbers of C. perfringens is not useful in determining the aetiology of sporadic diarrhoea. Diagnosis should be confirmed by the detection of enterotoxin, but further work is required to assess whether an acceptable accuracy is obtained with the RPLA kit or whether ELISA is needed in all cases.

Clostridium perfringens type A is a common cause of food-poisoning. Production of lecithinase (alpha toxin) is frequently used to identify the organism. Details of 10 outbreaks of food-poisoning caused by lecithinase-negative C. perfringens are reported here.

Eighteen isolates of Bacillus species and 15 of Clostridium perfringens, all of which had been associated with outbreaks of either food poisoning or non-gastrointestinal infection (NGI), were examined for relatedness by pyrolysis mass spectrometry (PyMS). The PyMS-analysis correctly clustered all the groups of epidemiologically related isolates of both genera, and distinguished all the single, epidemiologically unrelated isolates of the same species. PyMS is a simple, rapid and inexpensive technique which can provide useful and accurate inter-strain comparisons within both the Bacillus and Clostridium genera in complete accord with conventional serological typing results.

We report the results of a clinical trial. Patients enrolled had serum IgG titres against Pseudomonas aeruginosa above the control range. Assignment to the observation or treatment group was by minimisation. Significant signs or symptoms in any patient prompted antipseudomonal treatment. In addition, the treatment group received antipseudomonal treatment at intervals of four months until the serum IgG titre returned to the control range. P aeruginosa was isolated intermittently from patients in the main trial. Nineteen patients were enrolled (12 observation, seven treatment). After one year in the trial changes in parameters studied, including forced expiratory volume in one second, IgG titre, serum IgG concentrations, and frequency of P aeruginosa isolation had improved in the treated group and worsened in the observation group.

Aims: To determine the incidence of sporadic and apparently non-food related diarrhoea associated with Clostridium perfringens enterotoxin., Methods: Enzyme linked immunosorbent assay (ELISA) and reversed phase latex agglutination (RPLA) were used to detect C perfringens enterotoxin in faecal specimens from 818 sporadic cases of diarrhoea., Results: C perfringens enterotoxin was identified as a cause of sporadic diarrhoea in 56 of 818 (6.8%) cases. Diarrhoea was prolonged (three days or more) in most cases. Ages ranged from 3 months to 89 years, although most patients were over 60 years of age., Conclusions: These results suggest that C perfringens may be a cause of sporadic cases of diarrhoea when causes such as food consumption or cross-infection are absent, particularly in the elderly.

Serum IgA antibodies to Pseudomonas aeruginosa cell surface antigens were estimated by ELISA. Titres in patients with and without cystic fibrosis and with no pseudomonal infection were low (less than 105 to less than 261). Titres in patients with cystic fibrosis who were chronically infected with P aeruginosa were very high (1200-163,000), and patients who grew the organism intermittently had intermediate titres. Longitudinal studies suggested increasing tissue invasion or involvement of the lower respiratory tract, or both, with increasing time of infection and identified patients with a good prognosis after the onset of pseudomonal infection. Detection of an increased serum IgA titre can give an earlier indication than measurement of the serum IgG titre of the presence of P aeruginosa in the respiratory tract in a proportion of patients. IgA measurement seems to be better than IgG measurement at predicting the reappearance of P aeruginosa after apparent eradication of early infection. These results suggest that this assay may be a valuable additional indicator of the presence of P aeruginosa at the beginning of infection, and of the reappearance of the organism after treatment in the early stages of infection.

Serum IgG antibodies to Pseudomonas aeruginosa cell surface antigens were determined by enzyme linked immunosorbent assay. Titres in patients without cystic fibrosis were low (140-235). Those in patients with cystic fibrosis who were chronically infected by P. aeruginosa were very high (1100-20,500), while patients who grew the organism intermittently had lower titres (160-4400). Longitudinal studies showed that raised titres were observed at a very early stage of infection. High titres were associated with a poor clinical state, while low titres were associated with a better clinical state in both chronic and intermittently infected patients with cystic fibrosis. These results suggest that this test is a specific and sensitive measure of the severity and progress of the different stages of pulmonary infection by P. aeruginosa in patients with cystic fibrosis.

An enzyme linked immunosorbent assay (ELISA) to measure free serum IgA antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis is described. Results were reproducible and there was no interference from crossreacting antibodies directed against other Gram negative bacteria. Titres were high in patients with Pseudomonas aeruginosa infection and differed according to the stage of the infection. These preliminary results suggest that this assay may be of value in assessing the state of Pseudomonas aeruginosa infection in patients with cystic fibrosis.

An enzyme linked immunosorbent assay (ELISA) to measure free serum IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis was developed. Seven strains of P aeruginosa cells, treated with glutaraldehyde and representing the most commonly isolated serotypes in our cystic fibrosis unit, were used. The specificity of the test was confirmed by the absence of cross reacting antibodies to other Gram negative bacteria. The results showed differences in the titres of antibodies at different stages of P aeruginosa infection. Because of its reproducibility, specificity, and sensitivity these preliminary results suggest that this test may be of value in monitoring the progress of P aeruginosa infection in patients with cystic fibrosis.

Serum IgG antibodies to Pseudomonas aeruginosa surface antigens were measured by enzyme linked immunosorbent assay in all patients with cystic fibrosis from whom P. aeruginosa was isolated for the first time during a study period of 18 months. In 15 patients the titre of serum IgG antibodies was greater than control values before or at the time of the first bacteriological isolation of P. aeruginosa. The presence of serum antibodies specific to P. aeruginosa suggests exposure to infection by that organism for some months before its isolation in significant numbers from the respiratory tract. In the other two patients serum titres were within the control range before isolation of P. aeruginosa but had increased to above the control range within the next month. Longitudinal studies on the entire group of patients showed further increases in titre concurrently with further isolations of P. aeruginosa. These results suggest that this assay may be an indicator of the beginning of pulmonary infection by P. aeruginosa and may prove to be a sensitive monitor of the progress of infection, and response to treatment, during the first months of infection by that organism.

Immunoglobulin G (IgG) antibodies to Pseudomonas aeruginosa surface antigens in serum were estimated by enzyme-linked immunosorbent assay for all patients from whom P. aeruginosa was isolated for the first time during a study period of 3 years (33 patients). The titer of IgG antibodies was greater than control values at or up to 24 months before the first isolation of P. aeruginosa in 24 patients. Another five patients had titers that were within the control range before isolation of P. aeruginosa but increased to above the control range within the following 2 months. In these 29 patients, continuing intermittent isolations of P. aeruginosa were accompanied by further increases in titer. The presence of a systemic immune response above the control range indicates tissue invasion and hence infection. Four patients were deemed to have no infection: one or two isolations of P. aeruginosa were accompanied by no increase in specific antibodies to above the control range throughout the entire study period. Fifteen patients received intravenous antipseudomonal chemotherapy. Eradication of the organism and a return of titer to control values, suggesting complete removal of the organisms, occurred in 5 patients, while continued isolations and only a partial decrease in titer occurred in 10 patients. The 15 patients who received treatment improved clinically, in contrast to untreated patients, whose clinical state worsened during the study period. Continuous steroid treatment, given to two patients, was accompanied by a dramatic decrease in both serum IgG concentration and titer, despite continuing intermittent isolations of P. aeruginosa. These results confirm and extend our earlier finding that this assay appears to detect P. aeruginosa infection at a very early stage and helps in differentiating between early infection and harmless colonization. It also appears to be a useful monitor of the progress of infection and the response to intravenous antibiotic treatment in these early stages of infection, before any clinical changes are sufficiently large to be detected, in patients who were not on continuous steroid therapy. The effect of steroid treatment on the immunological response and clinical outcome of patients with early P. aeruginosa infection requires further investigation.