Evaluation of a novel molecular assay to diagnose toxigenic strains of Clostridium difficile.

The objective of this study was to evaluate the Amplidiag C. difficile+027® assay, a new molecular method that detects toxin B gene in stool samples and identifies the hypervirulent 027 strain, to diagnose Clostridium difficile infections. The assay was compared to the reference method i.e. toxigenic culture. Amplidiag C. difficile+027® assay was prospectively evaluated from 309 diarrheal stool specimens of patients suspected of C. difficile infection. Forty-five (14.6%) stools were positive by toxigenic culture and 11 (3.6%) stools gave discordant results with the molecular method. PR027 was not recovered during the study. After resolving the discrepant results, the sensitivity, specificity, positive and negative predictive values of Amplidiag C. difficile+027® assay were 91.1% [CI 95% 77.9-97.1], 99.6% [CI 95% 97.6-100], 97.6% [CI 95% 85.9-99.9] and 98.5% [CI 95% 96-99.5], respectively compared to toxigenic culture. This assay is sensitive compared to the toxigenic culture.

Clostridium difficile associated reactive arthritis: Case report and literature review.

INTRODUCTION: Extra-gastro-intestinal tract manifestations associated with Clostridium difficile infection (CDI), including reactive arthritis (ReA), are uncommon. METHOD: We report a case of ReA associated with a relapse of CDI in a 46-year-old woman. A toxigenic C. difficile strain was isolated from stools and characterized as PCR-ribotype 014/020/077. We conducted a comprehensive literature review of ReA associated with CDI (ReA-CDI). Diagnostic criteria for ReA-CDI were: (i) evidence of aseptic synovitis (confirmed by culture) developing during or immediately after colitis, (ii) presence of a toxigenic C. difficile strain in stool samples, and (iii) absence of other causes of colitis and arthritis. RESULTS: Forty-nine cases of ReA-CDI (excluding the present report) have already been described since 1976. Of these reports, Mean age of patients was 38 years (SD: 18.5), 46% were male, and 68% had HLA B27 genotype. Sixty-nine percent of patients received a ß-lactamin treatment before CDI. ReA-CDI occurred a median 10 days (range 0-55 days) after CDI. Outcome was favorable in 90% of patients and oral non anti-inflammatory drugs were required for 55%. CONCLUSION: ReA-CDI remains uncommon. Compared to the general population, it is more likely observed in younger patients with HLA B27-positive genotype.

[Clostridium difficile colitis]. / Colites à clostridium difficile.

C. difficile is a spore-forming anaerobic enteropathogen responsible for a wide range of clinical features ranging from mild uncomplicated diarrhoea to severe debilitating disease, toxic megacolon, or even perforation and sometimes death. Risk factors for C. difficile infection (CDI) include age > 65 years, previous hospitalization and recent antibiotic therapy. Main virulence factors of C. difficile are toxins A (TcdA) and B (TcdB). Since 2005, a new hypervirulent strain has emerged. This epidemic strain named 027/NAP/BI has been responsible for outbreaks worldwide, with increased mortality and severity. Antibiotic treatment of CDI is based on severity of the disease and relies on the use of oral metronidazole, vancomycin or fidaxomicin. Control of CDI needs an antimicrobial stewardship policy and the implementation of contact precautions for the infected patients.

Molecular test based on isothermal helicase-dependent amplification for detection of the Clostridium difficile toxin A gene.

The AmpliVue Clostridium difficile assay and a glutamate dehydrogenase (GDH)-illumigene algorithm were evaluated using 308 diarrheal stool specimens of patients suspected of having C. difficile infection. Compared to the enriched toxigenic culture method, the sensitivities, specificities, and positive and negative predictive values of the AmpliVue C. difficile assay and the GDH-illumigene-based algorithm were 91.7% (95% confidence interval [CI], 76.4 to 97.8), 100% (95% CI, 98.3 to 100), 100% (95% CI, 87 to 100), and 98.9% (95% CI, 96.6 to 99.7), respectively.

Contribution of MALDI-TOF mass spectrometry and machine learning including deep learning techniques for the detection of virulence factors of Clostridioides difficile strains.

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA+B+CDT+, 22 ToxA+B+CDT+ Hv+ and 105 ToxA+B+CDT-) and 50 non-toxigenic (ToxA-B-) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA-B- strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA+B+CDT- strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA+B+CDT+ strains. The classifiers' performances for Hv strain detection were linked to high specificity (≥0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

Diagnosis and treatment of bacterial peritonitis in patients with gastrointestinal cancer: an observational multicenter study.

Background: Bacterial peritonitis (BP) in patients with gastrointestinal (GI) cancer has been poorly described, and its prevalence is unknown. Objectives: This study aimed to evaluate in patients with both GI cancer and ascites the prevalence of BP, associated features, mechanisms, prognosis, and the diagnostic performance of neutrophil count in ascites. Design: A retrospective, multicenter, observational study. Methods: All patients with GI cancer and ascites who underwent at least one paracentesis sample analyzed for bacteriology over a 1-year period were included. BP was defined by a positive ascites culture combined with clinical and/or biological signs compatible with infection. Secondary BP was defined as BP related to a direct intra-abdominal infectious source. Results: Five hundred fifty-seven ascites from 208 patients included were analyzed. Twenty-eight patients had at least one episode of BP and the annual prevalence rate of BP was 14%. Among the 28 patients with BP, 19 (65%) patients had proven secondary BP and 17 (59%) patients had multi-microbial BP, mainly due to Enterobacterales. A neutrophil count greater than 110/mm3 in ascites had negative and positive predictive values of 96% and 39%, respectively, for the diagnosis of BP. The median survival of patients with BP was 10 days (interquartile range 6-40) after the diagnosis. Conclusion: BP is not rare in patients with GI cancer and is associated with a poor short-term prognosis. When a patient with GI cancer is diagnosed with BP, a secondary cause should be sought. Further studies are needed to better define the best management of these patients.

Evaluation of the chromogenic agar chromID C. difficile.

Three selective media (chromID C. difficile agar, taurocholate cycloserine cefoxitin agar [TCCA; homemade], and CLO medium) were compared from 406 stool samples of patients suspected of having Clostridium difficile infection. The sensitivities of chromID C. difficile agar at 24 h and 48 h, CLO medium, and TCCA were 74.1%, 87%, 85.2%, and 70.4%, respectively.

Accuracy of ICD-10 codes for surveillance of Clostridium difficile infections, France.

The sensitivity and specificity of surveillance for Clostridium difficile infections according to International Classification of Diseases, 10th revision, codes were compared with laboratory results as standard. Sensitivity was 35.6%; specificity was 99.9%. Concordance between the 2 methods was moderate. Surveillance based on ICD-10 codes underestimated the rate based on laboratory results.

[Clostridioides difficile: updated recommendations]. / Clostridioides difficile : des recommandations actualisées.

CLOSTRIDIOIDES DIFFICILE: UPDATED RECOMMENDATIONS Clostridioides difficile is a spore-forming anaerobic enteropathogen responsible for a wide spectrum of clinical features ranging from mild uncomplicated diarrhoea to severe debilitating disease, toxic megacolon, or even perforation and sometimes death. Risk factors for CDI include age >65 years, previous hospitalization and recent antibiotic therapy. Main virulence factors of C. difficile are toxins A (TcdA) and B (TcdB). The emergence and dissemination of a new hypervirulent strain (027/NAP/BI) in 2005 has stimulated clinical and basic research on C. difficile. Major advances have been made regarding the CDI epidemiology (better recognition of community acquired CDI), diagnosis (molecular tests) and therapy (new drugs such as fidaxomicin, bezlotoxumab, and fecal microbiota transplantation) aspects. These advances have allowed the updating of management recommendations, under the aegis of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Antibiotic treatment of CDI depends on both the severity of the infection, and the risk/number of recurrences. Prevention of CDI requires an antimicrobial stewardship policy and the implementation of contact precautions for the infected patients.

CLOSTRIDIOIDES DIFFICILE: DES RECOMMANDATIONS ACTUALISÉES Clostridioides difficile est un bacille à Gram positif anaérobie sporulé responsable d'un large spectre d'infections digestives : de la diarrhée simple spontanément résolutive à la colite pseudomembraneuse qui peut se compliquer de mégacôlon toxique, de perforation et entraîner le décès du patient. Les principaux facteurs de risque d'infections à C. difficile (ICD) sont l'âge supérieur à 65 ans, l'administration d'antibiotiques et les antécédents d'hospitalisation. La virulence des souches est liée à la production de deux toxines, TcdA et TcdB. L'émergence et la dissémination rapide d'un clone dit « hypervirulent ¼ (027/ NAP/BI) en 2005 a stimulé la recherche clinique et fondamentale. Des avancées majeures ont été réalisées tant sur le plan épidémiologique (meilleure reconnaissance des formes communautaires d'ICD), diagnostique (nouveaux tests moléculaires) que thérapeutique (nouveaux traitements comme la fidaxomicine, le bezlotoxumab, ou la transplantation de microbiote fécal) ; ces progrès ont permis la mise à jour des recommandations de prise à charge, sous l'égide de la Société européenne de microbiologie clinique et des maladies infectieuses (ESCMID). Le traitement antibiotique des ICD dépend à la fois de la sévérité de l'infection, du risque et du nombre de récidives. Le contrôle de l'infection requiert, d'une part, la maîtrise de la consommation d'antibiotiques et, d'autre part, la mise en œuvre de précautions « contact ¼ vis-à-vis des patients infectés.

Prevalence of Antimicrobial Resistance and Infectious Diseases in a Hospitalised Migrant Population in Paris, France, a Retrospective Study.

Objectives: The aim of this study was to estimate the prevalence of anti-microbial resistance (AMR) carriage and its risk factors in hospitalized migrants. Additionally, the prevalence of infectious diseases was evaluated, as well as symptoms of psychological trauma. Methods: We conducted a retrospective monocentric cross-sectional study including all migrant patients recently arrived and hospitalised over a one-year period. Results: Among 101 patients, seventy-nine percent originated from Sub-Saharan Africa. The overall AMR carriage rate was 20.7% [95% CI: 12.4; 28.9%]. We isolated 5/92 methicillin-resistant Staphylococcus aureus strains (5.4%) and 15/92 extended-spectrum beta-lactamase-producing Enterobacteriaceae (16.4%). AMR carriage was associated with older age, region of origin and length of migration. Rates of HIV, HBV, and HCV infection were 39.6%, 32.7%, and 5%, reflecting sampling bias linked to reasons for hospitalization. Eleven percent had serological evidence of treponemasis and 7.8% had Chlamydia trachomatis infection. Symptoms of depression or post-traumatic stress disorder were observed for more than half the patients. Conclusion: It appears essential to offer a systematic and comprehensive post-arrival screening of AMR carriage, infectious diseases and psychological trauma to subjects who experienced migration.

Differential Performance of the FilmArray Meningitis/Encephalitis Assay To Detect Bacterial and Viral Pathogens in Both Pediatric and Adult Populations.

Meningitis/encephalitis (ME) syndromic diagnostic assays can be applied for the rapid one-step detection of the most common pathogens in cerebrospinal fluid (CSF). However, the comprehensive performance of multiplex assays is still under evaluation. In our multisite university hospital of eastern Paris, France, ME syndromic testing has been gradually implemented since 2017 for patients with neurological symptoms presenting to an adult or pediatric emergency unit. We analyzed the results from the BioFire FilmArray ME panel versus standard routine bacteriology and virology techniques, together with CSF cytology and clinical data, over a 2.5-year period to compare the diagnostic accuracy of the FilmArray ME panel to that of the reference methods. In total, 1,744 CSF samples from 1,334 pediatric and 336 adult patients were analyzed. False-positive (mostly bacterial) and false-negative (mostly viral) cases were deciphered with the help of clinical data. The performance of the FilmArray ME panel in our study was better for bacterial detection (specificity >99%, sensitivity 100%) than viral detection (specificity >99%, sensitivity 75% for herpes simplex virus 1 [HSV-1] and 89% for enterovirus), our study being one of the largest, to date, concerning enteroviruses. The use of a threshold of 10 leukocytes/mm3 considerably increased the positive agreement between the results of the FilmArray ME panel and the clinical features, especially for bacterial pathogens, for which agreement increased from 58% to 87%, avoiding two-thirds of inappropriate testing. Based on this analysis, we propose an algorithm for the use of both syndromic and specific assays for the optimal management of suspected meningitis/encephalitis in adult and pediatric patients. IMPORTANCE Based on our comparative analysis of performances of the diagnostic assays, we propose an algorithm for the use of both syndromic and specific assays, for an optimal care of the meningitis/encephalitis threat in adult and pediatric patients.

Evaluation of a loop-mediated isothermal amplification assay for diagnosis of Clostridium difficile infections.

A new assay (illumigene C. difficile; Meridian Bioscience), based on the original loop-mediated isothermal amplification (LAMP) assay, was evaluated with 472 unformed stools from patients suspected of Clostridium difficile infection. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 91.8, 99.1, 91.8, and 99.1% for the illumigene C. difficile assay and 69.4, 100, 100, and 96.6% for the cytotoxicity assay, respectively.

Cutaneous diphtheria: three case-reports to discuss determinants of re-emergence in resource-rich settings.

Diphtheria is a re-emerging disease in resource-rich settings. We here report three cases of cutaneous diphtheria diagnosed and managed in our infectious disease department and discuss the determinants of its re-emergence. Migration, travel and vaccine scepticism are key factors not only for diphtheria re-emergence, but for the future of most preventable diseases.

Human intestinal microbiota gene risk factors for antibiotic-associated diarrhea: perspectives for prevention. Risk factors for antibiotic-associated diarrhea.

Antibiotic-associated diarrhea (AAD) is associated with altered intestinal microflora and other symptoms that may lead to possibly death. In critically ill patients, diarrhea increases rates of morbimortality. Assessing diarrhea risks is thus important for clinicians. For this reason, we conducted a hypothesis-generating study focused on AAD to provide insight into methods of prevention. We evaluated the hypothesis of predisposing factors within the resident intestinal microbiota in a cohort of outpatients receiving antibiotherapy. Among the pool of tested variables, only those related to bacterial 16S rRNA genes were found to be relevant. Complex statistical analyses provided further information: amid the bacteria 16S rRNA genes, eight were determined to be essential for diarrhea predisposition and characterized from the most important to the least. Using these markers, AAD risk could be estimated with an error of 2%. This molecular analysis offers new perspectives for clinical applications at the level of prevention.

Fecal microbiota transplantation to maintain remission in Crohn's disease: a pilot randomized controlled study.

BACKGROUND: The role of the gut microbiota in Crohn's disease (CD) is established and fecal microbiota transplantation (FMT) is an attractive therapeutic strategy. No randomized controlled clinical trial results are available. We performed a randomized, single-blind, sham-controlled pilot trial of FMT in adults with colonic or ileo-colonic CD. METHOD: Patients enrolled while in flare received oral corticosteroid. Once in clinical remission, patients were randomized to receive either FMT or sham transplantation during a colonoscopy. Corticosteroids were tapered and a second colonoscopy was performed at week 6. The primary endpoint was the implantation of the donor microbiota at week 6 (Sorensen index > 0.6). RESULTS: Eight patients received FMT and nine sham transplantation. None of the patients reached the primary endpoint. The steroid-free clinical remission rate at 10 and 24 weeks was 44.4% (4/9) and 33.3% (3/9) in the sham transplantation group and 87.5% (7/8) and 50.0% (4/8; one patient loss of follow-up while in remission at week 12 and considered in flare at week 24) in the FMT group. Crohn's Disease Endoscopic Index of Severity decreased 6 weeks after FMT (p = 0.03) but not after sham transplantation (p = 0.8). Conversely, the CRP level increased 6 weeks after sham transplantation (p = 0.008) but not after FMT (p = 0.5). Absence of donor microbiota engraftment was associated with flare. No safety signal was identified. CONCLUSION: The primary endpoint was not reached for any patient. In this pilot study, higher colonization by donor microbiota was associated with maintenance of remission. These results must be confirmed in larger studies (NCT02097797). Video abstract.

Campylobacter bacteremia: clinical features and factors associated with fatal outcome.

BACKGROUND: Campylobacter bacteremia is uncommon. The influence of underlying conditions and of the impact of antibiotics on infection outcome are not known. METHODS: From January 2000 through December 2004, 183 episodes of Campylobacter bacteremia were identified in 23 hospitals in the Paris, France, area. The medical records were reviewed. Characteristics of bacteremia due to Campylobacter fetus and to other Campylobacter species were compared. Logistic regression analysis was performed to identify risk factors for fatal outcome within 30 days. RESULTS: Most affected patients were elderly or immunocompromised. C. fetus was the most commonly identified species (in 53% of patients). The main underlying conditions were liver disease (39%) and cancer (38%). The main clinical manifestations were diarrhea (33%) and skin infection (16%). Twenty-seven patients (15%) died within 30 days. Compared with patients with bacteremia due to other Campylobacter species, patients with C. fetus bacteremia were older (mean age, 69.5 years vs. 55.6 years; P = .001) and were more likely to have cellulitis (19% vs. 7%; P = .03), endovascular infection (13% vs. 1%; P = .007), or infection associated with a medical device (7% vs. 0%; P = .02). Independent risk factors for death were cancer (odds ratio [OR], 5.1; 95% confidence interval [CI], 1.2-20.8) and asymptomatic infection (OR, 6.7; 95% CI, 1.5-29.4) for C. fetus bacteremia, the absence of prescription of appropriate antibiotics (OR, 12.2; 95% CI, 0.9-157.5), and prescription of third-generation cephalosporins (OR, 10.2; 95% CI, 1.9-53.7) for bacteremia caused by other species. CONCLUSIONS: Campylobacter bacteremia occurs mainly in immunocompromised patients. Clinical features and risk factors of death differ by infection species.

Clinical features of Clostridium difficile-associated infections and molecular characterization of strains: results of a retrospective study, 2000-2004.

BACKGROUND: Recent outbreaks of severe cases of Clostridium difficile-associated diarrhea (CDAD) reported in North America, the United Kingdom, and The Netherlands have emphasized the importance of an ongoing epidemiological surveillance of CDAD. OBJECTIVE: To determine the epidemiology of CDAD over the years 2000-2004 and the rate of nosocomial transmission of C. difficile. DESIGN: Retrospective survey of inpatients with CDAD and molecular characterization of the strains isolated. SETTING: A 760-bed teaching hospital. METHODS: All CDAD cases diagnosed from January 1, 2000, to December 31, 2004, were reviewed. A CDAD case was defined as diarrhea in a hospitalized patient who had a stool specimen that tested positive for C. difficile cytotoxin or had a positive toxigenic culture result. CDAD was considered to be severe if a patient fulfilled at least 1 of the following 3 criteria: (1) presence of a fever (defined as temperature higher than 38.5 degrees C), abdominal pain, and leukocyte count greater than 10,000 cells/mm(3); (2) endoscopically or histologically proven pseudomembranous colitis; or (3) complications (defined as death with C. difficile infection as the primary or a contributing cause, toxic megacolon, perforation, toxic shock, and/or colectomy). CDAD was considered community-acquired if the diarrhea occurred in the patient within 72 hours after admission and if the patient had no history of hospitalization in the previous month; otherwise, CDAD was considered healthcare-associated. All the strains isolated were serogrouped and were characterized by toxinotyping and PCR ribotyping. Detection of toxin A, toxin B, and binary toxin was performed by PCR. RESULTS: One hundred fifty-one cases of CDAD were diagnosed; 147 clinical records could be reviewed, and 131 strains were studied. The overall incidence of CDAD was 1.1 cases per 1,000 patients admitted, but incidence rates were higher in 2003-2004, compared with 2000-2002 (P=.017). Diarrhea was community acquired in 28 patients (19%). For patients with healthcare-associated CDAD, transmission of the strain from patient to patient (ie, infection with a strain of the same serogroup and PCR ribotype as the strain isolated from another patient hospitalized in the same ward or in a linked ward in the previous 2 months) was demonstrated in 12 cases (10.1%). Eleven percent of strains were positive for binary toxin. Binary toxin-positive strains were associated with more-severe diarrhea (P=.01) and with a higher case-fatality rate (P=.03). A specific clone of C. difficile (serogroup H, PCR ribotype sa026) accounted for 35 (26.7%) of all the strains isolated, but this clone was found both in healthcare-associated and community-acquired cases. Three strains belonged to toxinotype III, but only 1 was related to the hypervirulent clone involved in recent outbreaks. CONCLUSION: The incidence of CDAD is low in our hospital, and cross-infection is limited. These results also suggest that strains with binary toxin might be more virulent.

Clostridium difficile infection in acute flares of inflammatory bowel disease: A prospective study.

OBJECTIVES: Clostridium difficile infection (CDI) is a common complication in inflammatory bowel disease (IBD) and has been associated with poor IBD outcome. The aims of our study were to look for predictive factors of CDI in patients hospitalized for IBD flare and to evaluate a rapid testing strategy in this population. METHODS: Consecutive patients hospitalized for IBD flare in Saint-Antoine Hospital (Paris, France) were prospectively tested for CDI with a defined strategy involving rapid testing and reference methods. Risk factors for CDI were investigated and performances of diagnostic tests were evaluated. RESULTS: C. difficile testing was performed at admission in 461 hospitalizations for IBD flare. CDI was diagnosed in 35 cases (7.6%) and non-toxigenic C. difficile was identified in 10 cases (2.2%). In multivariate analysis, UC phenotype was associated with CDI (OR 2.2, 95% CI 1.03-4.6, p=0.047). Glutamate dehydrogenase (GDH) test had a 97.1% sensitivity and a 100% negative predictive value for CDI diagnosis but a positive predictive value of 79.1%. Enzyme immunoassay (EIA)-based toxin detection (C. Diff Quik Chek complete®, Alere) had a poor sensitivity and diagnosis was rescued by toxin PCR in 100% of cases. CONCLUSION: CDI is frequent in patients hospitalized for IBD flare. Clinical parameters do not help for the diagnosis and rapid testing should be performed in all patients. Currently, a negative result of an EIA-based toxin search associated with a positive GDH test cannot rule out a CDI and should not delay initiation of specific treatment in case of severe symptoms or high presumption.

Clinical features of Clostridium difficile-associated diarrhoea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains.

Toxins A and B are known to be the primary virulence factors of Clostridium difficile. Other potential virulence factors have been identified such as binary toxin (actin-specific ADP-ribosyltransferase toxin, or CDT). A retrospective case-control study was performed in order to identify clinical features and risk factors of C. difficile-associated diarrhoea due to binary toxin-producing strains. Each case (a patient with diarrhoea due to binary toxin-producing strain) was compared with two controls (patients with diarrhoea due to a C. difficile strain that did not produce binary toxin) matched for ward and date of hospitalization. cdtA and cdtB genes were screened by PCR. Production of CDT was studied by Western blotting using an antiserum against Ia and Ib from the Clostridium perfringens iota toxin, and the activity of the binary toxin was assessed using an ADP-ribosyltransferase assay. Twenty-six cases (14 males and 12 females) were identified in 1999 and 2000. Cases and controls did not differ significantly for sex, age, previous administration of antibiotics or frequency of endoscopic examination. Diarrhoea was community-acquired more often in cases than in controls (65.4 vs 35.7 %, P = 0.017) and more often represented the cause of hospitalization (61.5 vs 26.2 %, P = 0.003). Moreover, diarrhoea in cases was more frequently associated with abdominal pain (63.6 vs 39.4 %, P = 0.07) and with liquid stools (76.9 vs 59.5 %, P = 0.14) than in controls. These results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea.