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.     

Epidemic Clostridium difficile Ribotype 027 in Chile

TO THE EDITOR: The increased severity of Clostridium difficile infection is primarily attributed to the appearance of an epidemic strain characterized as PCR ribotype 027 (1). The only report that identified epidemic C. difficile ribotype 027 in an American country outside of North America comes from Costa Rica, raising the possibility that strains 027 might also be present in other countries of Latin America (2). Several studies between 2001 and 2009 have been conducted in South American countries to detect the incidence of C. difficile infection in hospitalized patients, but they did not identify which C. difficile strains were causing these infections (3).     

Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB₍₀₂₇₎). TcdB₍₀₂₇₎ is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB₍₀₀₃₎). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB₍₀₀₃₎, TcdB₍₀₂₇₎ and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB₍₀₀₃₎/TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB₍₀₀₃₎/TcdA/TcdB₍₀₂₇₎) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI).     

Clostridium difficile ribotype 027, toxinotype III, the Netherlands

Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A-negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control.     

中国部分地区艰难梭菌PCR 核糖体分型及毒素基因多态性研究

目的了解中国部分地区艰难梭菌聚合酶链反应（PCR） 核糖体型别分布及其A、B毒素基因多态性,为建立适宜中国的艰难梭菌分子检测和分子分型技术提供基础数据,同时在基因水平上为艰难梭菌感染导致的复杂临床表现提供依据。方法对中国3个城市（北京、广州、济南）分离的64株艰难梭菌临床株进行PCR 核糖体分型,并对不同型别的26株代表菌株的A、B毒素基因进行扩增测序。结果64株艰难梭菌中,毒素基因型以A+B+型（45株,70.31%）为主,A-B+型19株（29.69%）。共存在9种PCR 核糖体型别,以017型（21株,32.81%）为主要型别,其次为001型（13株,20.31%）、012型（11株,17.19%）。A-B+菌株中,14株(73.68%)是017型,1株是001型。A、B毒素基因呈现一定的多态性,其中有7种A毒素序列型别（TSTA）,6种B毒素序列型别(TSTB),8种A、B毒素序列型别组合（TSTG）。结论我国部分地区的艰难梭菌可能以PCR 核糖体017型为主,A、B毒素基因在菌株间存在多态性,且核糖体型别与毒素基因多态性间存在相对应的关联。应进一步扩大菌株数量和范围,探寻适合我国的分子检测和分子分型方法,从而帮助医院更好地预防和控制艰难梭菌感染。     

Life-threatening infections with a new strain of Clostridium difficile

Three men, aged 39, 73, and 66 years, respectively, developed an infection with a new strain ofClostridium difficile, ribotype 027.C.difficile-associated diarrhoea (CDAD) occurred in two patients after multiple abdominal surgery and in the third patient one week after autologous haematopoietic cell transplantation. Within a few days, despite antibiotic therapy, all three patients developed severe (pseudomembranous) colitis with sepsis for which admission to the Intensive Care Unit was required. Two patients underwent (sub)total colectomy and received an intensive course of oral and/or rectal vancomycin. In all patients who develop diarrhoea in hospital, especially during or after treatment with antibiotics or chemotherapeutic agents, an infection with C. difficile ribotype 027 should be suspected. Recent outbreaks of this hypervirulent strain of C. difficile have been reported in Canada, the United States, United Kingdom, and The Netherlands. Demonstration of C. difficile toxin in faeces confirms the clinical suspicion of CDAD and ribotyping of the strain may reveal whether the 027 strain is present. For treatment of these 027 infections, vancomycin is preferred to metronidazole. After a severe course of colitis or in case of recurrence a 'tapering and pulse' course ofvancomycin can be prescribed; alternatively, treatment with bovine antibody-enriched whey may be considered. The introduction of this hypervirulent strain has led to reinforcement of the hygienic measures in accordance with the recommendations of the Dutch Working Party on Infection Prevention and a policy to deter the use of fluoroquinolones.     

Epidemiology of Clostridium difficile PCR ribotype 027 in the Netherlands 2005-present and the emergence of other subtypes

Outbreaks of Clostridium difficile associated diarrhoea (CDAD) involving the virulent PCRribotype 027, toxinotype III were first reported in the Netherlands in 2005. This ribotype has now been detected in 26 of the 97 hospitals in the Netherlands. In 13 of the hospitals, the introduction of ribotype 027 was linked to increased CDAD incidence; this was found in 2 hospitals since December 2006. Ribotype 027 has also been detected in to nursing homes. In 2007, no evidence of ribotype 27 was found in 6 of the 12 hospitals in which ribotype 027 was confirmed in 2005-2006 and an outbreak of CDAD had occurred. The incidence of CDAD increased again in 2 hospitals that had previously had the epidemic well under control. Meanwhile, other PCR ribotypes appear to be gaining ground in the Netherlands, some of which have the same virulent characteristics as ribotype 027. Notably, ribotype 078, which appears to be associated with livestock, is becoming increasingly common.     

Morbidity and mortality associated with Clostridium difficile ribotype 078: a case-case study

The morbidity and mortality associated with Clostridium difficile ribotype 078 were examined by comparison with other known outbreak strains. A healthcare interaction within eight weeks of a positive specimen significantly increased the likelihood of ribotype 078 compared with ribotype 027. Individuals with ribotype 078 also tended to come from community sources, have a hospital stay post specimen similar to ribotype 027 and a lower 30-day mortality, but these differences were not statistically significant. This study generates several hypotheses and a methodological platform to explore this unique profile.     

Difference in the incidence of Clostridium difficile among patients infected with human immunodeficiency virus admitted to a public hospital and a private hospital.

OBJECTIVE: To compare the occurrence of Clostridium difficile among inpatients infected with human immunodeficiency virus (HIV) in two different hospitals. DESIGN: Prospective, observational study. SETTING: Specialized HIV inpatient units. PATlENTS: HIV-infected inpatients at Cook County Hospital (CCH) and Rush Presbyterian St. Luke's Medical Center (RPSLMC). INTERVENTIONS: A clinical and epidemiologic assessment of patient risk factors for C. difficile was performed. C. difficile isolates found on stool, rectal, and environmental cultures were typed by pulsed-field gel electrophoresis. RESULTS: Twenty-seven percent of patients admitted to CCH versus 4% of patients admitted to RPSLMC had positive cultures for C. difficile (P = .001). At CCH, 14.7% of environmental cultures were positive versus 2.9% at RPSLMC (P = .002). Risk factors for C. difficile acquisition included hospitalization at CCH, more severe HIV, use of acyclovir and H2-blockers, and longer hospital stay. Patients admitted to CCH were taking more antibiotics, had longer hospital stays, and more frequently had a history of C. difficile infection. During the study, two strains (CD1A and CD4) extensively contaminated the CCH environment. However, only CD1A caused an outbreak. CONCLUSIONS: The C. difficile acquisition rate at CCH was sevenfold higher than that at RPSLMC, and CCH had a more contaminated environment. Differences in patient acquisition rates likely reflect a greater prevalence of traditional C. difficile risk factors and a concurrent outbreak at CCH. Although two strains heavily contaminated the environment at CCH, only one caused an outbreak, suggesting that factors other than the environment are important in initiating C. difficile outbreaks.     

Treatment of recurrent Clostridium difficile-associated diarrhoea with a suspension of donor faeces

OBJECTIVE: To study the effect of treating recurrent Clostridium difficile-associated diarrhoea (CDAD) with a suspension of donor faeces. DESIGN: Uncontrolled interventional study. METHOD: Patients that, despite adequate antibiotic therapy, had developed at least 2 recurrences ofCDAD, including at least one recurrence that had been treated with a vancomycin tapering regimen, were included in the study. Relatives or volunteers served as faeces donor. All donors were previously examined for the presence of HIV, hepatitis B- and C-virus, and acute infection with cytomegalovirus or Epstein-Barr virus. The donor faeces were examined for the presence of C. difficile, Yersinia, Campylobacter, Shigella, Salmonella, and parasites. Before the infusion of donor faeces, the patients were treated for 4 days with vancomycin 500 mg q.i.d., followed by colon lavage. The suspension of 150 g of donor faeces dissolved in 300-400 ml of NaCl was infused into the jejunum via a duodenal catheter or into the caecum via colonoscopy. RESULTS: 7 CDAD patients were included and treated, including 2 with the hypervirulent C. difficile-strain PCR ribotype 027, toxinotype III. In 5 patients, the defaecation frequency returned to normal almost immediately after treatment and the cultures and toxin tests for C. difficile were repeatedly negative. In the remaining 2 patients, the treatment was successful after a repeated infusion of faeces from a different donor. CONCLUSION: Treatment with donor faeces seems promising for patients who develop repeated recurrences despite adequate therapy and could be valuable in the future during (local) epidemics of the PCR ribotype 027 strain. A randomised nationwide study (FECAL trial) has been started in order to determine the efficacy of this treatment.     

Epidemiological survey of Clostridium difficile ribotypes in the North East of England during an 18-month period

During an 18-month period, 1606 stool specimens from laboratory-confirmed cases of Clostridium difficile infection in the North East of England were ribotyped using unrestricted polymerase chain reaction. Of these, 87.6% grew C. difficile on culture; 70% had one of 10 recognizable ribotypes of which 001, 106 and 027 were the most prevalent. The proportions of ribotypes 002 and 015 declined during the study period, whereas ribotypes 016 and 023 increased. Ribotype 005 was significantly more numerous in males and ribotype 027 was associated with significantly higher mean age. Our findings differ from national data derived from more selective testing.     

Emergence and control of fluoroquinolone-resistant, toxin A-negative, toxin B-positive Clostridium difficile.

BACKGROUND: Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Between August 2003 and January 2004, we experienced an increase in the incidence of C. difficile-associated disease. We describe the investigation into and management of the outbreak in this article. METHODS: A total of 73 consecutive patients with nosocomial C. difficile-associated diarrhea were identified. C. difficile isolates were characterized using toxin-specific enzyme immunoassays, a tissue-culture fibroblast cytotoxicity assay, polymerase chain reaction (PCR), and antimicrobial susceptibility tests. Rates of recurrence and of C. difficile colitis were recorded. Changes in antibiotic use and infection control policies were documented. RESULTS: The incidence of C. difficile-associated diarrhea peaked at 21 cases per 1,000 patient admissions. Of the C. difficile isolates recovered, 85 (95%) were identical toxin A-negative and toxin B-positive strains, corresponding to toxinotype VIII and PCR ribotype 017. All clonal isolates were resistant to multiple antibiotics, including ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin (minimum inhibitory concentrations [MICs] of greater than 32 micro g/mL) and erythromycin, clarithromycin, and clindamycin (MICs of greater than 256 micro g/mL). Recurrent C. difficile-associated disease occurred in 26 (36%) of the patients. At least 10 (14%) of the patients developed C. difficile colitis. Additional infection control measures introduced included the use of ward memos, a hand-hygiene awareness campaign, increased environmental cleaning, attention to prescribing practices for antibiotics, increased awareness of diarrheal illness, and early isolation of affected patients. Total use of fluoroquinolones did not change throughout the study period. Despite persistence of this toxin-variant strain, the incidence of C. difficile-associated disease in our institution decreased to fewer than 5 cases per 1,000 admissions. CONCLUSIONS: We report on the emergence of a fluoroquinolone- and clindamycin-resistant, toxin A-negative, and toxin B-positive strain of C. difficile associated with an outbreak of C. difficile-associated disease in our institution during a 6-month period. We found that careful attention to improvement of infection control interventions was the most important means of controlling this nosocomial pathogen.     

Laboratory-based surveillance for vancomycin-resistant enterococci: utility of screening stool specimens submitted for Clostridium difficile toxin assay.

OBJECTIVE: To study vancomycin-resistant enterococci (VRE) gastrointestinal colonization prevalence in high-risk hospitalized patients and to assess the cost and utility of this laboratory-based surveillance. SETTING: Large university teaching hospital. DESIGN: Quarterly prevalence culture survey of 50 stool specimens submitted for Clostridium difficile toxin A assay from October 1996 through June 1999 (n=526). Screening culture survey of all C difficile-positive stool specimens from July 1998 through June 1999 (n=140). PATIENTS: Specimens for analysis were collected from patients who were admitted to the hospital and who had C difficile toxin A testing ordered. Patient samples were excluded from analysis if they were obtained from patients not hospitalized at UCLA Medical Center, if the C difficile toxin assay result was indeterminate, or if the patient was known to have previous VRE colonization or infection. RESULTS: During quarterly surveillance, VRE was detected in 19.8%, C difficile toxin A in 9.5%, and both VRE and C difficile toxin A in 3.2% of stool specimens submitted for C difficile toxin assay. Patients whose stool specimens were positive for C difficile toxin A were significantly more likely than those whose specimens were negative to have VRE detected (odds ratio, 2.3; 95% confidence interval, 1.2-4.5). Based on these findings, in July 1998, we began routine screening of all C difficile-positive stool specimens for VRE. From July 1998 through June 1999, 58 (41.4%) of 140 patients with C difficile-positive specimens had VRE newly detected in the stool. The combined cost of the two laboratory-based surveillance strategies was approximately $62 per VRE-positive patient identified and $5,800 per year. CONCLUSION: Quarterly surveillance of stool submitted for C difficile assay combined with screening all C difficile-positive stools is a cost-effective and efficient strategy for detecting VRE stool colonization among high-risk hospitalized patients. Such a laboratory-based surveillance should be included as part of a comprehensive program to limit nosocomial VRE transmission.     

Enteric carriage of vancomycin-resistant Enterococcus faecium in patients tested for Clostridium difficile.

OBJECTIVE: To identify independent risk factors for enteric carriage of vancomycin-resistant Enterococcus faecium (VREF) in hospitalized patients tested for Clostridium difficile toxin. DESIGN: Retrospective case-cohort study. SETTING: Tertiary-care teaching hospital. PATIENTS: Convenience sample of 215 adult inpatients who had stool tested for C difficile between January 29 and February 25, 1996. RESULTS: 41 (19%) of 215 patients had enteric carriage of VREE Five independent risk factors for enteric VREF were identified: history of prior C difficile (odds ratio [OR], 15.21; 95% confidence interval [CI95], 3.30-70.10; P < .001), parenteral treatment with vancomycin for > or = 5 days (OR, 4.06; CI95, 1.54-10.73; P = .005), treatment with antimicrobials effective against gram-negative organisms (OR, 3.44; CI95, 1.20-9.87; P = .021), admission from another institution (OR, 2.95; CI95, 1.21-7.18; P =.017), and age > 60 years (OR 2.57; CI95, 1.13-5.82; P = .024). These risk factors for enteric VREF were independent of the patient's current C difficile status. CONCLUSIONS: Antimicrobial exposures are the most important modifiable independent risk factors for enteric carriage of VREF in hospitalized patients tested for C difficile.     

住院腹泻患者艰难梭菌检测与分析

目的通过对住院腹泻患者粪便标本中的艰难梭菌进行筛查和不同时期检出率的比较,了解某院腹泻患者艰难梭菌的感染情况。方法收集该院2009年2—12月和2011年4—7月住院腹泻患者粪便标本106份,进行厌氧培养和API鉴定,对培养鉴定获得的菌株应用聚合酶链反应（PCR）扩增法进行A、B毒素及二元毒素基因检测;酶联荧光免疫法检测毒素A/B。结果 106份标本中,厌氧培养艰难梭菌阳性16株（15.09%）。16株菌经PCR扩增,A、B毒素均阳性,二元毒素均阴性。直接毒素A/B检测阳性率为12.26%（13/106）,与厌氧培养阳性率比较,差异无统计学意义（χ^20.16,P〉0.05）。2009年2—12月和2011年4—7月两个时期的标本厌氧培养艰难梭菌阳性率分别为22.81%（13/57）、6.12%（3/49）,两者比较,差异有统计学意义（χ^25.73,P〈0.05）;毒素A/B检出率分别为17.54%（10/57）、6.12%（3/49）,差异无统计学意义（χ^23.18,P〉0.05）。艰难梭菌检测阳性患者住院期间均使用过头孢类、喹诺酮类、碳青霉烯类、广谱青霉素、克林霉素等其中一种或多种抗菌药物。结论该院艰难梭菌相关性腹泻比较严重,抗菌药物的使用是诱使艰难梭菌感染的重要因素。     

Seasonality of Clostridium difficile infections in Southern Germany

Between 2000 and 2009, the total number of patients with Clostridium difficile infections increased considerably in Southeastern Germany. A clear seasonality was observed with a higher number of affected patients occurring in the winter months (January-March). Moxifloxacin and erythromycin-resistant C. difficile PCR ribotypes 001 (72%) and 027 (4·6%) were the most commonly isolated strains.     

High case-fatality rates of meningococcal disease in Western Norway caused by serogroup C strains belonging to both sequence type (ST)-32 and ST-11 complexes, 1985-2002

A total of 293 meningococcal disease (McD) patients from Western Norway hospitalized during 1985-2002 were examined for risk factors related to death. The case-fatality rate (CFR) increased from 4% during 1985-1993 to 17% during 1994-2002. We analysed the phenotypic and genotypic characteristics of the meningococcal patient isolates, with the aim of identifying whether highly virulent meningococcal strains contributed to the increased CFR. The Norwegian epidemic strain B:15:P1.7,16/ST-32 complex was overall the most common phenotype/genotype (n=75) and caused most deaths (n=9; CFR 12.0%). However, fatality was significantly associated with disease caused by serogroup C meningococcal strains; C:15:P1.7,16/ST-32 and C:2a/ST-11 complex strains, which had the highest CFRs of 21.1% and 18.2% respectively. Serogroup B strains of the ST-32 complex differing by serotype and/or serosubtype from the epidemic strain had a CFR of 5.1%, while the CFR of disease caused by other strains (all phenotypes and genotypes pooled) was 2.2%. The distribution of phenotypes/clonal complexes varied significantly between 1985-1993 and 1994-2002 (P<0.001); B:15/ST-32 complex strains decreased whereas both C:15:P1.7,16/ST-32 complex strains and strains with other phenotypes/clonal complexes increased. Our results indicate that C:15:P1.7,16/ST-32 and C:2a/ST-11 complex strains were highly virulent strains and contributed to the high CFR of McD in patients from Western Norway. To reduce fatality, rapid identification of such virulent strains is necessary. In addition, early and specific measures should include public information, vaccination of populations at risk of disease and carriage eradication, when clustering of patients occurs.     

Toxinotype V Clostridium difficile in humans and food animals

Clostridium difficile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. difficile-associated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identified as toxinotype V; before 2001, 7 (<0.02%) of approximately 6,000 isolates were identified as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39-bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. difficile, including the potential of foodborne transmission to humans.     

Clostridium difficile in Ready-to-Eat Salads, Scotland

Of 40 ready-to-eat salads, 3 (7.5%) were positive for Clostridium difficile by PCR. Two isolates were PCR ribotype 017 (toxin A–, B+), and 1 was PCR ribotype 001. Isolates were susceptible to vancomycin and metronidazole but variably resistant to other antimicrobial drugs. Ready-to-eat salads may be potential sources for virulent C. difficile.     

"Second-look" cytotoxicity: an evaluation of culture plus cytotoxin assay of Clostridium difficile isolates in the laboratory diagnosis of CDAD.

Clostridium difficile is one of the most frequent causes of hospital-acquired diarrhoea. Our objective was to prove that some stool samples with a direct negative cytotoxicity assay may indeed harbour toxigenic C. difficile and that this can be demonstrated by performing a "second-look" cytotoxicity assay using the isolated C. difficile strains. Over an eight-year period (1992-1999), the 8241 stool samples submitted for direct cell culture from patients with suspected C. difficile-associated diarrhoea (CDAD) were simultaneously plated on cycloserine cefoxitin fructose agar. C. difficile strains isolated from samples with a negative direct cell culture assay were re-tested for toxin production "second-look" cell culture assay). Using both methods 6423 samples (78%) were negative. Of the remaining 1818 samples, 127 (7%) yielded C. difficile isolates which were confirmed as non-producers of toxin by both methods, 1437 (85%) were positive in direct cell culture assay, and 254 were positive only after the "second-look" cell culture assay. Thus, our approach allowed us to detect an extra 15% of toxin-producing strains that could have gone undetected otherwise.The combination of direct-cell culture assay, culture for toxigenic C. difficile and "second-look" cell culture assay enhances the potential for diagnosis of CDAD and enables us to be more efficient with our patient care resources.