B-positive Strains Research Articles

Epidemiology of Clostridium (Clostridioides) difficile Infection in Southeast Asia.

This review describes the current understanding of Clostridium (Clostridioides) difficile infection (CDI) in southeast Asia regarding the prevalence of CDI, C. difficile detection methods, antimicrobial susceptibility profiles, and the potential significance of a One Health approach to prevention and control. Our initial focus had been the Indochina region, however, due to limited studies/surveillance of CDI in Indochina, other studies in southeast Asian countries and neighboring Chinese provinces are presented here for comparison. Clostridium (Clostridioides) difficile infection is one of the most common causes of hospital-acquired gastroenteritis worldwide. Since its discovery as a cause of pseudomembranous colitis in 1978, C. difficile-related disease has been more prevalent in high-income rather than low-income countries. This may be because of a lack of knowledge and awareness about the significance of C. difficile and CDI, resulting in underreporting of true rates. Moreover, the abuse of antimicrobials and paucity of education regarding appropriate usage remain important driving factors in the evolution of CDI worldwide. The combination of underreporting of true CDI rates, along with continued misuse of antimicrobial agents, poses an alarming threat for regions like Indochina. C. difficile ribotype (RT) 027 has caused outbreaks in North America and European countries, however, C. difficile RT 017 commonly occurs in Asia. Toxin A-negative/toxin B-positive (A-B+) strains of RT 017 have circulated widely and caused outbreaks throughout the world and, in southeast Asia, this strain is endemic.

Rare toxin A-negative and toxin B-positive strain of Clostridioides difficile from Japan lacking a complete tcdA gene

IntroductionClostridioides difficile (C. difficile) produces three kinds of toxins: toxin A (enterotoxin), toxin B (cytotoxin), and C. difficile transferase (CDT), a binary toxin. Some strains show positivity only for toxin B. These strains reportedly possess a gene for toxin A, tcdA. However, toxin A production is inhibited due to a mutated stop codon and/or deletion within the tcdA gene. Here for the first case in Japan, we describe toxin genomes and proteins of a strain possessing only toxin B and lacking a complete tcdA gene, along with clinical manifestations. MethodsC. difficile was isolated from the bloody stool of a 60-year-old female patient treated with meropenem. Although a rapid detection kit of toxins (C. DIFF QUIK CHEK COMPLETE®, TechLab, Blacksburg, VA, USA) showed positivity, Western blotting detected no toxins. Therefore, we explored the strain's toxin genes and their sequences to determine whether the strain possessed a toxin. ResultsPolymerase chain reaction did not identify toxin genes. Whole-genome sequencing analysis showed that a gene for toxin A, tcdA, was completely deleted in the strain. Moreover, 701 mutations and some deletions/insertions were identified on the tcdB gene. ConclusionsWe isolated a rare strain of C. difficile producing only toxin B and lacking a complete tcdA gene herein Japan. The possibility of a false negative needs to be considered with a genetic method for a diagnose of C. difficile infection.

Applicability of commercial multiplex kit for the diagnosis of: a multicenter investigation Clostridium difficile infections

Objective To evaluate the performance of Xpert C. difficile multiplex real-time PCR assay for diagnosis of Clostridium difficile infections in Chinese hospital settings. Methods This study was performed in Huashan Hospital, Ruijin Hospital, Beijing Hospital, Nanfang Hospital and Sir Run Run Shaw Hospital using a standard study protocol. Unique unformed stools from patients with acute hospital-acquired diarrhea were simultaneously analyzed by toxigenic anaerobic cultures and the Xpert C. difficile assay. All specimens displaying discordant results between the Xpert assay and toxigenic culture were sent for Sanger tcdB gene sequencing. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), total concordance rate, and 95% confidence interval (CI) were calculated before and after resolution of discordant results using SAS 9.3. Results A total of 745 stool specimens were collected and 46 were excluded due to failure of C. difficile recovery. The remaining 699 specimens were included. Compared to the results of toxigenic culture, the sensitivity, specificity, PPV, and NPV of Xpert C. difficile assay were 94.1% (144/153)(95%CI:89.1%-97.3%), 93.2% (509/546)(95%CI:96.7%-99.2%), 79.6% (144/181)(95%CI: 72.9%-85.2%)and 98.3% (509/518)(95%CI: 96.7%-99.2%), respectively. Both methods had a Kappa of 0.819. Xpert C. difficile assay showed sensitivity of 98.4%(62/63) (95% CI: 90.3%-99.9%) and specificity of 93.2%(509/546) (95% CI: 90.8%-95.2%) for toxin A-negative toxin B-positive strains. After the discordant results resolved by tcdB gene sequencing, PCR assay provided better performance with high sensitivity, specificity, positive predictive value, and negative predictive value [98.8% (171/173), 98.1% (516/526), 94.5% (171/181) and 99.6% (516/518), respectively]. Conclusions Compared to the results of toxigenic culture, the sensitivity, specificity and NPV of Xpert C. difficile assay were 94.1% (144/153) and 93.2% (509/546), respectively. With the results available within 1 h, Xpert C. difficile assay provides prompt and precise laboratory diagnosis in Chinese clinical settings. Key words: Clostridium difficile; Clostridium infections; Real-time polymerase chain reaction

Noncontiguous Finished Genome Sequence of Staphylococcus aureus KLT6, a Staphylococcal Enterotoxin B-Positive Strain Involved in a Food Poisoning Outbreak in Switzerland

We present the first complete genome sequence of a Staphylococcus aureus strain assigned to clonal complex 12. The strain was isolated in a food poisoning outbreak due to contaminated potato salad in Switzerland in 2009, and it produces staphylococcal enterotoxin B.

Epidemiology of Clostridium difficile infection in Asia

While Clostridium difficile infection (CDI) has come to prominence as major epidemics have occurred in North America and Europe over the recent decade, awareness and surveillance of CDI in Asia have remained poor. Limited studies performed throughout Asia indicate that CDI is also a significant nosocomial pathogen in this region, but the true prevalence of CDI remains unknown. A lack of regulated antibiotic use in many Asian countries suggests that the prevalence of CDI may be comparatively high. Molecular studies indicate that ribotypes 027 and 078, which have caused significant outbreaks in other regions of the world, are rare in Asia. However, variant toxin A-negative/toxin B-positive strains of ribotype 017 have caused epidemics across several Asian countries. Ribotype smz/018 has caused widespread disease across Japan over the last decade and more recently emerged in Korea. This review summarises current knowledge on CDI in Asian countries.

Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼ 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.

New types of toxin A-negative, toxin B-positive strains among clinical isolates of Clostridium difficile in Australia

A total of 817 human clinical isolates of Clostridium difficile from all Australian states were screened for A(-)B(+) strains by toxin gene PCR assays. Nine (1.1 %) strains were confirmed to be A(-)B(+) by enzyme immunoassay for toxin production. Of these, six (66.7 %) were binary toxin-positive by PCR. Using PCR ribotyping and toxinotyping, the A(-)B(+) strains could be grouped into seven ribotypes and three toxinotypes. Only one of the ribotypes had been reported previously (017). The prevalence of ribotype 017 was low in this study with only two strains detected. Two new A(-)B(+) toxinotypes were also defined (XXX, XXXI). Toxinotype XXX had a toxin B gene similar to that of toxinotype IV (A(+)B(+)) but with a novel cytopathic region. Toxinotype XXXI was similar to other A(-)B(+) types (X, XVII), but had a larger deletion to the toxin A gene than in either of those types. The types of A(-)B(+) strains identified in this study differed markedly from those described in other regions.

Epidemiology and Clinical Characteristics ofClostridium difficileInfection in a Korean Tertiary Hospital

In order to investigate the incidence, clinical and microbiologic characteristics of Clostridium difficile infection (CDI) in Korea, a prospective observational study was performed. From September 2008 through January 2010, all patients whose stool was tested for toxin assay A&B and/or C. difficile culture were studied for clinical characteristics. Toxin types of the isolates from stool were tested. The mean incidence of CDI per 100,000 patient-days was 71.6 by month (range, 52.5-114.0), and the ratio of CDI to antibiotic-associated diarrhea was 0.23. Among 200 CDI patients, 37.5% (75/200) was severe CDI based on severity score. Clinical outcome of 189 CDI was as followed; 25.9% (49/189) improved without treatment, 84.3% (118/140) achieved clinical cure and attributed mortality was 0.7% (1/140) with the treatment. Recurrence rate was 21.4% (30/140) and cure without recurrence was 66.4% (93/140). The most common type of toxin was toxin A-positive/toxin B-positive strain (77.5%), toxin A-negative/toxin B-positive strains or binary toxin-producing strains comprised 15.4% or 7.1%, respectively. In conclusion, the incidence of CDI in Korea is a little higher than other reports during the non-epidemic setting. We expect that the change of epidemiology and clinical severity in CDI can be evaluated based on these results.

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5'-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.

Emergence and Control of Fluoroquinolone-Resistant, Toxin A–Negative, Toxin B–PositiveClostridium difficile

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. 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. 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. 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.

Isolation and characterisation of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin B-positive (A- B+) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A- B+ strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified daily by review of ELISA results and were cultured on selective media. Following culture, toxin-specific immunoassays, IMR-90 cytotoxicity assays and PCR were used to analyse consecutive C. difficile isolates from 93 patients. Using a toxin A-specific ELISA, 52 samples produced detectable toxin. All isolates were positive using a toxin A/B ELISA. Similarly, all isolates were positive with the cytoxicity assay, although variant cytopathic effects were observed in 41 cases. PCR amplification of the toxin A and toxin B genes revealed that 41 of the previous A- B+ strains had a c. 1.7-kb deletion in the 3'-end of the tcdA gene. Restriction enzyme analysis of these amplicons revealed the loss of polymorphic restriction sites. These 41 A- B+ isolates were designated toxinotype VIII by comparison with C. difficile strain 1470. PCR ribotyping revealed that all A- B+ isolates belonged to PCR-ribotype 017. A- B+ C. difficile isolates accounted for 44% of the isolates examined in this study, and appeared to be isolated more frequently in Dublin, Ireland, than reported rates for other countries.

Clostridium difficile toxoid vaccine in recurrent C. difficile-associated diarrhea

Recurrent C difficile -associated diarrhea (CDAD) is associated with a lack of protective immunity to C difficile toxins. A parenteral C difficile vaccine containing toxoid A and toxoid B was reported previously to be safe and immunogenic in healthy volunteers. Our aim was to examine whether the vaccine is also well tolerated and immunogenic in patients with recurrent CDAD. Subjects received 4, 50-microg intramuscular inoculations of the C difficile vaccine over an 8-week period. Serum antitoxin antibodies were measured by ELISA, and toxin neutralizing activity was evaluated using the tissue culture cytotoxin assay. Three patients with multiple episodes of recurrent CDAD were vaccinated. Two of the 3 showed an increase in serum IgG antitoxin A antibodies (3-fold and 4-fold increases, respectively) and in serum IgG antitoxin B antibodies (52-fold and 20-fold, respectively). Both also developed cytotoxin neutralizing activity against toxin A and toxin B. Prior to vaccination, the subjects had required nearly continuous treatment with oral vancomycin for 7, 9, and 22 months, respectively, to treat recurrent episodes of CDAD. After vaccination, all 3 subjects discontinued treatment with oral vancomycin without any further recurrence. A C difficile toxoid vaccine induced immune responses to toxins A and B in patients with CDAD and was associated with resolution of recurrent diarrhea. The results of this study support the feasibility of active vaccination against C difficile and its toxins in high-risk individuals but must be validated in larger, randomized, controlled trials.

Multiplex PCR Targetingtpi(Triose Phosphate Isomerase),tcdA(Toxin A), andtcdB(Toxin B) Genes for Toxigenic Culture ofClostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A-B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A-B-, and A-B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A-B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A-B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A(-)/B(+)) strains. Thirty-nine clinical toxin A(-)/B(+) isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A(-)/B(+) isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A(-)/B(+) isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A(-)/B(+) isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A(-)/B(+) strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.

A nosocomial outbreak of diarrhea caused by toxin A-negative, toxin B-positive Clostridium difficile in a cancer center hospital

Between February and July 2001, 15 patients were diagnosed as Clostridium difficile-associated diarrhea in a ward of hematological neoplasm and lung cancer in a cancer center hospital. Of these 15 patients, 10 had malignant lymphoma, and 12 and 11 had exposure to antimicrobial agents and cancer chemotherapy, respectively, before the onset of diarrhea. Toxin A-positive, toxin B-positive (A+ B+) C. difficile was recovered from five patients and the remaining 10 patients suffered from diarrhea caused by toxin A-negative, toxin B-positive (A- B+) strains. All of the 10A- B+ isolates represented an identical banding pattern by PCR ribotyping and classified into one type (two subtypes) by pulsed field gel electrophoresis typing, indicating that a nosocomial outbreak of diarrhea caused by A- B+ C. difficile occurred among the patients hospitalized on this ward. Detection of toxin A in stool specimens by a toxin A detection kit was performed on 14 patients. Although two patients who carried A+ B+ strains were positive for toxin A assay, toxin A detection test was negative in 12 patients including 10 patients with A- B+ C. difficile infection. Diagnosis of C. difficile-associated diarrhea by combination of toxin A assay in feces and culture of C. difficile could successfully lead to recognition of an outbreak caused by A- B+ C. difficile in a cancer center hospital.

Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine

Background & aims: Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis, diseases afflicting millions of people each year. Although C. difficile releases 2 structurally similar exotoxins, toxin A and toxin B, animal experiments suggest that only toxin A mediates diarrhea and enterocolitis. However, toxin A-negative/toxin B-positive strains of C. difficile recently were isolated from patients with antibiotic-associated diarrhea and colitis, indicating that toxin B also may be pathogenic in humans. Methods: Here we used subcutaneously transplanted human intestinal xenografts in immunodeficient mice to generate a chimeric animal model for C. difficile toxin-induced pathology of human intestine. Results: We found that intraluminal toxin B, like equivalent concentrations of toxin A, induced intestinal epithelial cell damage, increased mucosal permeability, stimulated interleukin (IL)-8 synthesis, and caused an acute inflammatory response characterized by neutrophil recruitment and tissue damage. Laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that intestinal epithelial cell-specific IL-8 gene expression also was increased significantly after luminal exposure to C. difficile toxins in vivo. Conclusions: We conclude that C. difficile toxin B, like toxin A, is a potent inflammatory enterotoxin for human intestine. Future therapeutic or vaccine strategies for C. difficile infection therefore need to target both toxins.

New types of toxin A-negative, toxin B-positive strains among Clostridium difficile isolates from Asia.

A total of 56 C. difficile strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of tcdA and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A(-)B(+) strain, C. difficile 8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A(+)B(+) strains. Toxinotypes XVI and XVII represent two new groups of A(-)B(+) strains. Strains of the well-known A(-)B(+) group from toxinotype VIII have a nonsense mutation at the beginning of tcdA gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5'-end sequence of tcdA in two newly described A(-)B(+) strains does not contain an identical mutation. The prevalence of variant C. difficile strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.

Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France.

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of Clostridium difficile have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (tcdA) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with C. difficile-associated diarrhea in France. The repeating segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol. 36:2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire tcdA gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11, C. difficile variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other tcdA variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of C. difficile-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion of C. difficile-associated diarrhea but a negative immunoassay for toxin A.

How to detect Clostridium difficile variant strains in a routine laboratory

Toxin A-negative, toxin B-positive strains (A–/B+) are the best studied examples of Clostridium difficile variant strains. In addition, there are some other groups of variant C. difficile strains that produce both toxins (A+/B+) or are non-cytotoxic (A-/B-) but differ from the reference strain VPI 10463 in their toxin genes. Here we describe two simple methods (amplification of the tcdA gene and amplification of the binary toxin gene cdtA) which can be used in rapid screening for variant C. difficile strains in collections or in routine laboratories.

Infection of hamsters with epidemiologically important strains of Clostridium difficile.

Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters. Three strains-types B1, J9, and K14-have caused hospital outbreaks. Type Y2 is associated with a high rate of asymptomatic colonization in patients. The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C. difficile-associated diarrhea. Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C. difficile. Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals. Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003). The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains.