Genes Encoding Toxin of Clostridium difficile in Children with and without Diarrhea (original) (raw)
Related papers
2002
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.
Diyala Journal of Medicine
Background: Clostridium difficile is a gram positive anaerobic spore forming bacteria. C. difficile-associated disease is a critical clinical issue that is accepted to happen mainly after hospitalization and used of expansive range anti-infection agents. Objective:To define the rate of C. difficile infections isolated from children patients suffering from diarrhea, detection profile toxigenicity of C. difficile strains for toxin A and toxin B by using of PCR, and revise different risk factors of C. difficile infections. Patients and Methods: This cross-sectional study included 50 patients who hospitalized for at least 2 days before the appearance of three or more unformed or liquid stools for 24h, genomic DNA was extracted by using 10% fecal supernatant and a ready kit was used for extraction according to manufacturer instructions. Molecular detection of toxigenic C. difficile done by using the specific primer sequences in polymerase chain reaction. Results: Current study showed diarrhea was the most prominent complain among the study population accounting for 41(82%), of whom 39(78%) presented with watery diarrhea. 38(76%) patients had no fever. The most comorbid disease was inflammatory bowel disease (IBD) with 7 (14%) patients. Forty-six (92%) cases had no history of hospitalization in the last 3 months versus only 8% had such history. PCR revealed that 16 (32%) samples were positive for tcdB gene, while all samples were negative for genes tcdA.
Memórias do Instituto Oswaldo Cruz, 2003
Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA + /tcdB + or tcdA -/tcdB + , and 1 strain as nontoxigenic (tcdA -/tdcB -). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.
Human pathology, 2010
Clostridium difficile toxin is frequently found in the stool of children; however, pseudomembranous colitis is rare. Studying the usefulness of Clostridium difficile toxin assays in pediatrics is required. We performed a correlation between presence of Clostridium difficile toxin in stool and evidence of Clostridium difficile in gastrointestinal pediatric tissue samples using immunohistochemistry (with a pan-clostridial antibody) and polymerase chain reaction (with primers for toxin genes). We studied 11 patients with a median age of 8 years (range, 4 weeks to 17 years); 4 (36%) were female. The median time between detection of Clostridium difficile toxin in stool and obtaining tissue was 3 days. Ten patients survived. Endoscopy was performed in 8 survivors and showed normal mucosa in 2, pseudomembranes in 2, erythema and friability in 1, aphthae in 1, increased mucous production in 1, and colitis in 1. Two survivors underwent laparotomy for either obstruction or resection of necrot...
Microorganisms
Detection of Clostridioides difficile toxins in patients with gastroenteritis has increasingly been accomplished through the use of enteric multiplex syndromic panels. Comparisons of the performance of these panels to both direct-from-stool (DFS) and culture-enriched stools followed by polymerase chain reaction (PCR) methods in pediatric populations are limited. Here, we compare the performance of the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to our DFS in-house real-time PCR (DFS RT-PCR) assay for the detection of C. difficile toxin gene, tcdB, using 2641 stool specimens collected from children enrolled in the Alberta Provincial Pediatric EnTeric Infection Team (APPETITE) study in Alberta, Canada. We used culture enrichment followed by in-house RT-PCR to resolve discordant results between the two assays. We found excellent agreement (k = 0.89) between the GPP and our DFS RT-PCR assay: the positive percent agreement between the two assays was 97%, and the negative percent ...
Journal of evolution of medical and dental sciences, 2015
BACKGROUND: Molecular methods for detection of toxigenic Clostridium difficile have been established in the developed countries though not very common in our country. AIMS: The study was intended to determine the presence of toxin A and toxin B genes of Clostridium difficile isolates by means of polymerase chain reaction (PCR) and analysis of clinical picture of the patients. MATERIALS AND METHODS: The prospective study was conducted in a tertiary care teaching hospital, South India from January 2012 to December 2014. Stool samples were collected consecutively from 563 in patients with diarrhoea from various wards. Clostridium difficile was isolated and identified by semi quantitative culture, latex agglutination and biochemical reactions. These isolates were then subjected to PCR for the detection of toxin A and toxin B genes. In addition, enzyme immunoassay was performed on stool samples for the detection of toxins A and B. The clinical spectrum of PCR positive patients was also analyzed. RESULTS: From 563 stool specimens, 113 (20.07%) Clostridium difficile isolates were grown by culture and identified by latex agglutination and biochemical reactions. Out of 113 isolates, 94 were subjected to PCR. 50 (53.19%) isolates out of 94 were found to be positive. Three toxigenic types obtained were A + B + , A-B + and A + Bwhich accounted for 6.38%, 42.55% and 4.26% respectively. A-Bisolates were 46.81%. 30 (26.55%) out of 113 stool samples (which were culture positive) was also enzyme immunoassay positive. 32 (64%) out of 50 PCR positive patients exhibited antibiotic usage (p<0.05) and 39(78%) revealed the presence of underlying illnesses/conditions (p<0.01). CONCLUSION: The study highlights the usefulness of PCR for detection of toxigenic Clostridium difficile and for determination of its molecular epidemiology.
Journal of Clinical Microbiology, 2009
Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.
Incidence and importance of Clostridium difficile in paediatric diarrhoea in Brazil
Journal of Medical Microbiology, 2003
Clostridium difficile strains were detected in 14 of 210 (6 . 7 %) faecal samples from children in Rio de Janeiro, Brazil, by cultivating faeces on cycloserine/cefoxitin/fructose agar after alcohol-shock. Two main groups of children were studied: inpatients (n ¼ 96) and outpatients (n ¼ 114). The inpatient group consisted of children on antibiotics or immunosuppressors who presented with diarrhoea and other children who did not present with diarrhoea and were not under an antibiotic or chemotherapeutic regimen. Among the outpatients, two groups were examined: namely, a group that comprised children who presented with diarrhoea and were occasionally under an antibiotic regimen and another group that comprised patients who were not taking antibiotics. After cytotoxic assay, toxigenic C. difficile (Cd tox þ ) strains were detected in 4 . 2 % of inpatients and 3 . 5 % of outpatients. Exclusion of other infectious causes of diarrhoea indicated a typical case of C. difficileassociated paediatric diarrhoea in the community. Among Cd tox þ isolates, no variations were detected by PCR for toxin A that employed primers NK9 and NKVO11. No resistance was found to metronidazole or vancomycin among strains that were isolated from children who presented with diarrhoea, but the MIC 50 and MIC 90 values for clindamycin were 6-8 and 16 ìg ml À1 , respectively. Resistance to clindamycin seems to be more disseminated in strains from outpatients than in those from inpatients (P , 0 . 05). In conclusion, these data suggest that investigation for C. difficile infection should be taken into account in paediatric diarrhoea in both inpatients and outpatients in developing countries.
Journal of Clinical Microbiology, 2009
Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n ؍ 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n ؍ 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n ؍ 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates. Clostridium difficile is one of the leading causes of infectious antibiotic-associated diarrhea and pseudomembranous colitis worldwide (2, 16). This is illustrated by the increased incidence and severity of C. difficile infection, suggesting the emergence of a new hypervirulent strain (5, 13-15, 17, 25, 32). While TcdB, a cytotoxin, is the known established virulence factor of C. difficile, toxin A (TcdA), a cytotoxic enterotoxin, works synergistically with TcdB, causing damage to the intestinal mucosa in cases of C. difficile infection (17). The genes that encode these toxins are located on the pathogenicity locus of C. difficile (4, 10, 24). Additionally, several deletions in the tcdC gene, a putative negative regulator of the expression of the toxin A (tcdA) and the toxin B (tcdB) genes, have been identified, and these deletions result in higher levels of cytotoxin expression (11). Furthermore, research has shown that some C. difficile strains produce another toxin, known as the binary toxin (19, 22, 28). The genes that encode this toxin, cdtA and cdtB, together produce an actin-specific ADP-ribosyltransferase that induces damage to the actin skeleton, leading to cytopathic effects in cell lines (1). It has been suggested that the binary toxin genes and deletions in the tcdC gene are potential virulence factors in the recent emerging hypervirulent strain (22, 29). The "gold standard" for the detection of C. difficile toxin production is a cytotoxin assay with stool specimens or isolates from anaerobic culture. The cytotoxin assay is difficult to perform and time-consuming, and it is often less sensitive than