Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.
Animals ; Classification ; Clone Cells ; Enterotoxins ; Epitopes ; Genetic Variation ; Genotype ; Humans ; Protein Folding ; Protein Processing, Post-Translational ; Rotavirus ; Sequence Analysis ; Viral Nonstructural Proteins ; Virulence

BACKGROUND: We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. METHODS: We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). RESULTS: Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). CONCLUSIONS: Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.
Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/*genetics/isolation & purification ; DNA, Bacterial/*analysis/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; *Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.
Bacterial Proteins/*analysis ; Bacterial Toxins/*analysis ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/enzymology/*isolation & purification/metabolism ; Enterotoxins/*analysis ; Feces/microbiology ; Glutamate Dehydrogenase/*analysis ; Humans ; *Immunoenzyme Techniques ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

BACKGROUND: ChromID Clostridium difficile agar (IDCd; bioMerieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). METHODS: A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMerieux SA), and multiplex PCR for tcdA, tcdB, and tpi. RESULTS: The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. CONCLUSIONS: The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.
Agar/*chemistry ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium difficile/genetics/*isolation & purification ; DNA, Bacterial/analysis ; Enterocolitis, Pseudomembranous/diagnosis/microbiology ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Triose-Phosphate Isomerase/genetics

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.
Aged ; Bacterial Proteins/analysis ; Bacterial Toxins/analysis ; Clostridium Infections/*epidemiology/physiopathology ; Clostridium difficile/*isolation & purification/*pathogenicity ; Diarrhea/epidemiology/microbiology ; Enterocolitis, Pseudomembranous/*epidemiology/microbiology/pathology ; Enterotoxins/analysis ; Feces/microbiology ; Female ; Hospitals ; Humans ; Incidence ; Male ; Metronidazole/therapeutic use ; Middle Aged ; Prospective Studies ; Recurrence ; Republic of Korea/epidemiology ; Treatment Outcome ; Vancomycin/therapeutic use

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I nuSabeta. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.
Animals ; Cattle ; Cluster Analysis ; DNA, Bacterial/chemistry/genetics ; Enterotoxins/chemistry/*genetics ; Female ; Genotype ; Mastitis, Bovine/epidemiology/genetics/*microbiology ; Microsatellite Repeats ; Milk/*microbiology ; Polymerase Chain Reaction/veterinary ; Prevalence ; Republic of Korea/epidemiology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus aureus/classification/genetics/*isolation & purification

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic

BACKGROUND/AIMS: The spectrum of Clostridium difficile-associated disease (CDAD) ranges from mild diarrhea to life-threatening colitis. Recent studies reported an increase in incidence and severity of CDAD and the presence of severe community-acquired CDAD (CA-CDAD). The aims of this study were to investigate the incidence of CA-CDAD and non-antibiotics-associated CDAD, and to compare the clinical characteristics between hospital-acquired (HA) and CA-CDAD. METHODS: The medical records of 86 patients who were diagnosed as CDAD in Hanyang University Guri Hospital between January 2005 and October 2007 were retrospectively reviewed. RESULTS: Of the 86 patients (mean age 64 years), 53 patients were women. The most frequently prescribed antibiotics were cephalosporins (67.4%), followed by aminoglycosides (38.4%) and quinolones (14%). Of the 86 patients, the average duration of treatment and recovery time of symptoms were 11.5 days and 4.6 days, respectively. Seven percent of patients experienced relapse treatment. The overall incidence rate of CA-CDAD and non-antibiotics-associated CDAD were 10.5% and 22.1%, respectively. CA-CDAD group had lower rate of antimicrobial exposure whilst showing higher rate of complications compared to HA-CDAD group. Three patients in the CA-CDAD progressed towards a severe complicated clinical course, including septic shock. CONCLUSIONS: The incidence rate of CA-CDAD and non-antibiotics-associated CDAD were 10.5% and 22.1%, respectively. CA-CDAD tends to have a higher complication rate compared to HA-CDAD. Community clinicians needs to maintain a high level of suspicion for CDAD, whilst coping with the ever evolving epidemiologic change.
Adult ; Aged ; Aged, 80 and over ; Aminoglycosides/therapeutic use ; Anti-Bacterial Agents/therapeutic use ; Bacterial Toxins/analysis ; Cephalosporins/therapeutic use ; *Clostridium difficile ; Community-Acquired Infections/epidemiology ; Cross Infection/epidemiology ; Enterocolitis, Pseudomembranous/*diagnosis/drug therapy/epidemiology ; Enterotoxins/analysis ; Female ; Humans ; Male ; Metronidazole/therapeutic use ; Middle Aged ; Quinolones/therapeutic use ; Retrospective Studies

The filtrate of Staphylococcus aureus culture has been used in an ampule form named as staphylococcal enterotoxin C injection for cancer therapy in clinic for ten years in China and proved to be effective. The active constituent of three kinds of injections is claimed to be staphylococcal enterotoxin C2 (SEC2), and the content of SEC2 is used as quality control. However, the correct content of SEC2 was not known and the relative amount of SEC2 was very low because of the complicated components of the filtrate. In this research, we established a proper ELISA system for the detection of SEC2 in staphylococcal enterotoxin C injection, which will improve the quality control of the injection. We produced and identified polyclonal and monoclonal antibodies of SEC2 and established BA-ELISA method based on the method of sandwich ELISA. It was found that the BA-ELISA method had good specificity, sensitivity and reproducibility, and being able to detect SEC2 at concentration from 2 to 20 ng x mL(-1), with an average CV value of 5.08%. The SEC2 content in staphylococcal enterotoxin C injection was calculated. There is some difference between the actual and labeled contents in the injections.
Animals ; Antibodies, Bacterial ; biosynthesis ; immunology ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antineoplastic Agents ; administration & dosage ; analysis ; immunology ; Enterotoxins ; administration & dosage ; analysis ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Hybridomas ; secretion ; Injections ; Mice ; Mice, Inbred BALB C ; Quality Control ; Rabbits ; Staphylococcus aureus ; chemistry

OBJECTIVETo clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap).

METHODSTwo couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced.

RESULTSThe ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis.

CONCLUSIONThe vector of pET28a ltb-fap was obtained.

Aggregatibacter actinomycetemcomitans ; Bacterial Toxins ; Cloning, Molecular ; Cloning, Organism ; Enterotoxins ; Escherichia coli ; Escherichia coli Proteins ; Hot Temperature ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Sequence Analysis