Umbral del ciclo de amplificación del gen de la toxina B de Clostridioides difficile como predictor de gravedad / Clostridioides difficile toxin B gen amplification cycle threshold as a predictor of severity

INTRODUCCIÓN: La infección por Clostridioides dfficile (ICD) es la principal causa de diarrea nosocomial, generalmente asociada al consumo de antimicrobianos. Esta infección puede causar desde diarrea no complicada hasta colitis pseudomembranosa o megacolon tóxico. Estudios recientes han intentado relacionar el valor el ciclo umbral (Ct) de la RT-PCR con la mortalidad, como un método rápido, sencillo, objetivo y eficaz. OBJETIVO: Evaluar el Ct como predictor de mala evolución en pacientes con y sin criterio clínico de dicha gravedad. PACIENTES Y MÉTODOS: Realizamos un estudio retrospectivo entre enero 2015 y diciembre 2018, incluyendo todos los pacientes del área de referencia del Hospital Universitario de Canarias en Tenerife (396.483 habitantes) en pacientes con criterios clínicos de gravedad (de acuerdo a la Guía para la Práctica Clínica de la enfermedad por C. dfficile de la Sociedad de Epidemiología del Cuidado de la Salud de América (SHEA) y la Sociedad de Enfermedades Infecciosas de Norteamérica (IDSA) y pacientes sin criterios clínicos de gravedad evaluando el Ct como predictor de mala evolución. RESULTADOS: Se diagnosticó un total de 202 episodios de ICD. El 77,7% (n = 157) presentó criterios clínicos de gravedad. La presencia de colitis ulcerosa (p < 0,001), fiebre (p < 0,001), leucocitosis (p < 0,001), neutrofilia (p < 0,001), creatininemia (p = 0,005) se presentaron como factores de riesgo para el desarrollo de ICD grave. El sexo femenino, la institucionalización, el ingreso previo y el exitus se describieron con mayor frecuencia en el grupo con ICD-G, no encontrando diferencias significativas. No encontramos diferencias respecto a los días de estancia previa, o de estancia post-ICD, aunque en este último, la media fue mayor en el caso de los pacientes con ICD-G. No se encontraron diferencias significativas en cuanto al Ct en ambos grupos; siendo sólo un punto menor en pacientes con criterio de gravedad (Ct = 26,1) que sin criterios de gravedad (Ct = 27,4) (p = 0,326).

BACKGROUND: Clostridioides dfficile infection (CDI) is the main cause of nosocomial diarrhea, generally associated with the use of antibiotics. This infection can cause uncomplicated diarrhea to pseudomembranous colitis or toxic megacolon. Recent studies have attempted to relate the threshold cycle (Ct) value of RT-PCR with mortality, as a fast, simple, objective and efficient method. AIM: To evaluate Ct as a predictor of poor outcome in patients with C. dfficile disease with/without clinical signs of severity. METHODS: We carried out a retrospective study between January 2015 and December 2018, including all patients in the reference area of the Hospital Universitario de Canarias in Tenerife (396,483 inhabitants) in patients with clinical criteria of severity and patients without clinical severity criteria (according to the guide for the clinical practice of CDI of the Society of Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of North America (IDSA). RESULTS: A total of 202 CDI episodes were diagnosed. 77.7% (n = 157) presented clinical severity criteria. The presence of ulcerative colitis (p < 0.001), fever (p < 0.001), leukocytosis (p < 0.001), neutrophilia (p < 0.001), creatininemia (p = 0.005) were presented as risk factors for the development of severe CDI (S-CDI). Female sex, institutionalization, previous admission and death were described more frequently in the group with S-CDI, not finding significant differences. We found no differences with respect to the days of previous stay, or of post-CDI stay, although in the latter, the mean was higher in the case of S-CDI patients. No significant differences were found in terms of Ct in both groups; being only one point lower in patients with severity criteria (Ct = 26.1) than without severity criteria (Ct = 27.4) (p = 0.326). CONCLUSION: Based on the results of our study, it has not been possible to systematically implement the Ct value as a predictor of severity to the clinical report, and it is not possible to extrapolate this predictive variable from S-CDI and standardize the Ct value as a predictor of severity. Conclusion: Basándonos en los resultados de nuestro estudio, no ha sido posible la implementación sistemática del valor Ct como predictor de gravedad al informe clínico, no siendo posible extrapolar esta variable predictora de enfermedad por C difficile-G y estandarizar el valor Ct como factor predictor de gravedad.

¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Clostridium difficile toxin B-induced apoptosis of colon cancer cells and related mechanisms / 中华微生物学和免疫学杂志

Objective To analyze the effects of Clostridium difficile toxin B (TcdB) on the prolif-eration and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis. Methods SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry. Re-sults TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent man-ner and the inhibition rate reached 46. 36％ at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20. 83％ apoptosis rate was in-duced by 800 ng/ml of TcdB at48 h. Conclusions TcdB could inhibit the proliferation and induce the ap-optosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mito-chondrial apoptosis pathway.

Clostridium difficile toxin B-induced apoptosis of colon cancer cells and related mechanisms / 中华微生物学和免疫学杂志

Objective@#To analyze the effects of Clostridium difficile toxin B (TcdB) on the proliferation and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis.@*Methods@#SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry.@*Results@#TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent manner and the inhibition rate reached 46.36% at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20.83% apoptosis rate was induced by 800 ng/ml of TcdB at 48 h.@*Conclusions@#TcdB could inhibit the proliferation and induce the apoptosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mitochondrial apoptosis pathway.

Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae

Background & objectives: Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 ?g/cassette, respectively. Interpretation & conclusions: The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.

Construction and anti-tumor efficacy of a peptide vaccine that targets calcium-binding protein S100A9 / 药学学报

Calcium-binding protein S100A9 is closely related to inflammation and tumor invasion, and is one of the specific markers of myeloid-derived suppressor cells (MDSC). In this study, a recombinant polypeptide vaccine CTB-S100A9 targeting mouse calcium-binding protein S100A9 was constructed by fusion cholera toxin B subunit (CTB) with S100A9 gene. The CTB-S100A9 fusion protein was expressed in E coli. and purified by Ni+ affinity chromatography. Vaccinate the purified recombinant CTB-S100A9 protein supplemented with aluminum hydroxide adjuvant can break the autoimmune tolerance and produce high titer of S100A9 antibody in mice. Moreover, the S100A9 antibody produced by CTB-S100A9 vaccination is more specific and does not cross-react with S100A8. In the mouse 4T1 breast cancer model, CTB-S100A9 vaccination not only has significant tumor prevention effects, but also has significant tumor therapeutic effects. In addition, CTB-S100A9 significantly inhibited lung metastasis in 4T1 mice breast cancer model. Further analysis by flow cytometry showed that CTB-S100A9 vaccination can significantly reduce the tumor induced Treg cells and granulocyte-derived MDSC in 4T1 mice model, and reverse the tumor immunosuppressive environment, thereby promote the anti-tumor efficacy. The animal experiments in this study were carried out under the animal care guidelines approved by the Animal Ethics Committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine. This study shows that CTB-S100A9 is a good recombinant vaccine that targets the tumor immune-suppression environment and has great potential for the future clinical application.

Construction and anti-tumor efficacy of a pentameric peptide vaccine that targets S100A8 / 药学学报

Myeloid-derived suppressor cells (MDSC) play critical roles in immune escape of tumor. We hypothesized that elimination of tumor-induced MDSCs might help to block tumor growth. Therefore, we constructed a cholera toxin B based peptide vaccine that targets a MDSC surface marker S100A8. Immunized BALB/c mice with CTB-S100A8 plus aluminum hydroxide induced high titers of anti-S100A8 antibodies and reduced tumor burden significantly in 4T1 mice model. We also found the vaccination led to significant reduction of tumor-induced monocytic MDSC (M-MDSC), with no effect on innate MDSCs, dendritic cell (DC) and macrophage (Mφ), demonstrating that targeting tumor-induced MDSC may be a promising approach in cancer immunotherapy.

Increase of Clostridium difficile in Community; Another Worrisome Burden for Public Health / 대한임상미생물학회지

BACKGROUND: Increasing rates of Clostridium difficile infection (CDI) have been reported mainly in Europe and North America; however, only limited reports have originated in Korea. The current epidemiology of CDI in the community could help to understand the outpatient healthcare environment and to extend infection control measures to outpatient settings. METHODS: C. difficile isolates in NHIS Ilsan Hospital from 2012 to 2014 were included in this study. Clinical characteristics, acquisition types, and previous antimicrobial therapy were obtained via Electronic Medical Records. C. difficile culture was performed only in unformed stool. Toxin was positive by enzyme-linked fluorescent immunoassay (ELFA) in 247 specimens. In addition, toxin B and binary toxin gene were detected by PCR in 57 specimens. CDI was defined by toxigenic C. difficile isolation in unformed stool. RESULTS: In the previous 3 years, 251 unduplicated C. difficile cases have been detected; 168 healthcare facility- associated hospital onset (HCFA-HO), 45 healthcare facility-associated community onset (HCFA-CO), and 38 community-associated (CA). Toxin positive rates by ELFA for toxin A&B were HCFA-HO 50.6% (84/166), HCFA-CO 41.9% (18/43), and CA 42.1% (16/38). Toxin positive rate by PCR for tcdB were HCFA-HO 62.9% (22/35), HCFA-CO 69.2% (9/13), and CA 100% (9/9). No binary toxin (cdtA/cdtB) was detected in 57 cases. CONCLUSION: Community-associated CDI may be underestimated in Goyang province, Korea, especially by commonly used ELFA toxin assay. The spread of community-associated CDI should be recognized as an increasing burden of public health.

Evaluation of a Rapid Membrane Enzyme Immunoassay for the Simultaneous Detection of Glutamate Dehydrogenase and Toxin for the Diagnosis of Clostridium difficile Infection

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.

Effects of Peripheral Neuropathy on CTB-labeled Motor Neurons Following Ligation of the Tibial Nerve / 체질인류학회지

Changes in CTB labeled motor neurons of the spinal cord were observed after the induction of peripheral neuropathy by ligation of the tibial nerve. Rats were anesthetized and the tibial nerve was ligated with 3-0 silk. The rats were separated into three groups based on the length of time the tibial nerve was ligated (1, 2, or 4 weeks). After the ligation procedures were complete, the tibial nerve stumps were soaked in CTB solution. Tibial nerve segments and the spinal cord were then observed. In the control and experimental groups, CTB-labeled neurons formed a discrete population that was concentrated primarily at the L5 level, while the contributions from L4 and L6 were minor. According to the distributions, CTB-labeled neurons were divided into rostral and caudal groups. A selective decrease of CTB-labeled neurons was observed only in the caudal group, extending from the rostral L5 to one-half of the rostral L6. The total numbers of CTB-labeled motor neurons were 2,160+/-169.3, 1,002+/-245.1, 587.5+/-346.5, and 1,728+/-402.6 in the control group, 1 week group, 2 week group, and 4 week group, respectively. The selective decrease of CTB-labeled neurons in the caudal division was responsible for the decrease in the total number of labeled neurons in all groups. Following peripheral neuropathy caused by ligation of the tibial nerve, CTB-labeled neurons in the spinal cord decreased selectively. These results may provide important neuroanatomical data regarding the effects of peripheral neuropathy by ligation of the tibial nerve.

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.

Effect of Nasal Administration of Compound of Cholera Toxin B Subunit and Nerve Growth Factor on Spatial Learning and Memory Abilities in the Amnesic Mice / 中国康复理论与实践

@#ObjectiveTo explore the effects of the conjugate prepared from the cholera toxin B subunit(CB) and nerve growth factor(NGF) on the spatial learning and memory abilities and cholinergic function.MethodsThe conjugate of CB-NGF was prepared by the improved sodium metaperiodate method and nasally administrated to the β-amyloid protein(Aβ25-35) induced amnesic mice for 7 days with 2 dosage (7-5 μg/d、15 μg/d). Spatial learning and memory abilities were evaluated by Morris water maze and cholinergic function was assessed with the choline acetyl transferase (ChAT) immunohistochemical methods.ResultsMorris water maze test showed that the escape latency in Aβ25-35-treated mice prolonged and the staying time reduced in the crossed first quadrant where the platform had been located, compared with the control mice (P<0-01). In addition, the number of ChAT positive neuron declined in the model mice(P<0-001). CB-NGF nasal administration significantly shortened the escape latency and elevated the staying time and number of ChAT positive neuron(P<0-01).ConclusionCB-NGF treatment can improve the spatial and memory performance which may involve the neuroprotection to cholinergic system.

Investigation of Toxin Gene Diversity, Molecular Epidemiology, and Antimicrobial Resistance of Clostridium difficile Isolated from 12 Hospitals in South Korea / 대한진단검사의학회지

BACKGROUND: Clostridium difficile is a major cause of antibiotic-associated diarrhea. The objective of this study was to characterize clinical isolates of C. difficile obtained from various regions in Korea with regard to their toxin status, molecular type, and antimicrobial susceptibility. METHODS: We analyzed a total of 408 C. difficile isolates obtained between 2006 and 2008 from 408 patients with diarrhea in 12 South Korean teaching hospitals. C. difficile toxin genes tcdA, tcdB, cdtA, and cdtB were detected by PCR. Molecular genotyping was performed by PCR ribotyping. Antimicrobial susceptibilities of the 120 C. difficile isolates were assessed by agar dilution methods. RESULTS: Among 337 toxigenic isolates, 105 were toxin A-negative and toxin B-positive (A-B+) and 29 were binary toxin-producing strains. PCR ribotyping showed 50 different ribotype patterns. The 5 most frequently occurring ribotypes comprised 62.0% of all identified ribotypes. No isolate was susceptible to cefoxitin, and all except 1 were susceptible to piperacillin and piperacillin-tazobactam. The resistance rates of isolates to imipenem, cefotetan, moxifloxacin, ampicillin, and clindamycin were 25%, 34%, 42%, 51%, and 60%, respectively. The isolates showed no resistance to metronidazole or vancomycin. CONCLUSIONS: This is the first nationwide study on the toxin status, including PCR ribotyping and antimicrobial resistance, of C. difficile isolates in Korea. The prevalence of A-B+ strains was 25.7%, much higher than that reported from other countries. Binary toxin-producing strains accounted for 7.1% of all strains, which was not rare in Korea. The most prevalent ribotype was ribotype 017, and all A-B+ strains showed this pattern. We did not isolate strains with decreased susceptibility to metronidazole or vancomycin.

The effect of meningococcal serogroup A polysaccharide-protein conjugate vaccine prepared with TT and rCTB on mucosal immune responses / 中华微生物学和免疫学杂志

Objective To analyze the feasibility of the recombinant cholera toxin B subunit (rCTB) as a carrier protein candidate for the preparing of polysaccharide-protein conjugate, and to discuss the immune effects of tetanus toxoid (TT) as the carrier protein in mucosal delivery vaccine. Methods The refolded pentrumer protein, rCTB was obtained by genetic engineering methods. Then conjugated the refold-ed protein with group A meningococcal polysaccharide (GAMP) using the chemical method(ADH) ,the pol-ysaccharide-protein conjugates(GAMP-rCTB) were prepared. BALB/c mice were immunized either intraper-itoneally ( i. p. ) or intranasally ( i. n. ) with GAMP-rCTB. Moreover, GAMP-TT vaccine that TT as carrier proteins was i.n. immunized to the mice. The evaluation of immunology is performed. Results The conju-gates of polysaccharide-potein with the rCTB and TT as protein carrier both are able to elicit high level of GAMP specific IgG antibody in serum after i.n. immunization, and the conjugates can also elicit specific IgA antibody in lung lavage and intestinal mucosa. Conclusion rCTB and TT can both as the protein carri-er for polysaccharide-protein conjugate as mucosal vaccine. The route of intranasal may be more ways for im-mune function than i.p. immunization when rCTB is used as the carrier of the polysaccharide-protein conju-gates.

A Case of Palmoplantar Hyperhidrosis Successfully Treated with Botulinum Toxin B / 대한피부과학회지

Hyperhidrosis is an eccrine sweat gland disease that results from sympathetic hyperactivity, usually occurring on the axilla, palm, sole, or groin. It causes not only cosmetic problems, but also social stress in affected patients. Until now, several modalities have been used to treat focal hyperhidrosis, with variable clinical outcomes and complications, including skin irritation, neurological problems, and nonesthetic scar formation. Botulinum toxin type A has been used widely and successfully in the treatment of hyperhidrosis since 1981. Botulinum toxin type B has recently been introduced for off-label use after being approved by the Food and Drug Administration in 2000 for the treatment of cervical dystonia. However, there has been no report of Botulinum toxin type B treatment for palmoplantar hyperhidrosis in the Koreandermatologic literature. Herein, we report the first case of palmoplantar hyperhidrosis successfully treated with Botulinum Toxin B in Korea, along with a review of the literature.

Comparison of Simultaneous Use of C. DIFF QUIK CHEK and VIDAS C. difficile Toxin A&B to detect C. difficile in Fecal Specimen / 임상검사와정도관리

BACKGROUND: Clostridium difficile is the most common pathogen of antibiotic-associated diarrhea. Toxigenic strains produce toxin A and toxin B. The pathogenicity of C. difficile is due to the production of these two exotoxins. This study aimed to evaluate diagnostic value of two enzyme immunoassay by comparison of concordance rate to diagnose C. difficile-associated infection. METHODS: C. DIFF QUIK CHEK (TECHLAB, USA) that detect glutamate dehydrogenase antigen and VIDAS C. difficile Toxin A&B (BioMerieux, France) that detect toxin A and toxin B were done in 122 fecal specimens to detect C. difficile. RESULTS: In the total 122 stool specimens, 17 cases showed positive results in both tests. One specimen showed discrepancy that positive result in VIDAS C. difficile Toxin A&B (relative fluorescence value, RFV=2.93) but negative result in C. DIFF QUIK CHEK. Therefore, the concordance rate between two tests was 95.1% (116/122). Both anaerobic culture and in-house PCR for toxin B were negative in the discrepant fecal specimen and there was no clinical evidence that support C. difficile-associated diarrhea, so we concluded result in VIDAS C. difficile Toxin A&B as false positive. CONCLUSIONS: Although these two enzyme immunoassays targeted different antigen, they showed high concordance rate. The discrepant case was concluded to false positive in VIDAS C. difficile Toxin A&B test because it showed negative results in culture and PCR for toxin B and there were no clinical evidences of C. difficile-associated infection. It could be needed for analysis about conditions that cause false positive result in enzyme immunoassays to detect C. difficile toxin.

Comparison of Two Enzyme Immunoassay for Detection of Clostridium difficile Toxin A and Toxin B / 대한진단검사의학회지

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.

Clone and express ctb-stx2 b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeai toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity / 中华流行病学杂志

Objective To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157:H7(EHEC O157:H7)Shigela toxin 2B subunit(Stx2B)and vibrio cholera toxin B subunit (CTB)as well as to detect the immunogenicity and GM1-binding ability of fusion protein.Methods To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified,then to transform constructed plasmid into E.coliBL21(DE3)induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDSPAGE and Western-blot.Results The amplified ctb-stx2b fragments appeared to he 750 bp and gene sequence was identical to designed sequence.The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about Mr20×103and the expressed protein could react to CTB monoclone anti-body.The fusion protein CTB-Stx2B could bind GM1.Conclusion CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability.This study provided information on further EHEC O157:H7 vaccine research.

Evaluation of Rapid Assay (Tox A/B Quik Chek) for the Detection of Clostridium difficile Toxins A and B / 대한임상미생물학회지

BACKGROUND: Toxin immunoassay is widely used for rapid diagnosis of Clostridium difficile-associated diarrhea. The aim of this study was to evaluate the performance of Tox A/B Quik Chek test (TECHLAB, Blacksburg, VA, USA) compared to toxigenic culture. METHODS: From September 2006 to August 2007, 959 stools were examined by Tox A/B Quik Chek test and toxigenic culture (C. difficile culture plus tcdB PCR using colonies obtained from culture). RESULTS: Compared to the results of toxigenic culture, the sensitivity and specificity of Tox A/B Quik Chek test were 47.5% and 97.5%, respectively. CONCLUSION: The sensitivity of Tox A/B Quik Chek test was not high, but the specificity was high. Although Tox A/B Quik Chek test alone is not sufficient to diagnose Clostridium difficile-associated diarrhea, it may aid rapid diagnosis, early treatment and prevention of nosocomial spread of the infection, if supplemented by C. difficile culture or tissue culture cytotoxin assay.

Expression of clostridium difficile toxin B C-terminal repeated unit in E.coli and its immunogenicity / 基础医学与临床

Objective To perform expression and to detect immunogenicity of Clostridium difficile Toxin B receptor binding zone(CD3).Methods The clostridium difficile toxin B C-terminal repeated gene was amplified by PCR and cloned into the prokaryotic expression vector pET 22b(+),and the recombinant plasmid pET 22b(+)CD3 was then transformed into E.coli strainBL21(DE3).The E.coli strainBL21(DE3) containing pET22b(+)CD3 was induced with IPTG and analyzed with SDS PAGE.Results A 71.3 ku protein was harvested after inducing with IPTG and thin layer scanning which takes 34% of the total bacterial protein,22.7% of the supernatant and 24.9% of the inclusion body.The contentration of recombinant protein is 0.781 g/L identified by Coomassie brilliant blue G-250 chromatometry method.Conclusion The high expression and purification of clostridium difficile toxin B receptor gene lay a foundation for the further study on CD3 function and clostridium difficile vaccine.