Evaluation of a new chromogenic media for the isolation and culture of Clostridium difficile / 临床检验杂志

Objective@#To evaluate the performance of a chromogenic agar developed by our laboratory for the isolation and culture of Clostridium difficile (CDCA). @*Methods@#The chromogenic specificity of CDCA was evaluated by inoculation of C. difficile and other standard strains, and the sensitivities of CDSA (BD), CDIF (BioMérieux) and CDCA were determined by the C. difficile standard strains respectively. A total of 120 clinical stool specimens were cultured for C. difficile by three chromogenic media respectively. The colonies were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tpi gene was also detected. The sample which could be identified as C. difficile in any of the three chromogenic medium was defined as true positive. @*Results@#Most of standard strains were inhibited by CDCA, however some Clostridium species including C. clostridiiforme, C. bifermentans, C. tertium and Bacteroides fragilis grew lightly with chromogenic reaction. The sensitivities of CDSA, CDIF and CDCA were 2.0×105 CFU/mL, 8.0×101 CFU/mL and 4.0×10 2 CFU/mL, respectively. Among the 120 samples, 31 (25.8%) were defined as true C. difficile positive samples, while the positive rate of CDSA, CDIF and CDCA were 25 (20.8%), 28 (23.3%) and 26 (21.7%), respectively. There was no significant difference for clinical diarrhea specimens among the three chromogenic media (χ 2 =0.418, P=0.811). In comparison to the standard, the sensitivity, specificity, positive predictive value and negative predictive value were 83.8%, 100%, 100% and 94.7% for CDCA; 90.3%, 98.9%, 96.6% and 96.7% for CDIF; and 80.6%, 100%, 100% and 93.7% for CDSA. @*Conclusion@#The CDCA developed by our laboratory could be used to preliminarily isolate C. difficile with good specificity and sensitivity.

Simultaneous detection and characterization of toxigenic Clostridium difficile directly from clinical stool specimens / 医学前沿

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.

Evaluation of clinical application of two molecular assays for detection of toxigenic Clostridium difficile / 中华检验医学杂志

Objective In comparison with Xpert C.difficile/Epi through detection of Clostridium difficile toxin genes from clinical stool , the performance of a laboratory-developed ( LD) assay was evaluated in detail.Methods A total of 176 stool specimens collected from patients with diarrhea in the First People′s Hospital of Yuhang District and the People′s Hospital of Yingzhou , Ningbo from August 1 to December 30 were detected by the two assays in parallel , and meanwhile the C.difficile strains will be isolated and identified for C.difficile toxin genes by a conventional PCR assay .The Cross-tabs Analysis was used for the results by using SPSS20.0 software.Results In comparison with the results of Xpert C.difficile/Epi as the standard, the LD assay had a sensitivity of 91.7％(22/24), a specificity of 100％(152/152), a positive predictive value (PPV) of 100％(22/22), and negative predictive value (NPV) 98.7％(152/154).The results of two assays were statistically coherent (Kappa=0.950, P<0.001).In comparison with culture and detection of toxin genes results , the LD assay had a sensitivity of 90.0％ ( 18/20 ) , a specificity of 97.0％(152/156), a PPV of 81.8％ (18/22), and NPV of 98.7％ (152/154)(Kappa=0.838, P<0.001), and the Xpert C.difficile/Epi assay had a sensitivity of 90.0％ (18/20), a specificity of 96.0％(150/156), a PPV of 75.0％(18/24), and NPV of 98.7％ (150/152)(Kappa=0.792, P<0.001). Conclusions The performance of the LD assay was similar to that of the Xpert C .difficile/Epi kit in detection of toxigenic C.difficile.The LD assay could be directly applied to detection of toxigenic C.difficile from clinical stool samples .The clinical application of this LD assay will also provide a domestic and promising diagnostic assay for diagnosis of C.difficile infection in China.

Evaluation of a real-time PCR assay for the qualitative detection of Clostridium difficile toxin gene and its clinical application / 中华检验医学杂志

Objective In comparison of the performances for the detection of Clostridium difficile toxin B genes from stool between BD MAX Cdiff assay and a laboratory-developed (LD) assay.The LD assay was evaluated in clinical application.Methods This study was a clinical application research.A total of 147 stool specimens from patients with diarrhea in Hangzhou First Hospital affiliated with Zhejiang Chinese Medical University were detected by the two assays from 1 July to 30 September 2014.DNA extraction and amplification of the tcdB gene were performed automatically on the BD MAX platform.Meanwhile, the tcdA and tcdB gene were detected by the LD real-time PCR assay after DNA extraction.Then, the results were analyzed by use of SPSS 10.0.Results A total of 147 stool samples were collected.There were 33 C.difficile positive cases and 114 negative cases detected by both of two assays.However, there were four stool samples had incongruent results.In comparison with BD MAX, the LD assay had a sensitivity of 93.94% (31/33), a specificity of 98.25% (112/114), a positive predictive value of 93.94% (31/33), and negative predictive value 98.25% (112/114).Furthermore, the results of the LD assay were statistically coherent with that of the BD assay (Kappa=0.922, P<0.01).Conclusions The LD assay was highly sensitive and accurate as BD MAX Cdiff assay in the detection of toxigenic Clostridium difficile.Furthermore, this LD assay could be also applied to detection of clinical stool samples directly with low cost.The assay will be more promising in diagnosis of toxigenic C.difficile in clinical application in China due to no additional instrument needed.

Research progress in Clostridium difficile tcdC gene / 军事医学

TcdA and B toxins secreted by Clostridium difficile( CD) are two important causes of diseases in organisms. The expression of tcdA and tcdB genes is regulated by a few factors located in the pathogenicity locus ( PaLoc) .Studies have indicated that the tcdC gene is likely to act as a negative regulator of toxin gene expression.So far, it has been debatable whether tcdC gene is regarded as a negative regulator.The mechanism of tcdC gene in pathogenesis remains unclear.In this paper, the structure and function of the tcdC gene are summarized, which will help study the mechanism of tcdC gene and obtain optimal drug targets.

Expression and antigenicity analysis of Clostridium difficile glutamate dehydrogenase / 军事医学

Objective To construct prokaryotic expression vectors for glutamate dehydrogenase(GDH)of Clostridium difficile(CD),and express recombinant GDH in Escherichia coli,and identify its antigenicityed.Methods The entire gene of GDH was cloned from ATCC43255 genome DNA.The recombinant antigens were expressed in E.coli with IPTG induction and purified by Ni-NATBeads.The antigenicity was detected using CD Qick Chek Complete dual-antigen EIA. Results Prokaryotic expression vectors of CD GDH were constructed successfully.The antigen could be identified by specific anti-GDH antibodies.Conclusion The GDH antigen can be used to prepare corresponding antibodies,which facilitate the development of immunoassay for CD GDH.

Establishment and evaluation of identification method for Yersinia pestis and Yersinia pseudotuberculosis / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish a gene identification method of Yersinia pestis and Yersinia pseudotuberculosis for plague surveillance.</p><p><b>METHODS</b>According to the specific genomic sequences of Y. pestis and Y. pseudotuberculosis, i.e. "pestis Island (PeI)" and "pseudotuberculosis Island (PsI)" and the published genomic sequences of 12 strains of Y. pestis and 4 strains of Y. pseudotuberculosis, the specific identification primers of these sequences were designed.</p><p><b>RESULTS</b>A total of 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis and other intestinal bacteria strains were tested with PCR. Of the 5 pairs of Y. pestis identification primers, PeI2 and PeI11 were specific for Y. pestis. Besides Y. pestis, the primers PeI1, PeI3 and PeI12 could detect part of 57 Y. pseudotuberculosis strains. Of the 5 pairs of Y. pseudotuberculosis identification primers, PsI1 could detect all the 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis. PsI7, PsI16, PsI18 and PsI19 were specific for Y. pseudotuberculosis.</p><p><b>CONCLUSION</b>The primers PsI1, PeI 2 and PeI11, PsI7, PsI16, PsI18 and PsI19 can be used in the rapid identification of Y. pestis and Y. pseudotuberculosis, which can be also used to explore the circulation of atypical Y. pestis in quiescent plague foci.</p>

Detection and identification of Escherichia coli O157:H7 by multiplex real-time PCR / 中华微生物学和免疫学杂志

Objective To develop a rapid, sensitive and specific assay based on multiplex real-time PCR for detecting and identifying Escherichia coli O157: H7. Methods The lipopolysaccharide gene (rJbE) and H7 flagellar antigen gene(fliC) of Escherichia coli O157:H7 was chosen as targets, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and HEX fluo-resceins respectively; the 3'end of probes was labeled with MGB. The PCR reaction was optimized systemati-cally. Then the specificity, sensitivity and reproducibility of multiplex real-time PCR were estimated. Final-ly, multiplex real-time PCR was applied to detected clinical specimens. Results Escherichia coil O157:H7 were detected by multiplex real-time PCR accurately and quickly, which could distinguish Escherichia coli O157:H7 from O157: non-H7. Meanwhile, none of other bacteria could be identified. The sensitivity was 10 CFU/ml in pure culture. The coefficient of variation of intra-assay and inter-assay was less than 5%. When this assay was applied directly to identify 66 clinical specimens, the results showed that t5 were positive to Escherichia coil O157:H7 and 2 were positive to Escherichia coil O157: non-H7, in which 16 was the same to the results obtained from the conventional assays. The coincidence was 98.49%. Conclusion It is showed that multiplex real-time PCR is a reliable, accurate and feasible assay for detecting and identifying Escherich-ia coli Oi57: H7, The assay reported here provided a tool for analysis and diagnosis in the field of detecting clinical pathogens, epidemiologic survey and food safety monitoring.

Detection and identification of viable Listeria monocytogenes by real-time PCR / 中华微生物学和免疫学杂志

Objective To establish a rapid,sensitive and specific assay based on real-time PCR combined with reverse transcription for detecting and identifying viable Listeria monocytogenes.Methods The hlyA gene of Listeria monocytogenes was chosen as target,and then the primers and TaqMan probe were designed.Both ends of probe were modified with two different fluorescence groups.The PCR reaction was optimized systematically.The mRNA of Listeria monocytogenes was extracted,and then reverse transcription was performed through random primer.The cDNA Was detected by real-time PCR.Then the specificity,sensitivity and reproducibility of real-time PCR were estimated.In final,real-time PCR was applied to detect 20 mocked double-blind samplea.Results Viable Listeria monocytogenes were detected by real-time PCR accurately and quickly,and meanwhile,none of other bacteria and non-viable Listeria monocytogenes could be identified.The sensitivity was 10 CFU/ml in pure culture and 103CFU/ml for mocked samples respectively.The coefficient of variation of intra-assay and inter-assay Was less than 5%.When this assay was applied directly to identify 20 mocked double-blind samples,10 of these were positive to viable Listeria monocytogenes,5 were negative to non-viable Listeria monocytogenes,and 5 were negative to other pathogens.Conclusion It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for viable Listeria monocytogenes.The establishment of this assay provided complete data for analysis and diagnosis in the field of food safety and epidemiologic survey.