Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Comparison of ChromID Agar and Clostridium difficile Selective Agar for Effective Isolation of C. difficile from Stool Specimens

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.

Genotyping of Giardia duodenalis Isolates from Dogs in Guangdong, China Based on Multi-Locus Sequence

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.

First genotyping of Giardia lamblia from human and animal feces in Argentina, South America

The purpose of this study was to investigate the genotypes of Giardia lamblia from human and animal feces and their epidemiological and clinical characteristics in Argentina, South America. Seventy isolates, 60 from humans (adults and children), eight from dogs and two from cows were processed by polymerase chain reaction-restriction fragment length polymorphism. Data corresponding to demographic, socio-cultural and environmental variables and presence/absence of signs/symptoms were collected. The triosephosphate isomerase gene was amplified from 43 (71.66 percent) of the 60 human fecal samples. Among these, 3/43 (6.98 percent) were genotype AII and 40/43 (93.02 percent) were genotype B. Assemblage AII was detected in three children who lived together in a shantytown and they were oligosymptomatic and none had diarrhea. This genotype was not found in animals. Genotype B showed a high prevalence in both adults and children. It was also found in polysymptomatic people, many of whom presented diarrhea. It was also found only in one dog. The present study represents the first contribution to the knowledge of G. lamblia genotypes in Argentina.

The protective immunity of a DNA vaccine encoding Schistosoma japonicum Chinese strain triose-phosphate isomerase in infected BALB/C mice.

The development of a DNA vaccine for schistosomiasis japonica and testing the protective efficacy after challenge in BALB/c mice were performed. Thirty-nine female BALB/c mice were divided into three groups. Each mouse of the control group was injected intramuscularly with 100 microg of pcDNA3.1 DNA. In the TPI group, each mouse was injected with 100 microg of pcDNA3.1-SjCTPI DNA. The TPI+IL-12 group was injected with 100 microg of pcDNA3.1-SjCTPI DNA and 100 microg of the mixture of pcDNA3.1-P35 and pcDNA3.1-P40 DNA. Each mouse was immunized three times at two-week intervals and challenged with 45 cercariae of Schistosoma japonicum Chinese strain four weeks post-immunization. Then the mice were sacrificed and perfused at 45 days after challenge; the recovered worms and hepatic eggs were counted. Cytotoxic T lymphocyte (CTL) activity mediated by SjCTPI was detected with the 51Cr release assay. ELISA was performed for the detection of anti-rTPI antibodies. Anti-rTPI antibody detection with ELISA after immunization showed ten serum samples from the control group were negative, five of ten serum samples from the TPI group were weakly positive, six of ten from the TPI+IL-12 group were also weakly positive. The CTL activity of the control group was 9.1%, while CTL activities of the TPI group and the TPI+IL-12 group were 27.6% and 54.4%, respectively. The worm and egg reduction rates of TPI group and the TPI+IL-12 group were 30.2%, 52.9%, 32.7%, and 47.0%, respectively in comparison with the control group. This study further proved the possibility of the SjCTPI DNA vaccine as a potential DNA vaccine for schistosomiasis.

The protective immunity produced in infected C57BL/6 mice of a DNA vaccine encoding Schistosoma japonicum Chinese strain triose-phosphate isomerase.

The development of a SjCTPI DNA vaccine for Schistosoma japonicum and the detection of the immune responses to and the protective efficacy of immunization were performed and challenged in C57BL/6 mice. According to the gene sequence of SjCTPI and murine IL-12, three pairs of primers were designed. The full length cDNA encoding SjCTPI and P35, P40 amplified from pUC19-SjCTPI and murine IL-12 by PCR were subcloned into an eukaryotic expression vector (pcDNA3.1). Forty-five female C57BL/6 mice were divided into three groups; each mouse of the control group was injected with 100 pg of pcDNA3.1 by i.m. route; the TPI group was injected with 100 microg of pcDNA3. 1-SjCTPI; the TPI+IL- 12 group was injected with 100 microg of pcDNA3.1-SjCTPI and 100 pg of mixture of pcDNA3.1-P35 and pcDNA3.1-P40. Each mouse was immunized at weeks 1 and 5 and challenged with 45 cercariae of Schistosoma japonicum Chinese strain at week 9. The mice were killed and perfused 45 days after challenge; the numbers of recovered worms and hepatic eggs were counted. The expression of SjCTPI in muscle tissue was determined by an immunohistochemical method. Culture of spleen cells showed the production of IL-2, IL-4, IL-10 and IFN-gamma with the stimulation of specific antigen before and after challenge. Sera were collected from each group before immunization, before challenge and two weeks post challenge; ELISA and Western-blot tests were performed for detection of anti-rTPI antibodies. The antigen of SjCTPI was expressed in the membrane and plasma of the muscle cells of C57BL/6 mice. The obvious rising of IL-2 in TPI group and TPI+IL-12 group before and after challenge was seen. The anti-rTPI antibody detection with Western-blot showed that ten serum samples from the control group were negative; nine of ten serum samples from the TPI group were weakly positive, eight of ten from the TPI+IL-12 group were weakly positive. The worm and egg reduction rates of TPI group and TPI+IL- 12 group were 27.9% and 13.7%, 31.9% and 18.6% respectively in comparison with the pcDNA group. pcDNA3.1-TPI DNA vaccine could confer partial protection against a subsequent challenge of Schistosoma japonicum in C57BL/6 mice and might therefore be a potential DNA vaccine.