The prognostic value of toxin B and binary toxin in Clostridioides difficile infection.

To study the association between detection of the Clostridioides difficile gene encoding the binary toxin (CDT) and direct detection of toxinB (TcdB) from feces with the appearance of serious disease, complications, or recurrence in a prospective series of cases. A total of 220 confirmed cases were included, using a two-step algorithm: an initial study to detect the enzyme, glutamate dehydrogenase (GDH), followed, in cases of positivity, by detection of the tcdB. tcdB-positive patients were investigated for the presence of CDT and TcdB. Outcome variables were severe disease, the modified Illinois C. difficile infection (CDI) prognostic risk index (ZAR score), the appearance of complications (need for colectomy, CDI-related death, or toxic megacolon) and recurrence. Patients who tested positive for the presence of TcdB in feces were found to have greater disease severity than those who tested negative, with a ZAR score of 35.4% vs. 23% (p = .048), a higher recurrence rate (14.6% vs. 5.9%, p = .032), and a tendency for higher number of complications (20.7% vs. 11.5%), although without reaching statistical significance (p = .053). When presence of CDT was analyzed, higher frequencies of severe disease (39.2% vs. 21.2%, p = .005), complications and recurrence (21.6% vs. 10.9%, p = .037 and 14.9% vs. 5.8%, p = .029; respectively) were observed in patients where CDT was detected. TcdB and CDT act as prognostic markers of the appearance of serious disease, complications or recurrence in cases of CDI. Simultaneous detection of both markers, TcdB and CDT, had a greater impact on the prognosis than when they were detected separately.

Label-free detection of biotoxins via a photo-induced force infrared spectrum at the single-molecular level.

There is an increasing urge to investigate facile solutions for monitoring biotoxins, which are a major concern in both the food safety and the anti-terrorism fields. Current techniques, such as immunochromatographic tests (ICT), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry, are still insufficient to satisfy the needs for fast, label-free, and ultra-sensitive detection. Herein, a single-molecular, label-free detection method based on atomic force microscopy was employed to solve the abovementioned problem via a photo-induced force spectrum; typically, three important biotoxins, i.e. abrin toxin (ABR), ricin toxin (RT) and Clostridium perfringens exotoxin (ETX), were used for the demonstration of single molecule detection. The photo-induced force spectrum could be successfully obtained for each of the single protein particles with molecular weights down to 30 kDa. Furthermore, principal component analysis (PCA) was applied for each protein, resulting in a standard PCA identification database. Then, individual components in a mixture of these toxin proteins were well distinguished from each other via matching with the as-built database. Using this strategy, PiFM not only could be used as a powerful tool for single protein detection, but could also be used as a potential tool in protein structural analysis.

Toxigenic Clostridium difficile carriage in general practice: results of a laboratory-based cohort study.

OBJECTIVES: Reported rates of community-acquired Clostridium difficile infections (CDIs) have been increasing. However, the true burden of the disease in general practice is unknown in France. Our objective was to determine the incidence of toxigenic C. difficile carriage and the percentage of stool samples prescribed by general practitioners (GPs) which contained free C. difficile toxins. METHODS: During an 11-month period, all stool samples submitted for any enteric pathogen detection to 15 different private laboratories in Paris and the surrounding areas were tested for C. difficile, irrespective of the GPs' request. A clinical questionnaire was completed for each patient. Stool samples were screened using a rapid simultaneous glutamate dehydrogenase and toxins A/B detection test: any positive result (glutamate dehydrogenase or toxin) was further confirmed by the stool cytotoxicity assay (CTA) on MRC-5 cells and by toxigenic culture (TC) at a central laboratory. The C. difficile isolates were characterized by PCR ribotyping. RESULTS: A total of 2541 patients (1295 female, 1246 male) were included. The incidences of patients with a positive toxigenic culture and a positive CTA were 3.27% (95% CI 2.61%-4.03%) and 1.81% (95% CI 1.33%-2.41%), respectively. GPs requested C. difficile testing in only 12.93% of the stool samples, detecting 52.30% of all TC-positive patients. The 83 toxigenic C. difficile strains belonged to 36 different PCR ribotypes. CONCLUSIONS: Toxigenic C. difficile carriage is frequent in general practice but remains under-recognized. It may affect young patients without previous antimicrobial therapy or hospitalization.

Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme.

Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50ngmL-1 with the limit detection of 9.899ngmL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108CFUmL-1 in real samples with a detection limit of 320CFUmL-1.

Label-Free Optical Biodetection of Pathogen Virulence Factors in Complex Media Using Microtoroids with Multifunctional Surface Functionality.

Early detection of pathogens or their virulence factors in complex media has a key role in early diagnosis and treatment of many diseases. Nanomolar and selective detection of Exotoxin A, which is a virulence factor secreted from Pseudomonas aeruginosa in the sputum of Cystic Fibrosis (CF) patients, can pave the way for early diagnosis of P. aeruginosa infections. In this study, we conducted a preliminary study to demonstrate the feasibility of optical biodetection of P. aeruginosa Exotoxin A in a diluted artificial sputum mimicking the CF respiratory environment. Our surface engineering approach provides an effective biointerface enabling highly selective detection of the Exotoxin A molecules in the complex media using monoclonal anti-Exotoxin A functionalized microtoroids. The highly resilient microtoroid surface toward other constituents of the sputum provides Exotoxin A detection ability in the complex media by reproducible measurements. In this study, the limit-of-detection of Exotoxin A in the complex media is calculated as 2.45 nM.

miR-149 promotes human osteocarcinoma progression via targeting bone morphogenetic protein 9 (BMP9).

OBJECTIVES: To investigate the roles of miR-149 in the progression of human osteosarcoma (OS). RESULTS: miR-149 level was upregulated in tissues from OS patients more than in normal subjects. Cell proliferation and apoptosis assays revealed that miR-149 increased cell proliferation and inhibited cell apoptosis in OS cell line (MG63). An increase of Bcl-2 gene expression and a decrease of cleaved-caspase-3, and cleaved-PARP expression were observed in MG63 cells with transfection of miR-149. Additionally, bone morphogenetic protein 9 (BMP9) was identified as a target of miR-149 in MG63 cells, and BMP9 expression was negatively correlated with miR149 level in OS clinical samples. Co-overexpression of BMP9 with miR-149 in MG63 cells prohibited miR-149-mediated promotive effects on OS progression. Importantly, overexpression of miR-149 conferred chemoresistance in MG63 cells. CONCLUSIONS: miR-149 promotes OS progression via targeting BMP9.

An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines.

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

Comprehensive evaluation of chemiluminescent immunoassays for the laboratory diagnosis of Clostridium difficile infection.

For the microbiological diagnosis of a Clostridium (C.) difficile infection (CDI), a two-test algorithm consisting of a C. difficile glutamate dehydrogenase (GDH)-immunoassay followed by a toxin-immunoassay in positive cases is widely used. In this study, two chemiluminescent immunoassays (CLIAs), one for GDH and the other for the toxins A and B, have been evaluated systematically using appropriate reference methods. Three-hundred diarrhoeal stool specimens submitted for CDI diagnosis were analysed by the LIAISON CLIAs (DiaSorin). Toxigenic culture (TC) and cell cytotoxicity assay (CCTA) were used as "gold standard" reference methods. In addition, GDH and toxin A and B enzyme immunoassays (EIAs), C. diff Chek-60 and toxin A/B II (TechLab), and the Cepheid Xpert C. difficile polymerase chain reaction (PCR) were performed. C. difficile was grown in 42 (14%), TC was positive in 35 (11.7%) and CCTA in 25 (8.3%) cases. CLIAs were more sensitive but less specific than the respective EIAs. Using culture as reference, the sensitivity of the GDH CLIA was 100%. In comparison to CCTA sensitivity, specificity, positive predictive value and negative predictive value of the two-test algorithm were 88, 99.3, 91.7 and 98.9% by CLIAs and 72, 99.6, 94.7 and 97.5% by EIAs. Discrepant results by CLIAs were more frequent than that by EIAs (9% vs. 6.3%); in those cases, PCR allowed for the accurate detection of toxigenic strains. Due to performance characteristics and testing comfort, CLIAs in combination with PCR represent a favourable option for the rapid laboratory C. difficile diagnostics.

ART3 regulates triple-negative breast cancer cell function via activation of Akt and ERK pathways.

Triple-negative breast cancers (TNBCs) are defined by lack of expressions of estrogen, progesterone, and ERBB2 receptors. Because biology of TNBC is poorly understood, no targeted therapy has been developed for this breast cancer subtype and chemotherapy is its only systemic treatment modality. In this study, we firstly determined that the expression of human ecto-ADP-ribosyltransferase 3 (ART3) is significantly associated with the basal-like breast cancer subgroup, which is largely overlapped with TNBC, through analyzing published data sets. We also found that ART3 protein is significantly overexpressed in human TNBC tumors tissue and cell lines through using immunohistochemistry and immunoblotting. Overexpression of ART3 in MDA-MB-231 breast cancer cells increased cell proliferation, invasion, and survival in vitro and growth of xenograft tumors. Conversely, knockdown of ART3 in breast cancer cells inhibited cell proliferation and invasion. In addition, we showed that ART 3 overexpression activated AKT and ERK in vitro and in xenograft tumors. Together, our findings demonstrate that ART3 is a critical TNBC marker with functional significance.

Accurate detection of binary toxin producer from Clostridium difficile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Thirsty-six binary toxin producers were detected with 2 genotypes (cdtA(+) and cdtB(+)) among 265 Clostridium difficile isolates by multiplex PCR. The rate of accurate differentiation between these 2 genotypes was 100% by 6-peak cluster analysis of spectra generated by Bruker Biotyper matrix-assisted laser desorption ionization/time-of-flight mass spectrometry.

Comparison of BD Max Cdiff and GenomEra C. difficile molecular assays for detection of toxigenic Clostridium difficile from stools in conventional sample containers and in FecalSwabs.

In this study, the usability and performance of GenomEra™ C. difficile and BD Max™ Cdiff nucleic acid amplification tests (NAATs) for the detection of toxigenic Clostridium difficile were investigated in comparison with toxigenic culture and C. difficile toxin A- and toxin B-detecting immunochromatographic antigen (IA) test, the Tox A/B QuikChek®. In total, 302 faecal specimens were collected, 113 of which were in parallel to conventional sample containers and FecalSwab liquid-based microbiology (LBM) tubes. Seventy-nine specimens were considered true-positives for toxigenic C. difficile. The sensitivity and specificity were 97.5 % and 99.6 % and 93.7 % and 98.7 % for the GenomEra and BD Max assays respectively. Toxigenic culture and Tox A/B QuikChek had sensitivity and specificity of 91.1 % and 100 % and 34.2 % and 100 % respectively. Hands-on time for analysing 1 to 24 specimens using NAATs was 1 to 15 min. The rate of PCR inhibition was 0 % for both NAATs with faeces in LBM tubes, while with faeces in conventional sample containers the respective inhibition rates were 5.3 % and 4.4 % for the GenomEra and the BD Max assays. The NAATs demonstrated an excellent analytical performance, reducing significantly the overall workload of laboratory personnel compared with culture and IA test.

ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses.

Protein mono-ADP-ribosylation is a reversible post-translational modification of cellular proteins. This scheme of amino-acid modification is used not only by bacterial toxins to attack host cells, but also by endogenous ADP-ribosyltransferases (ARTs) in mammalian cells. These latter ARTs include members of three different families of proteins: the well characterised arginine-specific ecto-enzymes (ARTCs), two sirtuins, and some members of the poly(ADP-ribose) polymerase (PARP/ARTD) family. In the present study, we demonstrate that human ARTC1 is localised to the endoplasmic reticulum (ER), in contrast to the previously characterised ARTC proteins, which are typical GPI-anchored ecto-enzymes. Moreover, using the "macro domain" cognitive binding module to identify ADP-ribosylated proteins, we show here that the ER luminal chaperone GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) is a cellular target of human ARTC1 and hamster ARTC2. We further developed a procedure to visualise ADP-ribosylated proteins using immunofluorescence. With this approach, in cells overexpressing ARTC1, we detected staining of the ER that co-localises with GRP78/BiP, thus confirming that this modification occurs in living cells. In line with the key role of GRP78/BiP in the ER stress response system, we provide evidence here that ARTC1 is activated during the ER stress response, which results in acute ADP-ribosylation of GRP78/BiP paralleling translational inhibition. Thus, this identification of ARTC1 as a regulator of GRP78/BiP defines a novel, previously unsuspected, player in GRP78-mediated ER stress responses.

Clostridium difficile infection in hospitalized cancer patients in Beijing, China is facilitated by receipt of cancer chemotherapy.

The purpose of this study was to determine the presence of Clostridium difficile infection (CDI) and risk factors for infection in hospitalized patients with diarrhea in a cancer hospital in Beijing, China. A total of 277 patients with hospital-associated diarrhea (HAD) were studied of which 41 (15%) were positive for fecal C. difficile toxin A/B. For each CDI case identified, a control with HAD but negative C. difficile specimen was enrolled to look for CDI risk factors. Receipt of cancer chemotherapy occurred in 20 (49%) patients with CDI and 9 (22.0%) patients with non-CDI HAD (OR3.39, 95%CI 1.78-10.05). Median length of chemotherapy before HAD developed was 39 days for those with CDI and 22 days for patients with CDI-negative HAD (P = 0.0391). The study found that CDI is commonly seen in cancer patients in China with increasing risk for patients who receive chemotherapy.

Clostridium difficile binary toxin (CDT) and diarrhea.

Clostridium difficile is a major enteropathogen of humans. It produces two main virulence factors, toxins A and B. A third, less well known toxin, C. difficile toxin (CDT), is a binary toxin composed of distinct enzymatic (CdtA) and cell binding/translocation (CdtB) proteins. We used a novel enzyme linked immunoassay (EIA) to detect CdtB protein in feces and culture fluids. Additionally, PCR was used to assay C. difficile isolates from fecal samples for the CDT locus (CdtLoc). Although the results from 80 isolates suggest no relationship between toxin concentrations in situ and in vitro, there is a good correlation between PCR detection of the cdtB gene and EIA detection of CdtB protein in vitro. Possible implications of the detection of CDT in patients are discussed.

What is the current role of algorithmic approaches for diagnosis of Clostridium difficile infection?

With the recognition of several serious outbreaks of Clostridium difficile infection in the industrialized world coupled with the development of new testing technologies for detection of this organism, there has been renewed interest in the laboratory diagnosis of C. difficile infection. Two factors seem to have driven much of this interest. First, the recognition that immunoassays for detection of C. difficile toxins A and B, for many years the most widely used tests for C. difficile infection diagnosis, were perhaps not as sensitive as previously believed at a time when attributed deaths to C. difficile infections were showing a remarkable rise. Second, the availability of FDA-approved commercial and laboratory-developed PCR assays which could detect toxigenic strains of C. difficile provided a novel and promising testing approach for diagnosing this infection. In this point-counterpoint on the laboratory diagnosis of C. difficile infection, we have asked two experts in C. difficile infection diagnosis, Ferric Fang, who has recently published two articles in the Journal of Clinical Microbiology advocating the use of PCR as a standalone test (see this author's references 12 and 28), and Mark Wilcox, who played a key role in developing the IDSA/SHEA guidelines on Clostridium difficile infection (see Wilcox and Planche's reference 1), along with his colleague, Tim Planche, to address the following question: what is the current role of algorithmic approaches to the diagnosis of C. difficile infection?

[Expression and implication of base excision repair genes in human nasopharyngeal carcinoma and non-tumor nasopharyngeal tissues].

BACKGROUND & OBJECTIVE: Base excision repair (BER) genes play important roles in maintaining genomic stability and their abnormal expression are associated with several cancers. This study was to investigate the expression of 7 important BER genes (hOGG1, ADPRT, APE1, MBD4, POLB, XRCC1 and LIG3) in nasopharyngeal carcinoma (NPC) and non-tumor nasopharyngeal tissues, and evaluate their clinical significance. METHODS: The expression of hOGG1, ADPRT, APE1, MBD4, POLB, XRCC1 and LIG3 in 24 specimens of NPC and 24 specimens of non-tumor nasopharyngeal tissues was detected by reverse transcription-polymerase chain reaction (RT-PCR). The differential expression of hOGG1 and ADPRT was further detected by immunohistochemistry in 99 specimens of NPC and 28 specimens of non-tumor nasopharyngeal tissues. RESULTS: hOGG1, ADPRT, APE1, MBD4, POLB, XRCC1 and LIG3 were expressed in both NPC and non-tumor nasopharyngeal tissues. Among them, the mRNA levels of hOGG1 and ADPRT were significantly lower in NPC than in non-tumor nasopharyngeal tissues (P<0.001). The protein levels of hOGG1 and ADPRT in NPC were also reduced. The high expression rates of hOGG1 were 50.5% in NPC and and 92.8% in non-tumor nasopharyngeal tissues (P<0.001), and those of ADPRT were 53.5% and 96.4%, respectively (P<0.001). However, the expression levels of hOGG1 and ADPRT had no correlations to the clinical stage and prognosis of NPC. CONCLUSION: The decreased expression of hOGG1 and ADPRT might be closely related to the development of nasopharyngeal carcinoma.

Comparison of phenotypic and genotypic methods for the detection of environmental isolates of Pseudomonas aeruginosa.

During remediation processes, biological monitoring should be generally required. Hydrocarbon contaminated soils may provide favorable conditions for several opportunistic pathogenic microorganisms, thereby increasing their populations over risky levels. Therefore, during remediation processes of the subsurface medium biological monitoring is of prime importance. The accuracy, time and cost efficiency of the relevant identification method are major factors while monitoring these microbes. During previous years (2002-05), we collected 68 soil samples from 17 different oil contaminated sites, such as petrol stations, airfields and pipeline-breaks. We compared frequently applied detection methods of Pseudomonas aeruginosa, both traditional microbiological and molecular biological techniques, on 43 environmental isolates originating from these sites. The following methods were subjected to comparative analysis: (i) the Hungarian Standard method; (ii) the method described in "The Prokaryotes" handbook; (iii) the API 20NE biochemical fingerprinting, as well as PCR protocols aimed to amplify; (iv) the exotoxin-A gene; and (v) the 16S rDNA variable regions V2 and V8. In five cases, phenotypic methods gave false-negative results. 16S rDNA sequence analysis was done to confirm the identity of these five strains, which verified the results of molecular methods. In addition, faults were found in the evaluation of the originally described ETA PCR protocol, which was corrected by us.

Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system.

We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.