Cell Culture Cytotoxicity Assay Research Articles

Biofilm Expressing Genes in Bacteria Causing Urinary Catheter Associated Infections

Biofilm associated infection causes higher mortality in catheterized patients. Identification of cupA gene in Pseudomonas aeruginosa coding for biofilm formation was selected as a primary objective. Synergistic antibacterial drugs (moxifloxacin-metronidazole) were prepared using the selected concentrations for coatings onto urinary catheter samples. Antibacterial coatings of synergistic antibacterial drugs onto a urinary catheter and its antibacterial activity were evaluated using the standard diffusion method. The drug coated catheters were investigated for their biocompatible properties. The biocompatibility of the drug coated samples was investigated on L929 fibroblast cell lines using MTT assay. The biofilm expressing genes (cupA gene) of the selected organism was detected using molecular methods. During the analysis, the cupA gene from the target organism was confirmed by the PCR method aided with the DNA electrophoresis process. The study was confirmed from the obtained DNA fragments of 172bp by comparing with 170bp fragment of reference bacterial strain. Quantitative antibacterial activity results showed that the drug coated catheter samples were significantly reduced the bacterial numbers. Biocompatibility of coated catheter samples evaluated by in-vitro cell culture cytotoxicity assay exhibited no effect on cell viability of fibroblast cell lines. It is concluded that identifying biofilm expressing genes could help the health care workers to diagnose and treat the patients with an appropriate antibacterial agent.

Anticancer Effects & Comparative Study of Thymoquinone, Cordyceps, Spirulina, Ganoderma Lucidium, Poria Cocos, and Lion’s Mane on Human Nasal Cancer Cells (RPMI-2650)

Recently, many studies have been conducted to discover or improve cancers treatment. The current study aims to investigate the anticancer effect of thymoquinone, cordyceps, spirulina, ganoderma lucidium, poria cocos, and lion’s mane in four different concentrations 4, 8, 16, and 32 ug (equivalent to 1 mg/mL) in two different time treatments (48 and 96 hours) on human nasal epithelial cell line RPMI 2650. By using cell culture cytotoxicity techniques and assay, the highest anticancer effect on RPMI 2650 was obtained by thymoquinone. The lowest anticancer effect was demonstrated by poria cocos and cordyceps. However, these two medications showed higher anticancer effect when given in short-term treatment (48 hours) compared to long-term treatment (96 hours). Ganoderma lucidium and spirulina showed better impact than poria cocos, cordyceps, and lion’s mane in term of cells cytotoxicity. Mild to moderate antineoplastic effect was seen by utilizing lion’s mane treatment compared other drugs. Therefore, adopting a long-term treatment of high concentrations and doses of thymoquinone, cordyceps, spirulina, ganoderma lucidium, poria cocos, and lion’s mane can be more effective in the treatment of nasal cancer. In conclusion, these drugs were found to be a promising cancer remedy; therefore, they can be utilized as alternative treatment for nasal cancer or any other type of cancer therapy.

Investigation of community-associated Clostridium (Clostridioides) difficile infections in the Asia-Pacific region

Background: Clostridium (Clostridioides) difficile is the most common cause of nosocomial diarrhoeal infections in high-income countries, however, community-associated (CA)-C. difficile infections (CDIs) now make up about 30% of all CDIs. Antimicrobial use and advanced age are the most common risk factors for CDI, although CA-CDI often affects younger patients. Little is currently known about the epidemiology of CA-CDI in the Asia-Pacific region. Methods and materials: A prospective study of CDI in hospital inpatients was conducted from March 2014 to January 2015 at 70 sites in 13 Asia-Pacific countries. Recruited cases (n = 600) had ≥3 episodes of diarrhoea in a 24 h period and diagnosis of CDI by toxin enzyme immunoassay, cell culture cytotoxicity assay, real-time PCR or toxigenic culture. Demographic and clinical data were collected, stool samples were cultured for C. difficile, and PCR ribotyping of isolates was performed. Proportions were compared by Chi-square test for CA-CDI (no hospitalisation in previous 12 w) vs hospital-associated (HA)-CDI (discharged from hospital in previous 4 w) cases identified in the study. A generalised linear mixed model regression was performed to find risk factors for CA-CDI. Results: HA-CDI was determined in 350 (65.4%) and CA-CDI in 74/535 (13.8%) cases aged ≥2 y. The median age of CA-CDI cases was lower at 53.5 y vs 66.0 y in HA-CDI cases (p < 0.01). Recent antimicrobial use was less frequent among CA-CDI cases (63.5% vs 80.0%, p < 0.01) while death and recurrent CDI were rarer outcomes (1.7% vs 5.8% and 5.0% vs 9.8%, p > 0.05). The most common ribotypes (RTs) causing CA-CDI were RT 017 (17.0%) followed by RTs 012 (14.6%, primarily in China), 014/020 (9.8%, predominantly in Australia and China), and RTs 002, 018 and 070 (all 7.3%). Inflammatory bowel disease (odds ratio [OR] 5.26, p < 0.05) and infection with RTs 012 (OR 5.44, p < 0.05) and 070 (OR 6.19, p < 0.05) were significantly associated with CA-CDI. Conclusion: CA-CDI patients in the Asia-Pacific region were significantly younger and had less frequent history of antimicrobial use compared to HA-CDI patients. RTs 012 and 070 were associated with CA-CDI, highlighting a need for investigation of community-based reservoirs of C. difficile including animals and outdoor environments.

2368. Molecular Epidemiology of Clostridioides difficile Infections in Pediatric Oncology and Transplant Patients

BackgroundThe incidence of Clostridioides difficile infection (CDI) has been rising among children, both in the community and hospital setting. The genomic variability and molecular epidemiology of CDI in children, especially with cancer are poorly understood. We aim to evaluate the molecular epidemiology and relatedness of C. difficile isolates among inpatient and outpatient pediatric oncology and stem cell transplant patients (POTP) using whole-genome sequencing (WGS).MethodsThis was a retrospective cohort study of CDI in POTP in 2016. CDI cases were identified by electronic medical record search for positive C. difficile toxin PCR tests. Retrieved residual stool specimens were cultured anaerobically using pre-reduced CCMB-TAL broth (Anaerobe Systems, Inc.), toxin-producing C. difficile isolates were determined using a cell culture cytotoxicity assay with neutralization (TechLab, Inc.), and WGS was performed, followed by core genome MLST (cgMLST). Variability of the isolates was summarized by strain type (ST), and a minimum spanning tree was constructed, defining clusters of genomically related isolates as those with < 7 alleles difference. Plausible epidemiologic links among the closely related strains such as receiving care on the same inpatient unit on the same day or on the same unit but on different days within 8 weeks before CDI were evaluated to identify potential transmission events.ResultsAmong 141 CDI episodes in 89 patients; 103 stool samples were cultured, and 101 (98%) isolates were sequenced identifying 23 different strain types in 81 (80%) isolates. 34 (38%) patients had multiple episodes of CDI. 16 clusters of related isolates were identified (figure), 10 (62%) of which involved only multiple specimens from the same patient. For the 6 clusters involving multiple patients, epidemiologic investigation revealed only 2 (33%) potential transmission events.ConclusionWGS identified a highly diverse group of C. difficile isolates among POTP with CDI. Although WGS identified clusters of closely related isolates in multiple patients, epidemiologic investigation of shared inpatient exposures identified potential transmission in only two clusters. C. difficile transmission was uncommon. DisclosuresRandall Hayden, MD, Abbott Molecular: Advisory Board; Quidel: Advisory Board; Roche Diagnostics: Advisory Board.

2400. Are Multiple Clostridioides difficile Infections in Pediatric Oncology Patients Related to Recurrence of the Same Isolate or Re-infections with Different isolates?

BackgroundPediatric oncology and hematopoietic stem cell transplant patients (POTP) are at increased risk for Clostridioides difficile infection (CDI) and recurrence. It is unknown whether recurrent CDI is related to the same C. difficile strain as the initial CDI. We describe genomic relatedness of C. difficile strains in patients with multiple CDI using whole-genome sequencing (WGS).MethodsThis was a retrospective cohort study of CDI in POTP in 2016. CDI cases were identified by electronic medical record search for positive C. difficile toxin PCR tests. Patients with multiple CDI episodes were identified. CDI episodes were classified as incident, duplicate or recurrent using National Healthcare Safety Network (NHSN) definitions. Retrieved residual stool specimens were cultured anaerobically, toxin-producing C. difficile isolates were determined using a cell culture cytotoxicity assay with neutralization and WGS was performed followed by core genome MLST (cgMLST). Variability of the isolates was summarized by strain type (ST), and a minimum spanning tree was constructed, defining genomically related isolates as those with <7 allele difference.ResultsEighteen patients had 51 positive C. difficile samples. CDI were classified as incident in 31 (61%) episodes, recurrent in 18 (36%), and duplicate in 2 (3%). Isolates from 47 (92%) samples were sequenced, identifying 14 different strain types (ST) in 41 (87%) isolates. Of the 31 incident CDI, 13 (42%) episodes occurred 8 weeks or more after the initial incident CDI. Among those 13 incident CDI, 7 (54%) had prior CDI due to a related isolate. Of the 18 recurrent CDIs, 10 (55%) were due to an isolate related to the previous incident CDI and 5 (28%) were due to an isolate unrelated to the incident CDI. The relatedness of the remaining 3 recurrent episodes could not be evaluated because no isolate was available for sequence analysis.ConclusionCDI classification of incident vs. recurrent infection using NHSN definition might be not applicable in POTP. WGS showed that more than half of CDI episodes classified as incident were actually a recurrence of a previous C. difficile strain. Similarly, some CDI episodes classified as recurrent were actually re-infection with a different stain.DisclosuresRandall Hayden, MD, Abbott Molecular: Advisory Board; Quidel: Advisory Board; Roche Diagnostics: Advisory Board

Influence of Food Matrices on the Stability and Bioavailability of Abrin.

Abrin, a highly toxic plant toxin, is a potential bioterror weapon. Work from our laboratory and others have shown that abrin is highly resistant to both thermal and pH inactivation methods. We sought to evaluate the effectiveness of selected food processing thermal inactivation conditions against abrin in economically important food matrices (whole milk, non-fat milk, liquid egg, and ground beef). The effectiveness of toxin inactivation was measured via three different assays: (1) In vitro cell free translation (CFT) assay, (2) Vero cell culture cytotoxicity; and the in vivo mouse intraperitoneal (ip) bioassay. For both whole and non-fat milk, complete inactivation was achieved at temperatures of 80 °C for 3 min or 134 °C for 60 s, which were higher than the normal vat/batch pasteurization or the high temperature short time pasteurization (HTST). Toxin inactivation in liquid egg required temperatures of 74 °C for 3 min higher than suggested temperatures for scrambled eggs (22% solids) and plain whole egg. Additionally, the ground beef (80:20%) matrix was found to be inhibitory for full toxin activity in the mouse bioassay while retaining some activity in both the cell free translation assay and Vero cell culture cytotoxicity assay.

Abrin Toxicity and Bioavailability after Temperature and pH Treatment.

Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin’s toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin’s ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin’s ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays.

Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.

Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

Biotin-encoded Pullulan-Retinoic Acid Engineered Nanomicelles: Preparation, Optimization and In Vitro Cytotoxicity Assessment in MCF-7 Cells

Biotin and retinoic acid grafted pullulan conjugate was synthesized using carbodiimide activation ester bond formation strategy for biotin targeted delivery of doxorubicin in breast cancer chemotherapy using micelle formulation. The conjugate structure was evaluated and confirmed by proton nuclear magnetic resonance and Fourier transform infrared spectroscopy. Critical micelle concentration of final conjugate was determined using the pyrene fluorescence method. Doxorubicin-loaded micelles were prepared using the direct dissolution method. Physical parameters of optimized micelles comprising particle size, zeta potential, drug loading efficacy, and drug release profile were determined. The results reveal that the nanomicelles have formed with mean particle size of 191 nm and zeta potential of -9.45 mv. The drug loading efficacy of 92% and release efficiency of 81% during 24 h, are also obtained. The structure of the micelles was studied by transition electron microscopy technique. The cytotoxicity of doxorubicin-loaded micelles was studied in MCF-7 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results of in vitro cell culture cytotoxicity assay showed that the doxorubicin-loaded biotin grafted retinoic acid-pullulan micelles were more cytotoxic in comparison to the non-targeted pullulan and free doxorubicin.

Real-Time Polymerase Chain Reaction Detection of Asymptomatic Clostridium difficile Colonization and Rising C. difficile–Associated Disease Rates

To evaluate the accuracy of real-time polymerase chain reaction (PCR) for Clostridium difficile-associated disease (CDAD) detection, after hospital CDAD rates significantly increased following real-time PCR initiation for CDAD diagnosis. Hospital-wide surveillance study following examination of CDAD incidence density rates by interrupted time series design. Large university-based hospital. Hospitalized adult patients. CDAD rates were compared before and after real-time PCR implementation in a university hospital and in the absence of physician and infection control practice changes. After real-time PCR introduction, all hospitalized adult patients were screened for C. difficile by testing a fecal specimen by real-time PCR, toxin enzyme-linked immunosorbent assay, and toxigenic culture. CDAD hospital rates significantly increased after changing from cell culture cytotoxicity assay to a real-time PCR assay. One hundred ninety-nine hospitalized subjects were enrolled, and 101 fecal specimens were collected. C. difficile was detected in 18 subjects (18%), including 5 subjects (28%) with either definite or probable CDAD and 13 patients (72%) with asymptomatic C. difficile colonization. The majority of healthcare-associated diarrhea is not attributable to CDAD, and the prevalence of asymptomatic C. difficile colonization exceeds CDAD rates in healthcare facilities. PCR detection of asymptomatic C. difficile colonization among patients with non-CDAD diarrhea may be contributing to rising CDAD rates and a significant number of CDAD false positives. PCR may be useful for CDAD screening, but further study is needed to guide interpretation of PCR detection of C. difficile and the value of confirmatory tests. A gold standard CDAD diagnostic assay is needed.

Study of microfluidic cell chip for the toxicity test of artemisinin on Hep-G2 cells

A novel microfluidic chip composed of a 6 × 6 array of cell culture microchambers was designed and fabricated in this article. The microchip analytical system combined with the designed microchip, measuring device and environmental control unit was established for cell culture and parallel drug cytotoxicity assay. Human hepatoma cell Hep-G2 and artemisinin were taken as the objective samples in the experiments. Hep-G2 cells were successfully cultured in the microchip for over 72 h. Moreover, the viability percentage of the Hep-G2 cells was higher than 90% with staining of trypan blue. The Hep-G2 cells were exposed to the artemisinin solutions at different concentrations (0, 10, 25, 50, 100 and 150 µmol L−1) for 12, 24 and 48 h. The viability of the Hep-G2 cells in chambers was measured by observing cellular morphology and detecting fluorescence intensity. Results showed that artemisinin could inhibit the viability of Hep-G2 cells, which depended on the dose of artemisinin and action time. The microchip analytical system could not only solve the shortcomings of conventional in vitro cell culture, such as labor intensive, reagent consuming and time consuming, but also improve great potential for the study of cell biology in the directions of miniaturization, automation, high efficiency and high sensitivity.

Correlation between Clostridium difficile Bacterial Load, Commercial Real-Time PCR Cycle Thresholds, and Results of Diagnostic Tests Based on Enzyme Immunoassay and Cell Culture Cytotoxicity Assay

The impact of Clostridium difficile fecal loads on diagnostic test results is poorly understood, but it may have clinical importance. In this study, we investigated the relationship between C. difficile fecal load and the results of four assays: a glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), a toxin A/B antigen EIA (ToxAB), a cell culture cytotoxicity assay (CCA), and PCR targeting the tcdB gene. We also compared the PCR cycle threshold (CT) with the results of quantitative culture using Spearman's rank correlation coefficient. Finally, we sequenced the genomes of 24 strains with different detection profiles. A total of 203 clinical samples harboring toxigenic C. difficile were analyzed and sorted into one of four groups: 17 PCR(+) (group 1), 37 PCR(+) GDH(+) (group 2), 24 PCR(+) GDH(+) CCA(+) (group 3), and 125 PCR(+) GDH(+) ToxAB(+) (group 4). The overall median fecal load in log10 CFU/g was 6.67 (interquartile range [IQR], 5.57 to 7.54). The median fecal bacterial load of groups 1, 2, 3, and 4 were 4.15 (IQR, 3.00 to 4.98), 5.74 (IQR, 4.75 to 6.16), 6.20 (IQR, 5.23 to 6.80), and 7.08 (IQR, 6.35 to 7.83), respectively. Group 1 samples had lower fecal loads than those from each of the other groups (P < 0.001). Group 2 samples had lower fecal loads than those from groups 3 and 4 (P < 0.001). There was a significant correlation between PCR CT and fecal loads (ρ = -0.697; P < 0.001). NAP1 strains were associated with the detection of toxins by EIA or CCA (P = 0.041). This study demonstrates an association between C. difficile fecal load and the results of routinely used diagnostic tests.

Clostridium difficile Infection: Epidemiology, Risk Factors, Pathogenesis, Clinical Features, Diagnosis and Therapy

Clostridium difficile is a gram-positive, spore-forming anaerobic bacterium. C.difficile is the leading cause of antibiotic associated diarrhea and colitis. The clinical spectrum of C.difficile infection (CDI) is highly variable, ranging from mild diarrhea to severe forms of intestinal illness including toxic megacolon, ileus, bowel perforation, and pseudomembranous colitis. Advanced age, long duration of hospitalization, and exposure to certain antimicrobial agents are the most common risk factors for CDI. The main virulence determinants of C.difficile are toxin A (enterotoxin) and toxin B (cytotoxin). Diagnosis of CDI is based on the identification of C.difficile toxin A or B in diarrheal stool. Various laboratory tests have been currently developed for the detection of C.difficile or its toxins in stool samples. The cell culture cytotoxicity assay and toxigenic culture have been regarded as the reference standard methods for the detection of C.difficile toxins. However, both of the reference methods are laborious, time consuming, and need expert personnel. Therefore, many microbiology laboratories use enzyme immunoassays, glutamate dehydrogenase antigen tests and real-time polymerase chain reaction (PCR) which are more rapid and practical. First-line antibiotics for CDI treatment are metronidazole and vancomycin. In this review, epidemiology, clinical spectrum, risk factors, pathogenesis, diagnostic methods and treatment of CDI have been summarized.

Diagnosis and Management of Clostridium difficile Infection

Clostridium difficile infection (CDI) is increasing in frequency and severity in and out of the hospital, with a high probability of recurrence after treatment. The recent literature on CDI was reviewed using PubMed to include recent publications dealing with diagnosis and therapy. Real-time polymerase chain reaction is a sensitive and useful diagnostic test for CDI but there are growing concerns of false-positive test results if the rate of CDI is low in the patient population providing samples and/or if the population being studied commonly includes people with C difficile colonization. Recommended therapy of CDI includes oral metronidazole for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases, each given for 10 days. Colectomy is being performed more frequently in patients with fulminant CDI. For treatment of first recurrences the drug used in the first bout can be used again and for second recurrences longer courses of vancomycin often are given in a tapered dose or intermittently to allow gut flora reconstitution, or other treatments including fidaxomicin may be used. Bacteriotherapy with fecal transplantation is playing an increasing role in therapy of recurrent cases. Metagenomic studies of patients with CDI during successful therapy are needed to determine how best to protect the flora from assaults from antibacterial drugs and to develop optimal therapeutic approaches. Immunotherapy and immunoprophylaxis offer opportunities to prevent CDI, to speed up recovery from CDI, and to eliminate recurrent infection. Humanized monoclonal antitoxin antibodies and active immunization with vaccines against C difficile or its toxins are both in development and appear to be of potential value.

Impact of the Type of Diagnostic Assay on Clostridium difficile Infection and Complication Rates in a Mandatory Reporting Program

Most Clostridium difficile infection (CDI) surveillance programs neither specify the diagnostic method to be used nor stratify rates accordingly. We assessed the difference in healthcare-associated CDI (HA-CDI) incidence and complication rates obtained by 2 validated diagnostic methods. This was a prospective cohort study of patients for whom a C. difficile test was ordered between 1 August 2010 and 31 July 2011. All specimens were tested in parallel by a commercial polymerase chain reaction (PCR) assay targeting toxin B gene tcdB, and a 3-step algorithm detecting glutamate dehydrogenase and toxins A and B by enzyme immunoassay and cell culture cytotoxicity assay (EIA/CCA). CDI incidence rate ratios were calculated using univariate Poisson regression. A total of 1321 stool samples were tested during a period totaling 95 750 patient-days. Eighty-five HA-CDI cases were detected by PCR and 56 cases by EIA/CCA (P = .01). The overall incidence rate was 8.9 per 10 000 patient-days (95% confidence interval [CI], 7.1-10.9) by PCR and 5.8 per 10 000 patient-days (95% CI, 4.4-7.4) by EIA/CCA (P = .01). The incidence rate ratio comparing PCR and EIA/CCA was 1.52 (95% CI, 1.08-2.13; P = .015). Overall complication rate was 27% (23/85) when CDI was diagnosed by PCR and 39% (22/56) by EIA/CCA (P = .16). Cases detected by PCR only were less likely to develop a complication of CDI compared with cases detected by both PCR and EIA/CCA (3% vs 39%, respectively; P < .001). Performing PCR instead of EIA/CCA is associated with a >50% increase in the CDI incidence rate. Standardization of diagnostic methods may be indicated to improve interhospital comparison.

Clostridium difficile: current approaches to laboratory testing

<i>Clostridium difficile</i> is the main aetiological agent of antibiotic-associated diarrhoea and pseudomembranous colitis. Infections with this organism have evolved from nuisance complications of antibiotic therapy to severe, sometimes fatal, events that are the scourge of hospitals worldwide. In the past decade there has been an increase in the incidence of <i>C. difficile</i> infection (CDI), coinciding with the emergence and spread of the hypervirulent ribotype 027 strain. The optimal laboratory diagnosis of CDI remains an area of complexity, with the availability of multiple tests with different <i>C. difficile</i> targets. Traditional tests have been based on the detection of the organism by culture and demonstration of toxin by either enzyme immunoassays or cell culture cytotoxicity assays. Recently, molecular assays targeting toxin genes present in <i>C. difficile</i> have become commercially available which deliver rapid results with a high degree of sensitivity and specificity but with a higher cost. Given the wide variety of tests for CDI available, there are multiple two- and three-step algorithms available, but the practicability of these will vary according to local expertise, facilities and finance.

Australasian Society for Infectious Diseases guidelines for the diagnosis and treatment of Clostridium difficile infection

Clostridium difficile is the most common cause of health care-associated and antibiotic-associated diarrhoea. These guidelines are intended to provide advice to clinicians on the clinical assessment, diagnosis and management of C. difficile infection (CDI). Hypervirulent strains of C. difficile, including PCR ribotype 027 strains recently identified in Australia, have been associated elsewhere with epidemic spread and high rates of severe disease and death. Diagnostic tests include stool culture, polymerase chain reaction-based assays, cell-culture cytotoxicity assays and enzyme immunoassays detecting C. difficile glutamate dehydrogenase, and/or toxin A and/or B. To treat an initial episode and a first recurrence, metronidazole is the preferred antibiotic, with oral vancomycin reserved for severe disease and subsequent recurrences. Surgery should be considered for fulminant disease.

Using an insect model to assess correlation between temperature and virulence in Bacillus weihenstephanensis and Bacillus cereus

The closely related bacterial species Bacillus cereus and Bacillus weihenstephanensis are adapted to the mesophilic and the psychrotrophic temperature range, respectively. While B. cereus strains are associated with foodborne diseases, B. weihenstephanensis strains are so far not, although similar virulence genes are found in both species. Our investigations show that both species were virulent in the insect model, Galleria mellonella, following infection via oral and haemocoel routes. However, virulence of B. weihenstephanensis was much higher at 15°C than at 37°C. Furthermore, a temperature-dependent difference between the species was seen in a cell culture cytotoxicity assay. In summary, our results demonstrate for the first time virulence of B. weihenstephanensis strains in an in vivo model. In addition, we found that G. mellonella is a useful model for studies of the psychrotolerant species of the B. cereus group, suggesting that insects might be an ecological growth niche for several members of this bacterial group.

Repeat PCR for Diagnosing

Methods for diagnosing Clostridium difficile infection include anaerobic toxigenic stool culture, cell culture cytotoxicity assay, enzyme immunoassay,

Frequency of Sample Submission for Optimal Utilization of the Cell Culture Cytotoxicity Assay for Detection of Clostridium difficile Toxin

We reviewed the results of repeated sample submissions within a 7-day time frame for Clostridium difficile toxin testing. A total of 2,940 samples were tested during a 3-month period using a cell culture cytotoxicity assay (CCCA). The results from all second samples (n = 1,101) were concordant with the original test result. In only two cases (0.8%; n = 247) was a third sample positive when the first two samples were negative. In this study, submission of multiple samples for CCCA did not increase detection of Clostridium difficile infection.