Kerrie Davies

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection

Summary Background Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. Methods In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12 420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. Findings Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12–2·31). Multistage algorithms performed better than did standalone assays. Interpretation We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection. Funding Department of Health and Health Protection Agency, UK.

Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013.

Clostridium difficile infection (CDI) is the major cause of infective diarrhoea in healthcare environments. As part of the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), the largest C. difficile epidemiological study of its type, PCR ribotype distribution of C. difficile isolates in Europe was investigated. PCR ribotyping was performed on 1,196 C. difficile isolates from diarrhoeal samples sent to the European coordinating laboratory in 2012-13 and 2013 (from two sampling days) by 482 participating hospitals from 19 European countries. A total of 125 ribotypes were identified, of which ribotypes 027 (19%, n =222), 001/072 (11%, n = 134) and 014/020 (10%, n = 119) were the most prevalent. Distinct regional patterns of ribotype distribution were noted. Of 596 isolates from patients with toxin-positive stools (CDI cases), ribotype 027 accounted for 22% (32/144) of infections in cases aged from 18 to less than 65 years, but the prevalence decreased in those aged ≥ 65 years (14% (59/412)) and further decreased in those aged ≥ 81 years (9% (18/195)). The prevalence of ribotype 027 and 176, but not other epidemic strains, was inversely proportional to overall ribotype diversity (R(2) = 0.717). This study highlights an increased diversity of C. difficile ribotypes across Europe compared with previous studies, with considerable intercountry variation in ribotype distribution. Continuous surveillance programmes are necessary to monitor the changing epidemiology of C. difficile.

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant.

Comparison of the Vidas C. difficile GDH Automated Enzyme-Linked Fluorescence Immunoassay (ELFA) with Another Commercial Enzyme Immunoassay (EIA) (Quik Chek-60), Two Selective Media, and a PCR Assay for gluD for Detection of Clostridium difficile in Fecal Samples

ABSTRACT Prevention and management of Clostridium difficile infection (CDI) can be improved by rapid and reliable diagnostics. The Vidas C. difficile glutamate dehydrogenase assay had performance comparable to that of the Quik Chek-60 assay (overall agreement, 95%) and a sensitivity of >93%; thus, it is suitable as the first test in two-stage algorithms for a CDI diagnosis.

Ribotypes and New Virulent Strains Across Europe

Clostridium difficile is a major bacterial cause of post-antibiotic diarrhoea. The epidemiology of C. difficile infections (CDI) has dramatically changed since the early 2000s, with an increasing incidence and severity across Europe. This trend is partly due to the emergence and rapid worldwide spread of the hypervirulent and epidemic PCR ribotype 027. Profiles of patients with CDI have also evolved, with description of community-acquired (CA) infections in patients with no traditional risk factors for CDI. However, recent epidemiological studies indicated that some European countries have successfully controlled the dissemination of the 027 clone whereas other countries recently reported the emergence of other virulent or unusual strains. The aims of this review are to summarize the current European CDI epidemiology and to describe the new virulent C. difficile strains circulating in Europe, as well as other potential emerging strains described elsewhere. Standardized typing methods and surveillance programmes are mandatory for a better understanding and monitoring of CDI in Europe.

Patient and Strain Characteristics Associated With Clostridium difficile Transmission and Adverse Outcomes

Combining epidemiological, clinical, and genomic data for Clostridium difficile cases offers fresh insight into characteristics associated with transmission and outcome. C. difficile lineages may have different reservoirs and modes of transmission, and recent strain acquisition is associated with poorer outcomes.

Simulation of enteric colonisation by and screening for Carbapenemase Producing Enterobacteriaceae using an in-vitro human gut model.

Background: Global spread of carbapenemase producing Enterobacteriaceae (CPE) is a critical threat to public health. Rapid, sensitive and specific methods of screening patients for CPE colonisation are paramount. Using a clinically reflective in-vitro human gut model we compared the relative accuracy of routine screening methods. Methods: Three gut models were seeded with pooled faeces from four healthy volunteers. Following an initial equilibration phase the models were spiked with increasing inocula of carbapenemase-producing (CP) Klebsiella pneumoniae (KPC) (range 1.8-8.9log10cfu/mL). Three clinical isolates with distinctive carbapenem genes (KPC, OXA48, NDM) were investigated. We compared the relative sensitivities of: two commercial multiplex real-time PCR assays, Cepheid Xpert®Carba-R (XCR) and the Check-Direct CPE Screen for BD MAX™(CDCPE); two commercial culture media, chromID® CARBA SMART and chromID® ESBL; and MacConkey agar containing 0.5mg/L imipenem poured in-house (MAC-IMI). Twice-daily sampling was performed for the first week, with subsequent reduction in sampling post inoculation. Each sample was tested in triplicate using all five methods. Results: 237 samples were tested. CPE were detected after an inoculation of ~4.9log10cfu/mL. After inoculation, populations stabilised at 3-5log10 cfu/mL. MAC-IMI agar was inferior to (lower limit of detection {LOD} 1.66log10cfu/ml) and less reliable than the commercial agars; these agars had similar sensitivity but both had a lower LOD of 0.82log10cfu/mL. XCR showed decreased sensitivity but increased specificity for KPC and OXA48 compared to CDCPE. Both methods had similar sensitivity for NDM, but XCR had higher specificity. CDCPE had a higher false positive rate. Conclusion: The in-vitro gut model is a useful approach to measure CPE screening efficacy. Selective media showed highest sensitivity, but does not provide gene identification and requires 24hrs incubation. CDCPE had lower specificity compared to XCR for individual gene identification with a higher false positive rate, which may undermine its use for tracking outbreaks and for infection control interventions.

The predictive value of quantitative nucleic acid amplification detection of Clostridium difficile toxin gene for faecal sample toxin status and patient outcome

Background Laboratory diagnosis of Clostridium difficile infection (CDI) remains unsettled, despite updated guidelines. We investigated the potential utility of quantitative data from a nucleic acid amplification test (NAAT) for C. difficile toxin gene (tg) for patient management. Methods Using data from the largest ever C. difficile diagnostic study (8853 diarrhoeal samples from 7335 patients), we determined the predicative value of C. difficile tgNAAT (Cepheid Xpert C.diff) low cycle threshold (CT) value for patient toxin positive status, CDI severity, mortality and CDI recurrence. Reference methods for CDI diagnosis were cytotoxicity assay (CTA) and cytotoxigenic culture (CTC). Results Of 1281 tgNAAT positive faecal samples, 713 and 917 were CTA and CTC positive, respectively. The median tgNAAT CT for patients who died was 25.5 vs 27.5 for survivors (p = 0.021); for toxin-positivity was 24.9 vs 31.6 for toxin-negative samples (p<0.001) and for patients with a recurrence episode was 25.6 vs 27.3 for those who did not have a recurrent episode (p = 0.111). Following optimal cut-off determination, low CT was defined as ≤25 and was significantly associated with a toxin-positive result (P<0.001, positive predictive value 83.9%), presence of PCR-ribotype 027 (P=0.025), and mortality (P=0.032). Recurrence was not associated with low CT (p 0.111). Conclusions Low tgNAAT CT could indicate CTA positive patients, have more severe infection, increased risk of mortality and possibly recurrence. Although, the limited specificity of tgNAAT means it cannot be used as a standalone test, it could augment a more timely diagnosis, and optimise management of these at-risk patients.

Impact of Clostridium difficile toxin gene PCR result on decisions to de-isolate patients: Do the ends justify the means?

We aimed to determine how often Clostridium difficile toxin gene PCR assay (CDPCR)-negative patients were appropriately removed from single room contact isolation. Hospital databases were used to collect information on glutamate dehydrogenase (GDH)-positive, toxin-negative inpatients (February–April 2015). Of 60 CDPCR-negative patients, only two (3%) were removed from single room isolation. At least 36% of 53 CDPCR-positive results did not influence bed management. In conclusion, identification of C. difficile toxigenic status did not impact significantly on decisions whether to continue single room isolation. Cost-benefit analysis should be undertaken before CDPCR testing is introduced.

Two Distinct Patterns of Clostridium Difficile Diversity Across Europe Indicates Contrasting Routes of Spread.

Whole-genome sequencing of Clostridium difficile across Europe demonstrates 2 distinct transmission patterns. Healthcare-associated ribotypes showed genetic clustering by country and region, and widespread fluoroquinolone resistance. However, multiple common ribotypes showed no within-country clustering, consistent with Europe-wide dissemination via nonhealthcare routes/sources.