Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England.

BACKGROUND: Marked increases in Clostridium difficile infection (CDI) incidence, driven by epidemic strain spread, is a global phenomenon. METHODS: The Clostridium difficile Ribotyping Network (CDRN) was established in 2007 as part of enhanced CDI surveillance in England, to facilitate the recognition and control of epidemic strains. We report on changes in CDI epidemiology in England in the first 3 years of CDRN. RESULTS: CDRN received 12,603 fecal specimens, comprising significantly (P < .05) increasing numbers and proportions of national CDI cases in 2007-2008 (n = 2109, 3.8%), 2008-2009 (n = 4774, 13.2%), and 2009-2010 (n = 5720, 22.3%). The C. difficile recovery rate was 90%, yielding 11,294 isolates for ribotyping. Rates of 9 of the 10 most common ribotypes changed significantly (P < .05) during 2007-2010. Clostridium difficile ribotype 027 predominated, but decreased markedly from 55% to 36% and 21% in 2007-2008, 2008-2009, and 2009-2010, respectively. The largest regional variations in prevalence occurred for ribotypes 027, 002, 015, and 078. Cephalosporin and fluoroquinolone use in CDI cases was reported significantly (P < .05) less frequently during 2007-2010. Mortality data were subject to potential reporting bias, but there was a significant decrease in CDI-associated deaths during 2007-2010, which may have been due to multiple factors, including reduced prevalence of ribotype 027. CONCLUSIONS: Access to C. difficile ribotyping was associated with significant changes in the prevalence of epidemic strains, especially ribotype 027. These changes coincided with markedly reduced CDI incidence and related mortality in England. CDI control programs should include prospective access to C. difficile typing and analysis of risk factors for CDI and outcomes.

The role of glutamate dehydrogenase for the detection of Clostridium difficile in faecal samples: a meta-analysis.

Clostridium difficile causes a serious, occasionally fatal, hospital-acquired infection. The laboratory diagnosis of C. difficile infection (CDI) needs to be accurate to ensure optimal patient management, infection control and reliable surveillance. Commercial enzyme-linked immunosorbent assays for C. difficile toxins have poor sensitivity when compared with cell culture cytotoxin assay (CTA) and toxigenic culture (TC). We performed a meta-analysis of the role of glutamate dehydrogenase (GDH) in diagnosis of CDI. We analysed 21 papers, of which eight were excluded. We included publications of original research that used a 'gold standard' reference test (either CTA or TC). We also included publications that used culture without toxin testing of the isolate as a reference test even though this is not recognised as a gold standard. Exclusion criteria were failure to use a gold standard reference test and where the index test was used as the gold standard. Significant heterogeneity between study results justified the summary receiver operating characteristic (SROC) analysis. The meta-analysis demonstrated high diagnostic accuracy of GDH for the presence of C. difficile in faeces; when compared with culture it achieved a sensitivity and specificity of >90%. The SROC plot confirmed this finding. As a surrogate for toxigenic strains the GDH yields a specificity of 80-100% with a false positivity rate of ∼20%, as it detects toxigenic and non-toxigenic strains of the organism. However, GDH test has high sensitivity and negative predictive value and would be a powerful test in a dual testing algorithm when combined with a test to detect toxin.

Laboratory diagnosis of clostridium difficile infection. An evaluation of tests for faecal toxin, glutamate dehydrogenase, lactoferrin and toxigenic culture in the diagnostic laboratory.

Faecal samples from 1007 patients suspected of having diarrhoea caused by Clostridium difficile infection are investigated for the presence of toxins A and B and for the presence of C. difficile-specific glutamate dehydrogenase (GDH). Toxigenic culture is performed on all samples and is used as the 'gold standard' for the purpose of the study. A marker for intestinal inflammation, faecal lactoferrin, is used on any samples that give a positive result in any of the above tests. Part of the study also involves an assessment of six commercial toxin kits to detect the presence of C. difficile toxins in faecal samples. This study revealed that the commercial toxin detection kits used can give rise to false-positive and false-negative results and that all demonstrated poor sensitivity when compared to the gold standard of toxigenic culture. Testing of faecal samples for GDH can be useful as a negative screening method as the results of this test show high correlation with culture. Faecal toxin testing can then be performed on all GDH-positive samples (GDH positivity is independent of toxigenicity in strains of C. difficile). The combined use of GDH and toxin testing, coupled with toxigenic culture, revealed that some patients with diarrhoea who harboured toxigenic strains of C. difficile were faecal toxin-negative. Lactoferrin appears to be a useful marker for the presence of inflammatory diarrhoea.

Detection of Clostridium difficile infection: a suggested laboratory diagnostic algorithm.

Currently, the diagnosis of Clostridium difficile infection (CDI) relies on the detection of toxins A and B in faeces but the sensitivity of these tests has been questioned, particularly in advanced disease. In this context, additional methods to enhance the diagnosis of C. difficile have been investigated. In this study, 1007 faecal samples are tested using toxigenic culture, an immunoassay for toxins AB and the C. difficile-specific glutamate dehydrogenase (GDH) test. Samples positive by any of the above tests are evaluated for the presence of faecal lactoferrin as an indicator of intestinal inflammation. Patients with evidence of inflammation but with negative toxin AB tests are followed up to assess clinical outcome. The toxin AB test was positive in 35 samples (3.4%), while 121 (12%) samples were culture-positive, 87 (8.6%) of which were toxigenic. Glutamate dehydrogenase proved to be a sensitive and specific marker of C. difficile with a negative predictive value of 99.3% (95% CI: 0.98-1.00). Faecal lactoferrin was positive in 52/129 (40.3%) samples tested. A cohort of 15 patients with a negative faecal toxin AB and a positive lactoferrin test was C. difficile culture-positive with a toxigenic isolate; clinically, all had advanced CDI. All demonstrated faecal toxin between five and 41 days later on repeat testing. It is suggested that a two-step algorithm be used to include screening faecal samples for GDH, with positive samples tested for faecal toxin AB and lactoferrin. Patients who present with a negative faecal toxin AB test and a positive lactoferrin test were serially tested for faecal toxin AB every five to seven days until a diagnosis was established. More sensitive tests than enzyme-linked immunosorbent assay (ELISA) for the detection of faecal toxin, or the use of a rapid specific test for the presence of a toxigenic strain, must be considered in such patients.

Removing bacteria from hospital surfaces: a laboratory comparison of ultramicrofibre and standard cloths.

We compared the ability of ultramicrofibre-woven cloths with conventional cloths moistened with water only, for their ability to remove several types of organisms relevant to hospital-acquired infections from a variety of surfaces in hospitals. We showed that ultramicrofibre cloths consistently outperformed conventional cloths in their decontamination ability, across all surfaces, and irrespective of whether the bacteria were coated on to the surfaces with phosphate-buffered saline (PBS) or PBS containing horse serum to simulate real-life soiling. The ability of the cloths to remove bacteria from surfaces was assessed by contact plating and colony formation, and by swabbing and measurement of ATP bioluminescence. The results suggest potential for use of ultramicrofibre in healthcare environments. Further studies are required, however, to define accurately how these cloths, which are designed to be used without detergent or biocides, might be capable of safe and effective deployment and recycling in the healthcare environment.

Fusobacterium necrophorum as the cause of recurrent sore throat: comparison of isolates from persistent sore throat syndrome and Lemierre's disease.

OBJECTIVE: Fusobacterium necrophorum is a well established cause of Lemierre's disease (LD); a syndrome characterised by severe sore throat, septicaemia, multiple abscesses and jugular vein thrombosis. There is no published data concerning the role of F. necrophorum in recurrent sore throats. As the result of an index case of persistent sore throat attributable to this organism being diagnosed in our laboratory, a subsequent case controlled study (not yet published) isolated F. necrophorum from 21% (P=0.0001) of cases of persistent, recurrent and chronic sore throats. The object of this study was to compare isolates of F. necrophorum from cases of systemic disease with isolates from cases of persistent sore throat syndrome (PSTS) to ascertain whether strains of similar type were responsible for both throat and systemic disease or whether different strains were involved in these presentations. METHODS: Throat swabs were cultured on GN anaerobe medium (Oxoid) and incubated at 37 degrees C for 5 days. Seventeen PSTS isolates were identified phenotypically. These were compared to 17 strains isolated from blood cultures which were referred to the Anaerobe Reference Unit, (ARU) cardiff, using enterogenic repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The control strains Fusobacterium necrophorum ssp. necrophorum (JCM 3718(T)) and Fusobacterium necrophorum ssp. funduliforme (JCM 3724(T)) from the Japanese Collection of Microrganisms (JCM) were tested in parallel with the clinical isolates. RESULTS: At least 12 separate types were identified. Four of 17 PSTS isolates and seven of 17 blood culture isolates grouped together with the F. necrophorum ssp. funduliforme control strain. There were also similarities between other proposed strains and clinical types but no comparison with the F. necrophorum ssp. necrophorum control. CONCLUSION: These results show that clinical disease caused by F. necrophorum has a wider spectrum than first anticipated. Similar strains are able to cause either chronic local or acute systemic disease suggesting that genetic factors such as those relating to major histocompatibility complex (MHC) class may be influencing the outcome of the disease in each patient. Further work is required to produce a more accurate typing scheme and to ascertain the mechanisms of disease caused by this organism. An age correlation between the high risk groups for onset of infectious mononucleosis (IM), peritonsillar abscess (PTA), LD and PSTS has been noted in adolescents and young adults. Further work is required to investigate whether IM is associated with the onset of PTA caused by F. necrophorum which may lead to either PSTS or LD.

Prevalence of Fusobacterium necrophorum and other upper respiratory tract pathogens isolated from throat swabs.

Fusobacterium necrophorum, an anaerobic, Gram-negative rod, has been identified recently as a significant cause of persistent sore throat syndrome (PSTS). This disease is characterised by chronic, recurrent or persistent sore throat, which is believed to respond poorly to penicillin in vivo. The aim of this study is to examine the prevalence of F. necrophorum in all throat swabs received in our diagnostic microbiology department and to compare the results with those for other recognised respiratory pathogens. All throat swabs received in the laboratory over a four-week period were cultured for beta-haemolytic streptococcus groups A, C and G, Corynebacterium diphtheriae, Arcanobacterium haemolyticum and F. necrophorum. Latex agglutination techniques, phenotypic reactions and antibiograms are used to identify these organisms. The age of the patient and the clinical details as stated on the request form were noted. Among a total of 248 samples, 27 were positive for beta-haemolytic streptococcus group A, two were positive for beta-haemolytic streptococcus group C, five were positive for beta-haemolytic streptococcus group G and 24 were positive for F. necrophorum. The most common isolate in the under 20 age group was beta-haemolytic streptococcus group A. In the over 20 age group, F. necrophorum was the pathogen most frequently isolated. A clinical diagnosis of 'sore throat' was most likely to be positive for beta-haemolytic streptococcus group A, a clinical diagnosis of PSTS was most likely to be positive for F. necrophorum and a clinical diagnosis of 'tonsillitis' was equally likely to be caused by beta-haemolytic streptococcus group A or F. necrophorum. beta-haemolytic streptococcus group A was present in 11% of the samples and F. necrophorum was present in 10% of the samples. In total, these two pathogens accounted for 18.5% of throat infections in the sampled group. The results show that F. necrophorum is as significant a cause of throat infection as is beta-haemolytic streptococcus group A. Examination of this provisional data suggests that targeting culture towards these two pathogens may be possible in certain cohorts of patients if more precise clinical data are received from medical staff. However, based on the clinical symptoms routinely provided by clinicians requesting microscopy, culture and sensitivity on throat swabs, F. necrophorum culture is required on all throat swabs received in the laboratory.

Infections with Corynebacterium diphtheriae: six years' experience at an inner London teaching hospital.

Recently, a preponderance of non-toxigenic strains of Corynebacterium diphtheriae has been reported, as has the broadening spectrum of disease caused by these strains. This study presents data on 85 isolates of C. diphtheriae over a six year period (1998-2003). Eighty were non-toxigenic isolates from patients with sore throat, and five (one toxigenic) were from cutaneous ulcers in travellers returning from endemic areas. When examined in relation to denominator data provided by the Health Protection Agency (HPA) for the whole of England and Wales, 1998-2002, 75% of all notifications of C. diphtheriae for England and Wales originated from the laboratories at University College London Hospitals (UCLH). In some years (1999 and 2001) 95-100% of isolates came from UCLH. We believe that national data do not reflect true incidence, as universal screening for these organisms is not routine policy in many laboratories. The results presented suggest the need for increased clinical and laboratory awareness of this important pathogen.

Evaluation of three commercial agar preparations for the presumptive identification of significant urinary isolates.

Three commercially available pre-poured chromogenic preparations--chromogenic urinary tract infection (UTI) medium (chromogenic UTI, Oxoid), CHROMagar Orientation (Becton Dickinson) and CPS ID2 (bioMérieux)--are evaluated in comparison to routine urine microbiology using cysteine lactose electrolyte-deficient (CLED) medium and conventional methods of identification and susceptibility testing by Vitek 1 for the majority of isolates. Most isolates were Escherichia coli, and a chromogenic medium has been shown to be a reliable, rapid and more economic medium on which to presumptively identify these organisms due to the substrates the strain utilises in the plate and the chromogen subsequently produced. However, the opacity of chromogenic UTI made the medium difficult to inoculate and read, although the colours were clear and strong. Although there was no statistical difference between CHROMagar Orientation and CPS ID2, the colours observed on the former were stronger. This meant that colony counting was possible at significant concentrations of 10(4) and 10(5) colony-forming units (cfu)/mL and it may be easier to detect mixtures that would indicate contamination. Chromogenic media are richer than CLED and a number of Lactobacillus spp. (normally regarded as normal flora) grew on this medium. These were not considered to be significant.

Use of bioinformatics and PCR in the search for ABC transporter homology among various bacteria.

Bioinformatics databases and search tools are utilised to produce polymerase chain reaction (PCR) primers for the amplification of an ABC transporter gene from the clinically important anaerobe Finegoldia magna. On sequencing, a 450 base pair amplicon showed homology with the amino acid transporter of Enterococcus faecalis. Little sequence data is available for F. magna and the newly isolated DNA could be a useful tool in the identification of this organism in clinical specimens.

Screening for methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: an evaluation of three selective media and Mastalex-MRSA latex agglutination.

Laboratory confirmation of MRSA is important for the implementation of infection control; conventional screening culture methods take up to five days for confirmation. The purpose of this study is to ascertain the efficiency of three selective media for growth of methicillin-resistant Staphylococcus aureus (MRSA) before and after enrichment in salt broth, and to evaluate the Mastalex-MRSA latex agglutination kit for detection of methicillin resistance. Screening swabs were collected from 63 patients, yielding 125 S. aureus isolates and 40 coagulase-negative staphylococcus (CNS) isolates. Selective media used were mannitol salt agar (MSA), Baird-Parker agar with ciprofloxacin (BPC) and bromocresol purple (BCPA). Polymerase chain reaction (PCR) for mecA gene detection was used as the reference standard for evaluation of the Mastalex-MRSA assay, which was also evaluated on colonies of S. aureus from the selective media. No significant difference was found in efficiency of MRSA isolation among the selective media. Pre-enrichment in the salt broth did not enhance isolation of MRSA. Methicillin-sensitive S. aureus and CNS were significantly inhibited in all selective media (P<0.05). Only BPC significantly selected out methicillin-resistant CNS (P<0.01). Mastalex-MRSA was 97% specific and sensitive for the detection of MRSA. It was 65% sensitive and 100% specific in detecting methicillin resistance in CNS. In conclusion, all selective media performed equally well (MRSA isolation rate of approximately 80%). Mastalex-MRSA provided rapid and reliable detection of MRSA from selective media, reducing the time required for confirmation of this organism.