Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of community-acquired gastroenteritis.

The European, multicentre, quarterly point-prevalence study of community-acquired diarrhoea (EUCODI) analysed stool samples received at ten participating clinical microbiology laboratories (Austria, Finland, France, Germany, Greece, Ireland, Italy, Portugal, Romania, and the UK) in 2014. On four specified days, each local laboratory submitted samples from ≤20 consecutive patients to the Austrian Study Centre for further testing with the FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA). Of the 709 samples from as many patients received, 325 (45.8%) tested negative, 268 (37.8%) yielded only one organism, and 116 (16.4%) yielded multiple organisms. Positivity rates ranged from 41% (30 of 73 samples) in France to 74% (59 of 80 samples) in Romania. With the exception of Entamoeba histolytica and Vibrio cholerae, all of the 22 targeted pathogens were detected at least once. Enteropathogenic Escherichia coli, Campylobacter species, toxigenic Clostridium difficile, enteroaggregative E. coli, norovirus and enterotoxigenic E. coli were the six most commonly detected pathogens. When tested according to local protocols, seven of 128 positive samples (5.5%) yielded multiple organisms. Overall, the FilmArray GI Panel detected at least one organism in 54.2% (384/709) of the samples, as compared with 18.1% (128/709) when testing was performed with conventional techniques locally. This underlines the considerable potential of multiplex PCR to improve routine stool diagnostics in community-acquired diarrhoea. Classic culture methods directed at the isolation of specific pathogens are increasingly becoming second-line tools, being deployed when rapid molecular tests give positive results. This optimizes the yield from stool examinations and dramatically improves the timeliness of diagnosis.

Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture.

The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine toxigenic culture. In total, 300 faecal samples from Danish hospitalised patients with diarrhoea were included consecutively. Culture was performed in duplicate (routine and 'expanded toxigenic culture': prolonged and/or re-culture) and genotypic toxin profiling by polymerase chain reaction (PCR), PCR ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene C. difficile and PCRFast C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p < 0.05) and significantly more robust to inhibition compared to PCRFast (p < 0.001). Duplicate 'expanded toxigenic culture' increased the culture-positive rate by 29% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95%), and in-house PCR displayed 100% correct identification of PCR ribotypes 066 and 078. Furthermore, the presence of the PCR enhancer bovine serum albumin (BSA) was found to be related to high sensitivity and low inhibition rate. Rapid laboratory diagnosis of toxigenic C. difficile by RT PCR was accurate.

Risk factors for Clostridium difficile infection in the community: a case-control study in patients in general practice, Denmark, 2009-2011.

To identify risk factors for Clostridium difficile infection (CDI) in Danish patients consulting general practice with gastrointestinal symptoms, a prospective matched case-control study was performed; cases (N = 259) had positive cultures for toxigenic C. difficile and controls (N = 455) negative cultures. Data were analysed by conditional logistic regression. In patients aged ⩾2 years (138 cases), hospitalization [odds ratio (OR) 8·4, 95% confidence interval (CI) 3·1-23], consumption of beef (OR 5·5, 95% CI 2·0-15), phenoxymethylpenicillin (OR 15, 95% CI 2·7-82), dicloxacillin (OR 27, 95% CI 3·6-211), and extended spectrum penicillins (OR 9·2, 95% CI 1·9-45) were associated with CDI. In patients aged <2 years none of these were associated with CDI, but in a subgroup analysis contact with animals was associated with CDI (OR 8·1, 95% CI 1·0-64). This study emphasizes narrow-spectrum penicillins, and suggests beef consumption, as risk factors for CDI in adults, and indicates a different epidemiology of CDI in infants.

The incidence and clinical symptomatology of Clostridium difficile infections in a community setting in a cohort of Danish patients attending general practice.

Clostridium difficile infection (CDI) is gradually being recognised as a cause of morbidity in the community. We investigated the incidence and clinical characteristics of CDI in a community setting and characterised the C. difficile strains by toxin gene profiling and polymerase chain reaction (PCR) ribotyping. Patients included in the study had attended general practice, primarily because of diarrhoea; CDI patients (259 patients; 121 <2 years of age) had positive cultures for toxigenic C. difficile and non-CDI patients (455 patients) were culture-negative. Outcome variables included the frequency and duration of diarrhoea, vomiting, stomach ache, fever >38 °C, weight loss and sick leave. Data were analysed by logistic regression. CDI patients <2 and ≥2 years of age with C. difficile as the only enteropathogen in the faecal sample reported slimy stools (65% vs. 62%), stomach ache (60% vs. 75%), weight loss (50% vs. 76%) and duration of diarrhoea >15 days (59% vs. 73%) as the predominant symptoms. CDI patients ≥2 years old reported duration of diarrhoea >15 days more often compared to non-CDI patients (73% vs. 27 %, p < 0.0001). The annual incidence of CDI was 518 and 23/100,000 for patients <2 and ≥2 years of age, respectively, and 46/100,000 in the subgroup of patients ≥60 years of age. CDI was characterised by stomach ache and persistent diarrhoea, often leading to weight loss. This emphasises the importance of diagnosing CDI not only in hospitalised patients, but also in individuals ≥2 years of age attending general practice because of gastrointestinal symptoms, especially in the elderly, where the incidence of CDI is high.

Salmonella prevalence among reptiles in a zoo education setting.

Clinically healthy reptiles may shed Salmonella and therefore act as a potential zoonotic threat. Most people in Northern European countries are rarely exposed to reptiles, but many zoos have education departments where children have direct contact with this group of animals. The objectives of this study were to determine the prevalence and serotype distribution of Salmonella among reptiles in the Education Department (n = 55) at Copenhagen Zoo and compare it to the Zoo's main reptile collection (n = 145) to evaluate the zoonotic risk. Salmonella was isolated from cloacal swabs by selective enrichment, and a single isolate from each positive sample was further identified by biochemical tests and serotyped. The overall prevalence was 35% (69/200) with significant difference between the Education Department (64%, 35/55) and the main reptile collection (23%, 34/145). A total of 28 serotypes were detected. Ten serotypes were isolated from more than one specimen and four from more than one species. Salmonella enterica subsp. enterica serovar Eastbourne was the predominant serotype (32%, 22/69) and was also the serotype isolated from most reptile species (n = 7). Transmission of serotypes from one department to another was very limited indicated by the serotype distribution. Despite the relative high prevalence observed among the reptiles in the Zoo's Education Department compared to the reptiles in the Zoo's main reptile collection, no Salmonella cases have been linked to the Zoo, and Salmonella ser. Eastbourne is very rarely isolated from humans in Denmark. Simple hygienic procedures such as hand washing which is consistently carried out following handling of reptiles at the Education Department may reduce the risk and therefore contribute to this low prevalence.

A new real-time PCR method for the identification of Salmonella Dublin.

AIMS: Development of a real-time PCR method for the specific detection of Salmonella Dublin. METHODS AND RESULTS: The method was directed towards a Salm. Dublin-specific sequence of the vagC gene on the Salmonella virulence plasmid (pSDV) and towards Salmonella genus-specific sequence of the invA gene, serving as an internal amplification control. The method showed 100% inclusivity and exclusivity when tested on a strain collection containing 50 serotyped S . Dublin strains, 20 strains of other Salmonella serotypes and 10 non- Salmonella strains. The method also showed 100% inclusivity and 99% exclusivity in a collaborative study comprising eight laboratories, where each laboratory received ten different S . Dublin strains and 10 other Salmonella serotypes. CONCLUSIONS: The method showed excellent performance both when validated in the laboratory and in the collaborative study. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of the present method in food control, for example at slaughterhouses, can improve the contamination control of this veterinary and clinically important Salmonella serotype.

Antimicrobial susceptibility testing of Clostridium difficile using EUCAST epidemiological cut-off values and disk diffusion correlates.

With the emergence of reduced susceptibility of Clostridium difficile to metronidazole and vancomycin the value of antimicrobial susceptibility testing has increased. The aim of our study was to evaluate disk diffusion for susceptibility testing of C. difficile by comparing disk diffusion results with MICs from gradient tests and to propose zone diameter breakpoint correlates for the EUCAST epidemiological cut-off values (ECOFFs) recently published. We tested 211 clinical isolates of C. difficile, from patients with diarrhoea hospitalized at Aarhus and Odense University Hospitals, Denmark. Furthermore, ten clinical isolates of C. difficile from the Anaerobe Reference Laboratory, University Hospital of Wales, with known reduced susceptibility to either metronidazole or vancomycin, were included. Isolates were tested with Etest gradient strips and disk diffusion towards metronidazole, vancomycin and moxifloxacin on Brucella Blood Agar supplemented with hemin and vitamin K. We found an excellent agreement between inhibition zone diameter and MICs. For each MIC value, the inhibition zones varied from 0 to 8 mm, with 93% of values within 6 mm for metronidazole, 95% of values within 4 mm for vancomycin, and 98% of values within 4 mm for moxifloxacin. With proposed zone diameter breakpoints for metronidazole, vancomycin and moxifloxacin of WT ≥ 23 mm, WT ≥ 19 and WT ≥ 20 mm, respectively, we found no very major errors and only major errors below 2%. In conclusion, we suggest that disk diffusion is an option for antimicrobial susceptibility testing of C. difficile.

Bacteraemia with Campylobacter jejuni: no association with the virulence genes iam, cdtB, capA or virB.

The role of bacterial genes in the determination of the clinical spectrum of Campylobacter jejuni infection is unclear. We compared clinical isolates from invasive blood-stream infection with stool isolates from gastroenteritis and found no association of the putative virulence genes iam, capA, virB and cdtB with clinical presentation.

Outbreak of Clostridium difficile 027 in North Zealand, Denmark, 2008-2009.

We report an outbreak of Clostridium difficile PCR ribotype 027 in Denmark. The outbreak includes to date 73 cases from the area north of Copenhagen, but there may be related cases elsewhere in Zealand. Most infections are healthcare-associated and in patients who previously received antibiotic treatment. The strain is resistant to moxifloxacin, erythromycin, and clindamycin, and carries genes for toxin A, toxin B, and for the binary toxin. The antimicrobial pattern differs from that of the strain involved in a small cluster in Denmark in 2006-2007. Because of this outbreak, hygienic measures in the involved hospitals have been reinforced. Nationwide, microbiological laboratories were alerted to the outbreak and encouraged to send isolates for toxin profiling and PCR ribotyping.

Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite.

Two independent studies were conducted to describe symptoms and potential risk factors associated with Blastocystis infection. Isolates were subtyped by molecular analysis. In the NORMAT study (126 individuals randomly sampled from the general population) 24 (19%) were positive for Blastocystis. Blastocystis was associated with irritable bowel syndrome (P=0.04), contact with pigs (P<0.01) and poultry (P=0.03). In the Follow-up (FU) study (follow-up of 92 Blastocystis-positive patients), reports on bloating were associated with subtype (ST) 2 (P<0.01), and blood in stool to mixed subtype infection (P=0.06). ST1 was more common in FU individuals (32%) than in NORMAT individuals (8%), whereas single subtype infections due to ST3 or ST4 were seen in 63% of the NORMAT cases and 28% of the FU cases. Only FU individuals hosted ST7, and ST6/7 infections due to ST7 or ST9 were characterized by multiple intestinal symptoms. The data indicate subtype-dependent differences in the clinical significance of Blastocystis.

A foodborne outbreak of Cryptosporidium hominis infection.

Foodborne outbreaks of cryptosporidiosis are uncommon. In Denmark human cases are generally infrequently diagnosed. In 2005 an outbreak of diarrhoea affected company employees near Copenhagen. In all 99 employees were reported ill; 13 were positive for Cryptosporidium hominis infection. Two analytical epidemiological studies were performed; an initial case-control study followed by a cohort study using an electronic questionnaire. Disease was associated with eating from the canteen salad bar on one, possibly two, specific weekdays [relative risk 4.1, 95% confidence interval (CI) 2.1-8.3]. Three separate salad bar ingredients were found to be likely sources: peeled whole carrots served in a bowl of water, grated carrots, and red peppers (in multivariate analysis, whole carrots: OR 2.1, 95% CI 1.1-4.0; grated carrots: OR 2.1, 95% CI 1.2-3.9; peppers: OR 3.3, 95% CI 1.7-6.6). We speculate that a person excreting the parasite may have contaminated the salad buffet.

Outbreaks of Shigella sonnei infections in Denmark and Australia linked to consumption of imported raw baby corn.

We investigated an outbreak of Shigella sonnei infections in Denmark and Australia associated with imported baby corn from one packing shed in Thailand. We reviewed nationwide surveillance and undertook case finding, food trace-back and microbiological investigation of human, food and environmental samples. A recall of baby corn and sugar snaps was based on descriptive epidemiological evidence. In Denmark, we undertook a retrospective cohort study in one workplace. In total, 215 cases were laboratory-confirmed in Denmark, and 12 in Australia. In a multivariable analysis, baby corn was the only independent risk factor. Antibiotic resistance and PFGE outbreak profiles in Denmark and Australia were indistinguishable, linking the outbreaks. Although we did not detect S. sonnei in baby corn, we isolated high levels of other enteric pathogens. We identified a packing shed in Thailand that supplied baby corn to Denmark and Australia, and uncovered unhygienic practices in the supply chain. This outbreak highlights the importance of international communication in linking outbreaks and pinpointing the source.

A foodborne outbreak of enterotoxigenic E. coli and Salmonella Anatum infection after a high-school dinner in Denmark, November 2006.

In industrialized countries enterotoxigenic Escherichia coli (ETEC) is mainly diagnosed as a cause of travellers' diarrhoea, but it is also known to cause foodborne outbreaks. We report an outbreak of acute gastroenteritis caused by ETEC serotypes O92:H- and O153:H2 as well as Salmonella Anatum, which affected around 200 students and teachers after a high-school dinner in Greater Copenhagen, Denmark, November 2006. A retrospective cohort study showed that consumption of pasta salad with pesto was associated with an increased risk of illness (attack rate 59.4%; risk ratio 2.6, 95% confidence interval 1.2-5.7). Imported fresh basil used for preparation of the pesto was the most likely source of contamination. Although ETEC is associated with travellers' diarrhoea in Denmark, this outbreak suggests that a proportion of sporadic ETEC infections might be caused by contaminated imported foodstuffs. To improve food safety further, it is important to target this poorly regulated and researched area.

New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection.

Isolates of Clostridium difficile from 159 hospitalized Danish patients (2005) were analysed by a new 5-plex PCR method targeting the toxin genes tcdA, tcdB, cdtA and cdtB, and 16S rDNA as an internal positive control. Additionally, the toxin-regulating gene tcdC was partially sequenced by a new sequencing-based method that revealed genetic changes that may render the gene product inactive. Finally tcdA was analysed using a previously published method for the detection of internal deletions. The 5-plex PCR revealed four different toxin gene profiles: 36 tcdA+, tcdB+, cdtA+/cdtB+; one tcdA+, tcdB-, cdtA+/cdtB+; 98 tcdA+, tcdB+, cdtA-/cdtB-; and 24 non-toxigenic tcdA-, tcdB-, cdtA-/cdtB-. Deletion studies revealed that 26 strains contained a c. 700-bp deletion in tcdA, and 39 strains contained at least one possible inactivation feature in tcdC. The prevalence of the binary toxin genes was 23%. All strains with the tcdA+, tcdB+, cdtA+/cdtB+ profile were investigated by PCR ribotyping, and this revealed eight different ribotypes, none of which were 027. The 5-plex PCR method offers a one-step, rapid and specific screening method for C. difficile toxin genes. This toxin gene profiling, together with deletion studies in tcdA and tcdC, may allow an evaluation of the pathogenic potential of C. difficile.

Outbreak of group A streptococcal throat infection: don't forget to ask about food.

We report a large foodborne outbreak due to group A streptococci (GAS), which caused acute tonsillo-pharyngitis in 200-250 patrons of a company canteen in Copenhagen, Denmark, in June 2006. A retrospective cohort study of canteen users showed that consumption of cold pasta was associated with an increased risk of illness (attack rate 68%, risk ratio 4.1, P<0.0001). Indistinguishable GAS strains (emm89, T-type 3/13/B3264) were cultured from three cases and a cook, who had prepared the pasta. To our knowledge, pasta has previously only twice been incriminated as the source of a GAS outbreak. Only six foodborne GAS outbreaks have been reported in Europe since 1970, four of them in Sweden or Denmark. This geographical clustering suggests that foodborne GAS outbreaks are probably under-recognized elsewhere.

Attaching and effacing Escherichia coli isolates from Danish children: clinical significance and microbiological characteristics.

This study describes the prevalence, clinical manifestations and microbiological characteristics of attaching and effacing Escherichia coli isolates, i.e., enteropathogenic E. coli (EPEC) belonging to the classical EPEC serotypes, non-EPEC attaching and effacing E. coli (A/EEC) and verocytotoxin-producing E. coli (VTEC), isolated in a case-control study of Danish children aged <5 years. Among 424 children with diarrhoea and 866 healthy controls, EPEC and VTEC were more prevalent in cases (2.4% and 2.6%, respectively) than in controls (0.7% and 0.7%, respectively). There was a high frequency of A/EEC isolates (n = 121), but these were equally prevalent in cases (11.3%) and controls (12.5%), and comprised a heterogeneous distribution of O:H serotypes. The intimin (eae) subtypes in A/EEC isolates showed an even distribution; the eae-gamma subtype predominated in classical EPEC cases. The virulence genes encoding the bundle-forming pilus (bfpA) and enteroaggregative heat-stable enterotoxin (astA) were rare among all isolates, and seemed to be of limited pathogenic importance in this population. Virulence characterisation of A/EEC isolates did not reveal any significant differences between cases and controls. Colonisation of children with A/EEC was associated with contact with sheep or goats (OR 2.2). The role of A/EEC, not being VTEC or belonging to the classical EPEC serotypes, requires further clarification, but serotyping is useful in discriminating between EPEC and A/EEC strains.

The significance of the number of submitted samples and patient-related factors for faecal bacterial diagnostics.

The sensitivity of bacteriological testing of faecal samples from patients with diarrhoea has not been properly determined. The present study analysed the association between the results of stool sample examinations and the number of samples examined per patient and other patient-related factors. Data concerning faecal specimens referred for culture for enteric bacterial pathogens (Campylobacter, Salmonella, Shigella and Yersinia) to the central microbiological laboratory in Denmark between 1995 and 2003 were analysed. In total, 620 000 samples were sorted into 277 000 sample-series, i.e., samples submitted from the same individual on the same day. Data were analysed by multivariate logistic regression, with the outcome being a positive sample-series, i.e., one or more positive samples per series. Overall, 11.9% of the sample-series were positive. For adults (aged > or =18 years), the OR for a positive diagnosis was 1.20 (95% CI 1.18-1.21) for each additional sample. Positive diagnoses were also more likely during summer, if the patient was male, or if the patient was neither very young nor very old. The added diagnostic effect of additional samples was more pronounced for the group of patients with persistent (>2 weeks) diarrhoea. Overall, the probability of finding common pathogenic bacteria in faecal samples was found to vary according to the number of samples, the season and the patient's age and gender. Analysis of more than one sample improves the sensitivity of faecal culture by at least 20% for each additional sample.

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory.

A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.