K. E. P. Olsen

Blastocystis sp. Subtype 4 is Common in Danish Blastocystis-Positive Patients Presenting with Acute Diarrhea

Fecal samples from 444 Danish patients presenting with acute diarrhea were tested for Blastocystis and positive samples were subtyped to investigate the prevalence and subtype distribution of Blastocystis in this patient group. A total of 25 patients (5.6%) were positive, and 19 of these patients (76.0%) were positive for Blastocystis sp. ST4. Because the relative prevalence of ST4 in other patients presenting with other types of diarrhea (persistent, travel-related, and human immunodeficiency virus-related) in Denmark is low, the role of Blastocystis sp. ST4 in the etiology of acute diarrhea should be investigated further.

Eradication of Blastocystis carriage with antimicrobials: reality or delusion?

Metronidazole constitutes a mainstay in the antimicrobial therapy of intestinal protozoa, and is also traditionally considered first-line therapy in cases where there is a requirement to treat Blastocystis, a common protist of disputable clinical significance. Many compounds have been used in attempts to eradicate the parasite, and an accumulating body of data indicates that successful antimicrobial eradication of Blastocystis is far from straightforward. This review focuses on some issues that prevent us from reaching a clear understanding of how to eradicate Blastocystis based on chemotherapeutic intervention, by focusing on conflicting reports on the efficacy of metronidazole and other compounds and study design and data limitations. The review provides a comprehensive overview of antimicrobials used to target Blastocystis, and discusses issues pertaining to drug resistance, treatment failure, and reinfection. Finally, key methodological and molecular diagnostic tools that will assist in the generation of data required to improve current knowledge are identified and discussed.

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.

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.

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.

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 (pu2009<u20090.05) and significantly more robust to inhibition compared to PCRFast (pu2009<u20090.001). Duplicate ‘expanded toxigenic culture’ increased the culture-positive rate by 29xa0% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95xa0%), and in-house PCR displayed 100xa0% 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.

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 <2xa0years 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 >38xa0°C, weight loss and sick leave. Data were analysed by logistic regression. CDI patients <2 and ≥2xa0years of age with C. difficile as the only enteropathogen in the faecal sample reported slimy stools (65xa0% vs. 62xa0%), stomach ache (60xa0% vs. 75xa0%), weight loss (50xa0% vs. 76xa0%) and duration of diarrhoea >15xa0days (59xa0% vs. 73xa0%) as the predominant symptoms. CDI patients ≥2xa0years old reported duration of diarrhoea >15xa0days more often compared to non-CDI patients (73xa0% vs. 27xa0%, pu2009<u20090.0001). The annual incidence of CDI was 518 and 23/100,000 for patients <2 and ≥2xa0years of age, respectively, and 46/100,000 in the subgroup of patients ≥60xa0years 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 ≥2xa0years of age attending general practice because of gastrointestinal symptoms, especially in the elderly, where the incidence of CDI is high.

Diarrheagenic potential of Escherichia coli in children in a developed country.

Escherichia coli is an underestimated diarrheal pathogen in developed countries. This organism may cause gastroenteritis by many different mechanisms, and on the basis of this, diarrheagenic E. coli has been subdivided into different groups, of which the most important ones are Shiga-toxigenic E. coli, enteropathogenic E. coli (EPEC), other attaching and effacing E. coli (A/EEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli, and enteroinvasive E. coli. The latter two are associated with travel to less developed countries. Shiga-toxigenic E. coli is a well-established pathogen, whereas the pathogenic role of EPEC, A/EEC, and EAEC is less well established. We read the paper by Pabst et al. in the June issue of the Journal of Clinical Microbiology (1) with great interest. The authors try to shed light on the role of diarrheagenic E. coli in Switzerland by using a case-control setup and molecular diagnostic methods for the identification of the different E. coli groups. However, the study has a couple of shortcomings that makes us wonder if all of the conclusions presented are correct. Firstly, the authors do not take into account the potential bias of the travel history of the patients when comparing cases and controls. Travel may be an important confounder because none of the controls reported traveling, whereas travel was quite common among the patients; i.e., if a pathogen is more common in travelers than in patients with domestically acquired cases, it will not be possible to ascertain the relative importance of travel if the patients are compared with a group of controls who have not traveled in a univariate analysis. A way of circumventing the problem would be to exclude the travel-associated cases from the analysis. In the case of EAEC, the number of patients who had traveled was 9 or 47%; the total number of diarrheal patients who had traveled was 31. It is thus possible to compare the occurrence of EAEC in patients and controls with no travel history: The prevalence in patients was 9 of 156, and that in controls was 3 of 137. This difference is not statistically significant (P 0.21; Fisher’s two-tailed exact test). Secondly, the authors did not find any difference in the prevalence of EPEC between cases and controls. This conclusion may be questioned too, because the EPEC definition used is based solely on detection of the intimin gene (eae). In the definition of EPEC, the serotype of the strains should also be taken into account (2). We performed a study similar to that of Pabst et al. and found a strong correlation between disease in infants less than 2 years of age and EPEC, defined as eaepositive strains belonging to the classical EPEC O groups, whereas other A/EEC showed no correlation to disease (B. Olesen, P. Schiellerup, M. Helms, J. Neimann, F. Scheutz, and P. Gerner-Smidt, Abstr. 12th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P607, 2002). Classical EPEC is the most common bacterial cause of diarrhea in children less than 2 years old in Denmark. We wonder if Pabst et al. would have reached the same conclusion had they serotyped their eae-positive isolates.