Dennis Röser

DNA of Dientamoeba fragilis detected within surface-sterilized eggs of Enterobius vermicularis

With no evidence of a cyst stage, the mode of transmission of Dientamoeba fragilis, an intestinal protozoon of common occurrence and suggested pathogenicity, is incompletely known. Numerous studies have suggested that eggs of intestinal nematodes, primarily Enterobius vermicularis (pinworm), can serve as vectors for D. fragilis, although attempts to culture D. fragilis from pinworm eggs have been unsuccessful and data from epidemiological studies on D. fragilis/pinworm co-infection have been conflicting. The aim of this study was to investigate whether we could detect D. fragilis DNA from pinworm eggs collected from routine diagnostic samples (cellophane tape) and surface-sterilised by hypochlorite. DNA was extracted from individual eggs and tested by PCR using D. fragilis- and E. vermicularis-specific primers; amplicons were sequenced for confirmation. In cellophane tape samples from 64 patients with unknown D. fragilis status we detected D. fragilis DNA in 12/238 (5%) eggs, and in a patient known to harbour D. fragilis we detected D. fragilis DNA in 39/99 (39%) eggs. The finding of D. fragilis DNA within eggs of E. vermicularis strongly supports the hypothesis of D. fragilis-transmission by pinworm and has implications for antimicrobial intervention as well as control and public health measures.

Metronidazole Therapy for Treating Dientamoebiasis in Children Is Not Associated With Better Clinical Outcomes: A Randomized, Double-Blinded and Placebo-Controlled Clinical Trial

BACKGROUND There is a paucity of evidence documenting the pathogenicity of Dientamoeba fragilis, an intestinal protozoan common in children. As case reports on successful treatment are numerous, many authors advocate treatment, despite no placebo-controlled trials being available. Metronidazole is often used for treatment, though eradication rates are relatively low (60%-80%). In the present study we determined the clinical and microbiological efficacy of metronidazole in Danish children. METHODS In this parallel placebo-controlled double-blinded trial, children aged 3-12 years with >4 weeks of gastrointestinal symptoms were allocated using block randomization in a 1:1 ratio to a 10-day course of oral metronidazole or placebo. Primary outcome was change in level of gastrointestinal symptoms, measured on a visual-analog-scale (VAS), and secondary outcome was eradication of D. fragilis infection. Participants, caregivers, investigators, and sponsor were all blinded to group assignment. The trial was registered with clinicaltrials.gov (NCT01314976) prior to start. RESULTS Of 96 participants, 48 were allocated to the metronidazole and placebo group each. Mean VAS change from pre- to post-treatment did not differ significantly (P = .8) between the metronidazole (-1.8 CI, [-2.5, -1.1]) and the placebo group (-1.6 CI, [-2.3, -.9]). Eradication of D. fragilis was significantly greater in the metronidazole group, although it declined rapidly from 62.5% 2 weeks after end of treatment to 24.9% 8 weeks after end of treatment. CONCLUSIONS These findings do not provide evidence to support routine metronidazole treatment of D. fragilis positive children with chronic gastrointestinal symptoms. Study funded by Statens Serum Institut. CLINICAL TRIALS REGISTRATION Trial was registered with clinicaltrials.gov (NCT01314976).

Transmission of Dientamoeba fragilis: pinworm or cysts?

Recently, conflicting evidence has been published on the mode of transmission of the trichomonad Dientamoeba fragilis. Detection of D. fragilis DNA inside Enterobius vermicularis eggs agrees with the prediction of Dobell in 1940 that the eggs of a nematode act as a vector for transmission. However, the identification of a cyst stage of D. fragilis in the stool of rodents infected with a human isolate has also been reported, and this implies a life cycle similar to those of most other intestinal protistan parasites. Herein we discuss the recent data, identify gaps in the experimental evidence, and propose a method for determining which view of the life cycle of this organism is correct.

Is Supplementary Bead Beating for DNA Extraction from Nematode Eggs by Use of the NucliSENS easyMag Protocol Necessary

Recent studies have demonstrated the effectiveness and applicability of PCR-based methods for the detection of intestinal helminths in stool samples ([1][1][–][2][5][3]); however, successful DNA extraction is essential. We compared DNA extractions using NucliSENS easyMag, with and without a

Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes.

Dientamoeba fragilis is a common intestinal parasite of unsettled clinical significance. Differences in clinical outcome of parasitic infections may reflect parasite genetic diversity, and so tools to study intra-genetic diversity that could potentially reflect differences in clinical phenotypes are warranted. Here, we show that genetic analysis of three D. fragilis housekeeping genes enables clear distinction between the two known genotypes, but that integration of housekeeping genes in multi-locus sequencing tools for D. fragilis may have limited epidemiological and clinical value due to no further added genetic resolution.

A case of human babesiosis in Denmark

We report the first human case of Babesia microti infection imported to Denmark from the United States by a 64 year old female traveller with fever of unknown origin. The case raises the possibility that Babesia-infections may be under-diagnosed, illustrates the importance of a thorough travel history and discusses important diagnostic pitfalls.

Development and evaluation of a multiplex screening assay for Plasmodium falciparum exposure

Transfusion transmitted malaria (TTM) in non-endemic countries is reduced by questioning blood donors and screening of donated blood. Conventional screening is performed by Indirect Fluorescence Antibody Test (IFAT). This method is manual and difficult to standardize. Here we study the diagnostic performance of a multiplex assay for detection of antibodies against Plasmodium falciparum in donor blood using IFAT as a comparator. A multiplex assay (MPA) containing the antigens GLURP-R0, GLURP-R2, MSP3, MSP1 hybrid and AMA1 was constructed using xMAP® technology. A discrimination index for exposure to P. falciparum malaria was calculated by comparing travelers with clinical malaria (n=52) and non-exposed blood donors (n=119). The index was evaluated on blood donors with suspected malaria exposure (n=249) and compared to the diagnostic performance of IFAT. At a specificity of 95.8 %, the MPA discrimination index exhibited a diagnostic sensitivity of 90.4 % in travelers hospitalized with malaria. Percent agreement with IFAT was 92.3 %. Screening plasma from blood donors with suspected malaria exposure, we found 4.8 % to be positive by IFAT and 5.2 % by MPA with an agreement of 93.2 %. The calculated index from the MPA exhibits similar diagnostic performance as IFAT for detection of P. falciparum malaria. Combining the antibody response against multiple antigens in a discrimination index increased the sensitivity of the MPA and reduced the readout to a single value.

Dientamoeba fragilis, a Commensal in Children in Danish Day Care Centers

ABSTRACT Dientamoeba fragilis is an intestinal protozoan of debated clinical significance. Here, we present cross-sectional and longitudinal observations on D. fragilis in children aged 0 to 6 years from a 1-year multi-day-care-center cohort study set in Copenhagen, Denmark. The inclusion period for the cohort was 2009 through 2012. Stool samples collected from the children were accompanied by questionnaires completed by the parents or guardians of the children. Using real-time PCR, D. fragilis was detected in the first stool sample from 97 of 142 (68.3%) children. We evaluated the associations between seven plausible risk factors (age, sex, having siblings, having domestic animals at home, having had infant colic, recent history of intake of antibiotics, and recent history of travel abroad) as well as six reported symptoms (lack of appetite, nausea, vomiting, abdominal pain, weight loss, and diarrhea) and testing positive for D. fragilis. The final multivariable model identified being >3 years old and having a history of recent travel abroad as risk factors for testing positive for D. fragilis. Moreover, univariable analyses indicated that having siblings was a risk factor. There was no statistical association between a recent history of gastrointestinal symptoms and testing positive for D. fragilis. Among the 108 children who were represented by ≥2 samples and thus included in the longitudinal analysis, 32 tested negative on the first sample and positive later, and the last sample from each of the 108 children was positive. The results are in support of D. fragilis being a common enteric commensal in this population.

Childhood diarrhoea in Danish day care centres could be associated with infant colic, low birthweight and antibiotics.

Diarrhoea is very common in children attending day care centres. The aim of this study was to examine certain predisposing risk factors for an association with diarrhoea, including foreign travel, treatment with antibiotics, having household pets, infant colic, bottle feeding, using a pacifier and low birthweight.

History of antimicrobial use and the risk of Dientamoeba fragilis infection

Associations between antimicrobial use and risk of enteric infection with intestinal protozoa are scarcely studied. The aim of this study was to quantify the risk of Dientamoeba fragilis infection conferred by exposure to antimicrobials. We conducted a registry-based retrospective cohort study of 9,945 Danish patients investigated for D. fragilis infection between 2008 and 2011, using data from the Danish Register of Medicinal Product Statistics, and calculating relative risks (RR) for D. fragilis infection by stratified binary regression. Furthermore, we conducted a population based case–control study using controls sampled from the Danish Civil Registration System, calculating hazard ratios (HR) for D. fragilis infection by conditional logistic regression. Exposure to metronidazole was found to confer decreased risk of D. fragilis infection; however, similar associations were found for antimicrobials not commonly used to treat D. fragilis, such as broad-spectrum penicillin, fluoroquinolones, and macrolides. In contrast, mebendazole exposure was associated with increased risk. The intake of antimicrobials influences the risk of D. fragilis.