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Implementing molecular detection of gastro-intestinal protozoa and implications for an algorithm for complete parasitological diagnostics in the microbiological laboratory routine

Abstract number: O409

Bruijnesteijn van Coppenraet L.E.S., Wallinga J.A., Ruijs G.J.H.M., Bruins M.J., Wolfhagen M.J.H.M., Verweij J.J.

Introduction: Molecular assays for detection of Entamoeba histolytica, Giardia lamblia, Cryptosporidium hominis/parvum, and, respectively, Dientamoeba fragilis have already been described. They were more sensitive and/or specific than microscopy but are to date not routinely applied as replacement of the time-consuming microscopic analysis for protozoal gastrointestinal (GI) pathogens.

An internally controlled molecular assay for the simultaneous detection of all four protozoa in a single faeces sample was validated and compared to the Triple Feces Test (TFT) microscopic analysis. Also, an algorithm for the complete parasitological diagnosis after implementation of the real-time PCR assay in the laboratory routine was created.

Methods: TFT sets (2 fixed and 1 unpreserved faecal sample), were collected from 397 consecutive patients. The complete TFT set was examined for GI parasites by microscopy and compared to multiplex real-time PCR for E. histolytica, G. lamblia, C. hominis/parvum, and D. fragilis, applied on the unpreserved faeces samples. Faecal DNA was extracted by Nuclisens easyMAG (Biomerieux) with Phocid Herpes Virus added as internal inhibition control.

Also, microscopic results and clinical patient information of 2887 TFT sets were analysed retrospectively to determine local risk-factors for infection with non-protozoal GI parasites.

Results: Real-time PCR of 397 unpreserved samples yielded 169 (43%) positives (44 (11.1%) samples positive for G. lamblia, 122 (30.7%) positive for D. fragilis and 3 (0.8%) positive for C. hominis/parvum), while microscopy of the TFT samples yielded 100 (25%) positives (29 (7.3%) samples positive for G. lamblia, 69 (17.4%) positive for D. fragilis and 2 (0.5%) positive for C. hominis/parvum, respectively).

Analysis of the 2887 TFT sets showed eosinophilia, elevated IgE and travelling to (sub) tropical areas to be risk-factors for non-protozoal GI parasites. The resulting diagnostic algorithm includes application of real-time PCR on all samples, adding microscopy on an unpreserved faecal sample only in case of presence of a risk factor.

Conclusions: 1) Application of real-time PCR improved the diagnostic yield with 18%. 2) A single unpreserved faecal sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical patient information is applied.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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