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Clinical and Vaccine Immunology, July 2009, p. 1021-1024, Vol. 16, No. 7
1071-412X/09/$08.00+0     doi:10.1128/CVI.00031-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cytometric Approach for Detection of Encephalitozoon intestinalis, an Emergent Agent{triangledown}

Joana Barbosa,1,2* Acácio Gonçalves Rodrigues,1,3,4 and Cidália Pina-Vaz1,4,5

Department of Microbiology, Faculty of Medicine, University of Porto, Porto,1 Escola Superior de Saúde Jean Piaget, Vila Nova Gaia,2 Burn Unit, Department of Plastic and Reconstructive Surgery, Hospital S. João, Porto,3 Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, Porto,4 Department of Microbiology, Hospital S. João, Porto, Portugal5

Received 9 January 2009/ Returned for modification 20 March 2009/ Accepted 6 May 2009

Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 µg/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 x 104 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 x g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health.

* Corresponding author. Mailing address: Department of Microbiology, Porto Faculty of Medicine, University of Porto, Al. Prof. Hernani Monteiro, Porto 4200-319, Portugal. Phone and fax: 351 22 551 3662. E-mail: gui75{at}sapo.pt

{triangledown} Published ahead of print on 13 May 2009.

Clinical and Vaccine Immunology, July 2009, p. 1021-1024, Vol. 16, No. 7
1071-412X/09/$08.00+0     doi:10.1128/CVI.00031-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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