Cytometric approach for detection of Encephalitozoon intestinalis, an emergent agent

Abstract

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 microg/ml of Microspor-FA at 25 degrees C overnight provided the best results. The detection limit was 5 x 10(4) 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.

FIG. 1.

MIF of Encephalitozoon intestinalis spores labeled with serial concentrations of an Alexa Fluor 488 monoclonal antibody (Microspor-FA; 20× concentrate; A700 A488; Waterborne, Inc.). All experiments were performed twice.

FIG. 2.

Two-dimensional dot plot correlating FL1 (green fluorescence, 535 nm) with FL3 (red fluorescence, 620 nm) of Encephalitozoon intestinalis spores without staining (autofluorescence) (A), E. intestinalis spores stained with 10.0 μg/ml of specific antibody (Microspor-FA; A488; Waterborne, Inc.) (B), and E. intestinalis spores stained with 10.0 μg/ml of specific antibody and 5.0 μg/ml of PI (C).