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 g/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 ؋ 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 ؋ 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.

Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry

“…In a final set of experiments we tested whether the amount of dye required per sample can be reduced to lower the cost per sample for assessing Nosema spore viability, and whether different combinations of dyes can produce similar spore viability outcomes as the established combination of SG and PI (Table , Part II). Seven sets of reference samples were stained with the additional combinations and compared, those combinations included lower concentrations of SG and PI, SYBR and PI, SG and SR, or SYBR and SR (Table , Part III). Spore viability was determined according to the above protocols.…”

“…Similarly FCM can quantify effects of treatment on the viability of a wide range of human pathogens such as cytomegalovirus, herpes simplex virus, human immunodeficiency virus , and many others . FCM has also been used to determine the viability and abundance of microsporidia from humans ( Encephalitozoon intestinalis and Encephalitozoon cuniculi ) and fish ( Microsporidiun ovoideunm, Glugea stephani, Glugea atherinae and Spraguea lophii ) .…”

Mucosal Vaccination against Enteric Pathogens in the Developing World

“…After infection they are able to invade all organs and tissues. Infection with three Encephalitozoon species was shown to originate in either the intestinal or respiratory epithelia (Barbosa et al, 2009;Martinez et al, 2008). Dissemination to other parts of the body may occur, due to the ability of these species to infect macrophages.…”

Environmental Monitoring of Opportunistic Protozoa in Rivers and Lakes in the Neotropics Based on Yearly Monitoring

“…They are the most prevalent microsporidia identified in humans (Didier and Weiss 2006) and other mammals (Keeling and Fast 2002). The spores are the infective agent (Barbosa et al 2009) because they are resistant to environmental conditions (Stine et al 2005) and to the usual water treatment procedures (Hoffman et al 2003). Consequently, transmission involves waterborne routes (Karanis et al 2007).…”