Latent microsporidial infection in immunocompetent individuals - a longitudinal study.

BACKGROUND: Microsporidia (Fungi) have been repeatedly identified as the cause of opportunistic infections predominantly in immunodeficient individuals such as AIDS patients. However, the global epidemiology of human microsporidiosis is poorly understood and the ability of microsporidia to survive and multiply in immunocompetent hosts remains unsolved. AIMS: To determine the presence of latent microsporidia infections in apparently healthy humans in the Czech Republic, the authors tested sera, urine and stool originating from fifteen persons within a three month period examined on a weekly basis. METHODS: Sera, stool and urine samples originating from fifteen HIV-negative people at risk with occupational exposure to animals, aged 22-56 years, living in the Czech Republic were tested by indirect immunofluorescence assay (IFA) for the presence of specific anti-microsporidial antibodies, standard Calcofluor M2R staining for the detection of microsporidian spores in all urine sediments and stool smears and molecular methods for the microsporidial species determination. RESULTS: Specific anti-microsporidial antibodies were detected in fourteen individuals, asymptomatic Encephalitozoon spp. infection was found in thirteen and E. bieneusi infection was detected in seven of those examined. While E. hellem 1A and E. cuniculi II were the major causative agents identified, seven different genotypes of E. bieneusi were recorded. CONCLUSIONS: These findings clearly show that exposure to microsporidia is common and chronic microsporidiosis is not linked to any clinical manifestation in healthy population. Moreover, our results indicate much higher incidence of microsporidial infections among an apparently healthy population than previously reported. These results open the question about the potential risk of reactivation of latent microsporidiosis in cases of immunosupression causing life-threatening disease.

Cytometric approach for detection of Encephalitozoon intestinalis, an emergent agent.

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.

Use of a fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia.

Microsporidia are obligate intracellular protozoan parasites. Three species of the genus Encephalitozoon are among the microsporidia that infect immunodeficient humans. These species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, all develop in a parasitophorous vacuole within a host cell. The present study describes a method that uses the fluorescent probe calcein and confocal microscopy to detect drug-induced effects in Encephalitozoon-infected green monkey kidney cells. The effects were as follows: (i) changes in parasite organization within the parasitophorous vacuole; (ii) swelling and gross morphological changes of parasite developing stages in situ; (iii) killing of developing parasite stages in situ, detected by their uptake of the fluorescent probe; and (iv) reduction in the viability of the host cell population, assessed by the loss of the probe. Verapamil and itraconazole were used to increase the vital dye loading by both uninfected and infected cells. Agents with known antimicrosporidial activity, albendazole and fumagillin, caused all three types of parasite changes at concentrations that had no detectable effect on host cell viability. The effective doses of albendazole and fumagillin that caused swelling and disorganization of parasite developing stages were 5 x 10(-7) and 10(-6) M respectively. Killing of developing stages was detected at 10-fold-higher concentrations for these agents and at 10(-5) M for metronidazole. This method can be used to screen candidate antimicrosporidial agents in infected cultured cells.

Ultrastructural features of spindle microtubule organization during the nuclear division of Encephalitozoon hellem.

Ultrastructural studies were carried out to describe the nuclear division cycle of a strain of Encephalitozoon hellem isolated from an Italian AIDS patient. The nuclear division occurs during the proliferative vegetative phase and it is characterized by the intranuclear mitosis and by the lack of centrioles. The spindle termini are electron dense spindle plaques (ESPs), resembling to the spindle pole bodies (SPBs) of Saccharomycetes. The ESPs are bifacial organella forming microtubules on both nucleoplasic and cytoplasmic faces. In the outer layer of the spindle plaque are present vesicular elements lined by a double membrane of unknown function. The peculiar morphological features of E. hellem ESPs indicate that both intranuclear spindle and cytoplasmic microtubules are involved in the nuclear division.

The Warthin-Starry stain in the diagnosis of small intestinal microsporidiosis in HIV-infected patients.

A protocol for the handling of small intestinal biopsies from HIV-infected patients is presented. This protocol includes the Warthin-Starry stain for the detection of microsporidia. This stain has proved a reliable and sensitive diagnostic technique for microsporidial infections as it stains both Enterocytozoon bieneusi and Septata intestinalis in duodenal enterocytes. Because the stain demonstrates Septata intestinalis in lamina propria macrophages as well as enterocytes, it allows for the practical differentiation of these two microsporidial infections. The Warthin-Starry stain has also demonstrated Septata intestinalis in nasal and colonic biopsies in some of these patients. Since the completion of an earlier study, a further 40 cases of Enterocytozoon bieneusi and three cases of Septata intestinalis have been diagnosed in just over 240 consecutive duodenal biopsies from HIV positive patients presenting with diarrhoea and other gastrointestinal complaints. Other opportunistic infections include cytomegalovirus in four cases, mycobacteria in eight cases, cryptosporidia in nine cases, giardia in four cases and Isospora belli in one case. Since the ratio of these opportunistic infections has remained much the same as in the previous study of 180 consecutive duodenal biopsies, we suggest that these rates may reflect the actual prevalence of microsporidial infections in AIDS patients in Sydney, Australia.