Enerocytozoon bieneusi, Czech Republic | CDC EID

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Volume 16, Number 2–February 2010
Dispatch
Seropositivity for Enterocytozoon bieneusi, Czech Republic
Bohumil Sak, Zuzana Kučerová, Martin Kváč, Dana Květoňová, Michael Rost, and Evan W. Secor
Author affiliations: Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic (B. Sak, M. Kváč, D. Květoňová); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (Z. Kučerová, E.W. Secor); and University of South Bohemia in České Budějovice, České Budějovice (M. Kváč, M. Rost)

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Abstract
To determine seropositivity for Enterocytozoon bieneusi in the Czech Republic, we tested 115 serum samples from various groups. We found that 20% from HIV-positive persons, 33% from persons with occupational exposure to animals, and 10% from healthy persons were positive by indirect immunofluorescence assay. Proteins of 32 kDa were detected in serum samples from seropositive persons.

Microsporidia are small, single-celled, obligate intracellular parasites that were initially characterized as eukaryotic protozoa, but they have recently been reclassified as fungi. Since 1985, microsporidia have been identified as a cause of opportunistic infections associated with persistent diarrhea and weight loss in persons with AIDS (1). Because of heightened awareness and improved diagnostic methods, microsporidia infections have been recognized in a wide range of human populations, including organ transplant recipients, travelers, children, contact lens wearers, the elderly, and immunocompetent persons with no known risk factors (2).

Of the 14 species of microsporidia known to infect humans, Enterocytozoon bieneusi is the most common and is associated with diarrhea and systemic disease (3). Symptomatic E. bieneusi infections are primarily found in immunodeficient persons, although infection in immunocompetent populations is increasingly detected (4). It is unclear whether asymptomatic microsporidia infections persist in immunocompetent persons and can reactivate during conditions of immune compromise and are than able to be transmitted to others at risk, such as during pregnancy or through organ donation.

Studies focusing on risk factors associated with microsporidiosis will help define more clearly the sources of microsporidia that pose a risk for transmission in the environment so that preventive strategies can be implemented. To determine seropositivity for E. bieneusi in the Czech Republic, we used 2 serologic assays for detecting E. bieneusi–specific antibodies in serum specimens from HIV-positive and HIV-negative persons and from blood donors and persons with occupational exposure to animals.

The Study
The National Institute of Public Health in Prague provided anonymous serum samples, originally collected for HIV diagnostics in 2007, from HIV-positive persons (n = 70) and healthy blood donors (n = 30). In addition, serum specimens from persons who worked with animals and animal excrement were collected after informed consent was obtained in 2007 (n = 15). Every specimen in the study was supplemented with data on the patient's clinical symptoms (e.g., indigestion, abdominalgia). The study was approved by the Hospital České Budějovice, a.s. ethics committee (protocol no. 202/07). The serum specimens were frozen directly after recovery and were stored at –20°C. Patient identifiers were removed from the samples before testing.

E. bieneusi spores were purified from positive stool samples, originally obtained from an HIV/AIDS patient from Lima, Peru (provided by G.S. Visvesvara, Centers for Disease Control and Prevention, Atlanta, GA, USA), by using Percoll and cesium chloride gradient centrifugation as previously described (5). The spore suspension was stored in phosphate-buffered saline (PBS) supplemented with antimicrobial drugs at 4°C. The purity of spore suspension was tested by using light microscopy (optical brightener staining), and the background reactivity of serum specimens with bacteria was observed by using indirect immunofluorescence antibody (IFA) assay.

IFA was performed with purified whole E. bieneusi spores at a concentration of 105/well. The serum samples were diluted in PBS by serial dilution, 1:10, 1:50, 1:100, 1:200, and 1:400, and results were compared with negative (1:100) and positive (1:400) control serum specimens. Serum specimens with titers >100 were considered positive on the basis of positive control serum titration. A total of 115 human serum samples were examined by IFA for antimicrosporidial immunoglobulin G. Specific antibodies against E. bieneusi were detected for 22 persons (19%; 95% confidence interval [CI] 12%–28%); 20% of HIV-positive persons (CI 11%–31%), 10% of blood donors (CI 2%–26%), and 33% of persons with animal risk exposure were positive (CI 11%–61%). CIs were calculated by the Clopper-Pearson formula for binominal counts (Table). None of the persons had demonstrated any clinical symptoms (e.g., loose stool, indigestion). The titers were higher (400) for HIV-positive persons and 1 animal keeper; the highest titer in blood donors was 200. No background reactivity was observed in tested serum samples with bacteria present in spore suspension.

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