Printer Friendly

Seropositivity for Enterocytozoon bieneusi, Czech Republic.

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 Ceske Budejovice, a.s. ethics committee (protocol no. 202/07). The serum specimens were frozen directly after recovery and were stored at -20[degrees]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[degrees]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 [greater than or equal to] 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.

Proteins from 10 (10) purified spores were obtained by disruption of spores by using FastPrep 120 homogenizer and FastProtein Blue kit (both BIO-101, Inc., MP Biomedicals, Irvine, CA, USA) according to manufacturer's instructions. Proteins were separated by using preparative 4%-20% acrylamide gradient Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA, USA), electrotransferred onto nitrocellulose membranes (Schleicher and Schuell Bioscience, Inc., Keene, NH, USA), and cut into strips. Each strip was incubated with a 1:100 dilution of individual serum specimens in 0.3% Tween-PBS, peroxidase-conjugated goat antibody (Bio Source International Inc., Camarillo, CA, USA) diluted 1:4,000 in 0.05% Tween-PBS, and blots were developed by diaminobenzidine substrate solution containing [H.sub.2][O.sub.2]. Although several different proteins were identified in specimens from seropositive persons, a parasite protein with a molecular weight of [approximately equal to] 32 kDa was predominant, this protein was not identified in any of the negative serum specimens (Figure). The results of immunoblot testing correlated with those of IFA; all serum samples with a titer >200 showed a strong reaction with immunodominant antigen in immunoblot.

Conclusions

The epidemiology of human E. bieneusi infection is poorly understood, and environmental factors that affect transmission of the organism have not been fully elucidated. Most reports addressing prevalence of microporidiosis are based on coprologic or PCR diagnostics, and the serologic screening of humans for microsporidia infection has mostly been limited to species that can be cultured in vitro (6-8).

Our survey was performed on a limited sample size from highly selected populations, which resulted in decreased statistical power. Although our findings are likely minimal estimates, given the uncertain duration of serologic response and <100% sensitivity of testing, they showed a 33% seroprevalence of E. bieneusi among animal keepers and 20% among HIV-positive persons. In studies in which infection was diagnosed by detection of E. bieneusi spores or DNA in stool, infection rates ranged between 1.4% and 78% (9-12). However, PCR and coprology are not able to discriminate E. bieneusi spores that have simply been consumed and passed through the intestinal tract from those resulting from active infection. In contrast, the detection of specific antibodies indicates that these persons experienced infection.

In the healthy population represented by normal blood donors, we detected a prevalence of only 10%, which is similar to previously reported prevalences (1.3%-8.0%) of Encephalitozoon-specific antibodies among HIV-negative persons such as blood donors, slaughterhouse workers, dog breeders, forestry workers, and pregnant women (6-8). In other studies, microsporidia infection of immunocompetent travelers with self-limiting diarrhea has been reported (13). The persistence of microsporidia despite resolution of the intestinal disorder suggests microsporidia infection may cause clinical signs (e.g., diarrhea) during the early stages of infection that resolve even though the microsporidia persist. In our study, the highest seroprevalence was in the group with professional exposures (33%), concurrent with a high titer of specific antibodies. Some of these professionals cared for pigs on farms, where E. bieneusi spores have been found in the feces of up to 94% of pigs (14). Other studies also confirm the possibility of occupational risk exposure to microsporidia spores. An immunocompetent laboratory worker occupationally exposed to Encephalitozoon cuniculi remained seropositive 38 months after treatment (15). These results indicate the possible role of animals as a zoonotic source of microsporidia spores and show a possible occupational risk for persons who work with animals and animal excrement.

[FIGURE OMITTED]

Studies that focus on risk factors associated with microsporidiosis will more clearly define the environmental sources of microsporidia that pose a risk for transmission so that preventative strategies can be implemented. Because no data exist about latent infection in immunocompetent carriers, possible infection reactivation and person-to-person transmission risk through organ donation, our future studies will focus on detailed seroprevalence data among healthy populations, especially persons with occupational risk exposure, and will aim to elucidate the role of various animals in human infection. This information may lead to better identification of possible sources of microsporidial infections and help effect their prevention.

This study was funded by a grant of the Academy of Sciences of the Czech Republic (KJB500960701), the Grant Agency of the Czech Republic (project No. 523/07/P117), research project of the Institute of Parasitology, Academy of Sciences of the Czech Republic (Z60220518), and in part by National Institutes of Health grant R13AI078718 from the National Institute of Allergy and Infectious Diseases.

DOI: 10.3201/eid1602.090964

References

(1.) Weber R, Deplazes P, Schwartz D. Diagnosis and clinical aspects of human microsporidiosis. Contrib Microbiol. 2000;6:166-92. DOI: 10.1159/000060360

(2.) Deplazes P, Mathis A, Weber R. Epidemiology and zoonotic aspects of microsporidia of mammals and birds. Contrib Microbiol. 2000;6:236-60. DOI: 10.1159/000060363

(3.) Didier ES, Weiss LM. Microsporidiosis: current status. Curr Opin Infect Dis. 2006;19:485-92. DOI: 10.1097/01.qco.0000244055. 46382.23

(4.) Lores B, Lopez-Miragaya I, Arias C, Fenoy S, Torres J, Del Aguila C. Intestinal microsporidiosis due to Enterocytozoon bieneusi in elderly human immunodeficiency virus-negative patients from Vigo, Spain. Clin Infect Dis. 2002;34:918-21. DOI: 10.1086/339205

(5.) Kucerova Z, Moura H, Leitch GJ, Sriram R, Bern C, Kawai V, et al. Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques. J Clin Microbiol. 2004;42:3256-61. DOI: 10.1128/JCM.42.7.3256-3261.2004

(6.) Van Gool T, Vetter JC, Weinmayr B, Van Dam A, Derouin F, Dankert J. High seroprevalence of Encephalitozoon species in immunocompetent subjects. J Infect Dis. 1997;175:1020-4. DOI: 10.1086/513963

(7.) del Aguila C, Rueda C, De La Camara C, Fenoy S. Seroprevalence of anti-Encephalitozoon antibodies in Spanish immunocompetent subjects. J Eukaryot Microbiol. 2001;Suppl:75S-8S. DOI: 10.1111/ j.1550-7408.2001.tb00459.x

(8.) Kucerova-Pospisilova Z, Ditrich O. The serological surveillance of several groups of patients using antigens of Encephalitozoon hellem and E. cuniculi antibodies to microsporidia in patients. Folia Parasitol (Praha). 1998;45:108-12.

(9.) Cotte L, Rabodonirina M, Piens MA, Perreard M, Mojon M, Trepo C. Prevalence of intestinal protozoans in French patients infected with HIV. J Acquir Immune Defic Syndr. 1993;6:1024-9.

(10.) Coyle CM, Wittner M, Kotler DP, Noyer C, Orenstein JM, Tanow itz HB, et al. Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis among patients with AIDS-related diarrhea: determination by polymerase chain reaction to the microsporidian small-subunit rRNA gene. Clin Infect Dis. 1996;23:1002-6.

(11.) Molina JM, Sarfati C, Beauvais B, Lemann M, Lesourd A, Ferchal F, et al. Intestinal microsporidiosis in human immunodeficiency virus-infected patients with chronic unexplained diarrhea: prevalence and clinical and biologic features. J Infect Dis. 1993;167:217-21.

(12.) Sobottka I, Schwartz DA, Schottelius J, Visvesvara GS, Pieniazek NJ, Schmetz C, et al. Prevalence and clinical significance of intestinal microsporidiosis in human immunodeficiency virus-infected patients with and without diarrhea in Germany: a prospective coprodiagnostic study. Clin Infect Dis. 1998;26:475-80. DOI: 10.1086/516328

(13.) Wichro E, Hoelzl D, Krause R, Bertha G, Reinthaler F, Wenisch C. Microsporidiosis in travel-associated chronic diarrhea in immune-competent patients. Am J Trop Med Hyg. 2005;73:285-7.

(14.) Sak B, Kvac M, Hanzlikova D, Cama V. First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic. Vet Parasitol. 2008;153:220-4. DOI: 10.1016/j.vetpar.2008.01.043

(15.) Van Gool T, Vetter JC, Weinmayr B, Van Dam A, Derouin F, Dankert J. Serodiagnostic studies in an immunocompetent individual infected with Encephalitozoon cuniculi. J Infect Dis. 2004;189:2243-9. DOI: 10.1086/421117

Author affiliations: Biology Centre of the Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic (B. Sak, M. Kvac, D. Kvetonova); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (Z. Kucerova, E.W. Secor); and University of South Bohemia in Ceske Budejovice, Ceske Budejovice (M. Kvac, M. Rost)

Dr Sak is a research scientist at the Biology Centre of the Academy of Sciences of the Czech Republic. His research interests focus on microsporidia, including in vitro cultivation, immunologic studies, and molecular identification of human pathogenic microsporidia.

Address for correspondence: Martin Kvac, Biology Centre of the Academy of Sciences of the Czech Republic, Ceske Budejovice, Branisovska 31, Czech Republic; email: kvac@paru.cas.cz
Table. Seroprevalence of Enterocytozoon bieneusi in different groups,
Czech Republic

 95%
 No. positive confidence
 /no. interval, Maximum
Serum source examined (%) % * titer

HIV-positive persons 14/70 (20) 11-31 400
Blood donors 3/30 (10) 2-26 200
Persons with animal exposure 5/15 (33) 11-61 400
Total 22/115 (19) 12-28 400

* 95% Clopper-Pearson confidence interval for binomial counts.
COPYRIGHT 2010 U.S. National Center for Infectious Diseases
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2010 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:DISPATCHES
Author:Sak, Bohumil; Kucerova, Zuzana; Kvac, Martin; Kvetonova, Dana; Rost, Michael; Secor, Evan W.
Publication:Emerging Infectious Diseases
Date:Feb 1, 2010
Words:2067
Previous Article:Severe leptospirosis in hospitalized patients, Guadeloupe.
Next Article:Hendra virus outbreak with novel clinical features, Australia.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters