Chapter 71
Microsporidiosis
AYMAN NASERI
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TAXONOMY AND CLASSIFICATION
ECOLOGY AND EPIDEMIOLOGY
MICROBIAL AND CELLULAR PATHOGENESIS
IMMUNOLOGY AND ANIMAL MODELS
LABORATORY DIAGNOSIS
SUSCEPTIBILITY TO THERAPEUTIC AGENTS
REFERENCES

Microsporidia are obligate intracellular parasites that can infect both vertebrate and invertebrate hosts. Although the first human microsporidial infection was not recognized until 1959,1 14 different species have since been identified as human pathogens (Table 1). The human immunodeficiency virus (HIV) epidemic increased the recognition of microsporidiosis as a human disease after microsporidia were identified as the cause of chronic diarrhea in patients with acquired immunodeficiency syndrome (AIDS). Although microsporidiosis is generally considered an emerging opportunistic infection of immunocompromised individuals, microsporidia can also infect immunocompetent hosts.

TABLE 1. Most Common Clinical Presentation of Human Pathogenic Microsporidia


OrganismReported ocular diseaseClinical presentation
Brachiola algeraeYes35Keratitis, myositis
Brachiola connoriNoDD
Brachiola vesicularumYes28Myositis
Encephalitozoon cuniculiYes8Keratoconjunctivitis, DD
Encephalitozoon hellemYes7Keratoconjunctivitis, DD
Encephalitozoon intestinalisYes6Diarrhea, DD
Enterocytozoon bieneusiNoDiarrhea, DD
Microsporidium africanumYes20Keratitis
Microsporidium ceylonensisYes36Keratitis
Nosema ocularumYes20Keratitis, DD
Pleistophora ronneafieiNoMyositis
Trachipleistophora anthropophtheraNoDD
Trachipleistophora hominisYes37Myositis, keratoconjunctivitis
Vittaforma corneaeYes 37Keratitis, DD

DD, disseminated disease.

 

In 1973, Ashton and Wirasinha2 published the first report of a well-documented case of human ocular microsporidial infection, which had been identified in the cornea of a young boy who had been injured by a goat 6 years earlier. Since then, cases of ocular microsporidiosis have been reported in both immunocompetent persons3–5 and immunocompromised persons.3,6–8 Ocular microsporidial infection typically presents either as chronic epithelial keratoconjunctivitis or as deep stromal keratitis, but one case of sclerouveitis with retinal detachment has also been reported.9

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TAXONOMY AND CLASSIFICATION
Microsporidia are unicellular spore-forming eukaryotic parasites. The taxonomic classification of these organisms has been controversial during the past few decades. As molecular phylogenetic data have emerged, classification schemes based on morphologic and physiologic characteristics of these parasites have been reconsidered. In 1857, when Nageli10 named Nosema bombycis as the causative agent of pepper disease in silkworms, he believed that the organism was a yeast and placed it among the schizomycetes, which were an assortment of both yeasts and bacteria. In 1882, Balbiani1 recognized these unusual organisms as a distinct group, and he proposed the name microsporidia to accommodate the only known microsporidium at the time, N. bombycis.

As eukaryotes lacking identifiable mitochondria, microsporidia were later considered to be ancient protists that had diverged before the evolution of mitochondria. This theory placed microsporidia together with other amitochondriate protists, and they were collectively known as Archezoa.10 Early molecular genetic analyses of ribosomal RNA also supported the Archezoan hypothesis, as did the absence of organelles such as peroxisomes and Golgi apparati.

However, further analysis of microsporidian phylogeny contradicts the notion that microsporidia derive from a primitive origin. Recent scientific discoveries, including the sequencing of the full genome of Encephalitozoon cuniculi, support the relation of microsporidia to fungi.11,12 Rather than emerging from primitive origins, microsporidia may be highly reduced, sophisticated fungi that contained mitochondria at one time.10,13

Although the first microsporidial organism was not discovered until the mid-1800s, since then approximately 150 genera with more than 1,200 different species have been described within the phylum Microspora. Fourteen microsporidian species have been identified as human pathogens (Table 1): Brachiola algerae, B. connori, B. vesicularum, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Microsporidium africanum, M. ceylonensis, N. ocularum, Pleistophora ronneafiei, Trachipleistophora anthropophthera, T. hominis, and Vittaforma corneae. Species from several genera have caused documented ocular infection (Table 1), with E. hellem being the most commonly identified organism in reported cases.

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ECOLOGY AND EPIDEMIOLOGY
Microsporidia infect organisms of many different types, especially animals. Insects and fish are particularly common hosts for microsporidial infection, but a definitive reservoir of human pathogenic species has not been identified. The prevalence and distribution of microsporidiosis in humans is not fully known. Factors that limit our understanding of the epidemiology of microsporidiosis include subclinical infection, diagnostic inconsistency, diverse clinical manifestations of disease, nonrandom study design, and uncertain sources of transmission.

Because both domestic and wild animals are common hosts for microsporidia, zoonotic transmission from animal reservoirs is likely. However, recent evidence suggests that some strains of microsporidia may also be waterborne pathogens. Cotte et al14 reported an outbreak of intestinal microsporidiosis that was linked to local water distribution subsystems. In 1998, scientists in Arizona identified three different pathogenic species of microsporidia in surface water, groundwater, and tertiary effluent by using polymerase chain reaction (PCR) amplification.15 That same year, Enriquez et al16 reported a link between community water sources in Mexico and the presence of E. intestinalis spores in the feces of local residents.

Molecular methods have similarly confirmed the presence of E. intestinalis in water used for drinking.17 Although the evidence is accumulating for waterborne transmission of microsporidiosis, respiratory tract infection with microsporidia suggests potential airborne transmission and ocular infection may be caused by autoinoculation with contaminated fingers.

Although microsporidia can infect immunocompetent persons, microsporidiosis is more common in immunosuppressed populations. In 1985, E. bieneusi was detected in a patient with AIDS with chronic diarrhea.1 Since then, several published studies have identified microsporidial infection as a cause of chronic diarrhea in patients with AIDS.18 Ocular microsporidial infection has also been reported in both immunocompromised and immunocompetent patients.4–8,19,20 Risk factors for ocular disease include AIDS, prednisone use, and trauma. However, in 2003, Lewis et al4 reported a case of microsporidial keratoconjunctivitis in an immunocompetent patient without a history of trauma or contact lens wear. A case of corneal microsporidiosis has also been reported in a healthy patient who had undergone laser in situ keratomileusis 3 years earlier.21

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MICROBIAL AND CELLULAR PATHOGENESIS
Not only are there more than 100 genera containing more than 1,000 different species of microsporidia, there is also a wide range of potential hosts for infection that extends from other protists to higher vertebrates. Therefore, one might expect some variability in the microbial pathogenesis between various species and hosts. However, one cellular pathogenic mechanism is common to all microsporidia: the extrusion of a coiled polar tube as a means of penetrating and infecting adjacent cells.

The production and distribution of the microsporidian spore define the life cycle of microsporidia. Microsporidian spores are unicellular, ranging in size from 1 μm to 10 μm. The size and morphologic characteristics of these spores are the criteria for the differentiation and classification of the various species of microsporidia. The complex structure of the cell wall of the microsporidian spore provides resistance to many environmental stresses. Each mature microsporidian spore comprises an extrusion apparatus consisting of a polaroplast, a polar tube or filament, and a posterior vacuole. The unique microsporidial mechanism of infection involves the rapid extrusion and uncoiling of the polar tube through the anterior end of the spore. The tube can then penetrate an adjacent host cell (Fig. 1) and inoculate the host cytoplasm directly with its infective sporoplasm.22,23

Fig. 1. Scanning electron micrograph of a microsporidian spore with an extruded polar tubule inserted into a larger eukaryotic host cell. Reprinted from the Parasite Image Library of the Centers for Disease Control and Prevention Website on Laboratory Identification of Parasites of Public Health Concern (DPDx). (Available at http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Microsporidiosis_il.htm accessed February 17, 2005.)

Host cells can also be infected with microsporidian spores by ingesting spores via phagocytosis. Microsporidia have been identified as persisting within macrophages. Confined to a specialized compartment known as a parasitophorous vacuole, microsporidia can avoid the destructive enzymes of a macrophage's lysosomal network. A spore can also escape hostile lysosomal enzymes by using its polar tube to penetrate the wall of the lysosome and injecting its sporoplasm into the free cytoplasm of its host macrophage. Once inside the cytoplasm of the host cell, the microsporidium goes through two major life cycle stages: multiplication (merogony or schizogony) and maturation (sporogony). These developmental phases are species- and host-specific.

Many microsporidian genera multiply and mature while floating free among the intracellular organelles within the host cell's cytoplasm. Other genera develop within either a host-produced vacuole (Encephalitozoon) (Fig. 2) or a parasite-produced envelope (Pleistophora).

Fig. 2. Electron micrograph of a eukaryotic host cell that contains Encephalitozoon intestinalis spores and developing forms inside a septated parasitophorous vacuole. Reprinted from the Parasite Image Library of the Centers for Disease Control and Prevention Website on Laboratory Identification of Parasites of Public Health Concern (DPDx). (Available at http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Microsporidiosis_il.htm accessed February 17, 2005.)

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IMMUNOLOGY AND ANIMAL MODELS
Little is known about the immunologic characteristics of microsporidiosis in humans. Although it has primarily been considered an infection of persons who are immunocompromised, microsporidiosis can also infect persons with competent immune systems. Animal models have offered some clues about the immunologic characteristics of microsporidiosis. In general, chronic infections may develop in adult animal hosts with an intact immune system without the hosts showing many clinical signs of disease, however, microsporidial infection is often lethal in immunocompromised animals.24

Numerous animal models exist for the human pathogenic species of microsporidia. Nonhuman primates, pigs, mice, rabbits, fish, birds, and insects have all been used as experimental hosts for microsporidia, but most of the immunologic information about microsporidiosis is derived from research with mice.25 Studies of cell-mediated immunity suggest that T lymphocytes are key players in the defense against microsporidiosis. Evidence from experiments with severe combined immunodeficient mice points to CD8+ T lymphocytes as central to the prevention of microsporidial infection.24,26 However, the mechanisms of both cell-mediated and humoral immunity may vary, depending on the species of both the parasite and the host as well as the route of infection.

E. hellem is the most commonly reported cause of ocular infection. In fact, it was first isolated from the corneas of patients with AIDS and keratoconjunctivitis. Athymic mice can serve as an animal model for this disease after they are infected with E. hellem spores either by inhalation or by intraperitoneal injection. Although E. hellem is a common cause of microsporidial keratoconjunctivitis, it is primarily a respiratory pathogen that causes disseminated disease. Athymic mice can also serve as a model for infection with another reported ocular pathogen, V. corneae.

The role of humoral immunity in human microsporidiosis is unclear. Studies of antibody responses to microsporidial infection in humans have produced variable results. This variability may be attributable to differences in the immune status of the populations being studied, subclinical microsporidial infection, or cross reactivity with other pathogens. Although a specific antibody response may contribute to host resistance to microsporidial infection, antibodies alone are not protective. In fact, in some domestic dogs, the antibody response to microsporidiosis may contribute to disease by causing immune complex renal failure.24

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LABORATORY DIAGNOSIS
The laboratory methods used to confirm microsporidial disease in humans depend on the tissue source and the technical capacity of the laboratory. Because the treatment of microsporidiosis is not always genera specific, basic diagnostic techniques often simply confirm the presence or absence of microsporidia.27 Light microscopy with special stains can quickly reveal intracellular spores in conjunctival swab specimens or corneal biopsies. More sophisticated testing, such as electron microscopy and molecular genetic analysis, can be helpful in identifying the genus and species of pathogenic microsporidia.

In the first reported case of ocular microsporidiosis, Ashton and Wirasinha2 described the histopathologic characteristics of spores within the cornea as “refractile oval bodies” having an average dimension of 3.5 × 1.5 μm. They reported that microsporidia from ocular tissue can be well visualized with Giemsa staining, but that Giemsa stain is less useful with stool specimens because of the presence of debris and artifact material.

On Gram's stain, microsporidia will stain weakly positive. Spores can also be visualized using acid-fast stains or phase-contrast microscopy, but they are sometimes missed on routine hematoxylin-and-eosin stain. If microsporidial infection is suspected, a modified trichrome stain is quite reliable and usually readily available. Chemofluorescent agents such as Calcofluor White 2MR (American Cyanamid Corp, Princeton, NJ) can also enhance detection sensitivity in cases missed by modified trichrome staining.28

Electron microscopy can identify microsporidia when traditional light microscopy fails to reveal spores. Although ultrastructural detail provided by electron microscopy can help distinguish between microsporidia of different genera, it is time consuming and not always readily available. Molecular methods of identifying microsporidia that use PCR have been incorporated in research, but their clinical application is not yet widespread. In one recent case report, a PCR-based assay was used to facilitate the diagnosis of microsporidial keratitis in an HIV-positive patient.29 PCR-based diagnostic assays hold much promise for improvement in the sensitivity of tests for microsporidiosis.

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SUSCEPTIBILITY TO THERAPEUTIC AGENTS
The pharmacologic treatment of microsporidiosis depends in part on the tissues that are infected. Some species tend to cause localized infection, whereas others typically cause disseminated disease. Numerous medications have been used to treat ocular microsporidiosis, including trimethoprim-sulfisoxazole, metronidazole, propamidine isethionate (Brolene), and thiabendazole. However, the two most common medications used to treat microsporidiosis in humans are fumagillin and albendazole.

In 1993, there were at least two reports about the treatment of microsporidial keratoconjunctivitis with topical fumagillin.30,31 This crystalline antibiotic is produced from the fungus Aspergillus fumigatus. Although it originally was used to treat microsporidiosis in honeybees infected with N. apis, a fumagillin analogue has been studied as an angiogenesis inhibitor and as a potential anticancer agent.32 Topical fumagillin appears to be the treatment of choice for microsporidial keratoconjunctivitis, but its systemic use has been limited because of the medication's side effect of bone marrow toxicity (e.g., thrombocytopenia and neutropenia). Nonetheless, oral fumagillin is a promising treatment of recalcitrant intestinal E. bieneusi infection in patients with AIDS.33

Albendazole is used to treat microsporidiosis because it inhibits microtubule polymerization and interferes with cellular division. It has been used to counteract infections with echinococcus, Taenia solium, and Strongyloides stercoralis. Albendazole is administered orally and has shown particular efficacy in treating E. intestinalis infection.32 For ocular microsporidiosis, albendazole is effective in both immunocompromised and immunocompetent patients. The side effects of albendazole are usually reversible but can include hepatotoxicity, neutropenia, and alopecia. In cases of ocular microsporidiosis when albendazole is ineffective, the antifungal agent itraconazole may also be considered.34

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REFERENCES

1. Wittner M, Weiss LM: The Microsporidia and Microsporidiosis, Washington, D.C.: ASM Press, 1999

2. Ashton N, Wirasinha PA: Encephalitozoonosis (Nosematosis) of the cornea. Br J Ophthalmol 57:669, 1973

3. Franzen C, Muller A: Microsporidiosis: human diseases and diagnosis. Microbes Infect. 3:389, 2001

4. Lewis NL, Francis IC, Hawkins GS, et al: Bilateral microsporidial keratoconjunctivitis in an immunocompetent non-contact lens wearer. Cornea 22:374, 2003

5. Theng J, Chan C, Ling ML, et al: Microsporidial keratoconjunctivitis in a healthy contact lens wearer without human immunodeficiency virus infection: Ophthalmology 108:976, 2001

6. Lowder CY, Meisler DM, McMahon JT, et al: Microsporidia infection of the cornea in a man seropositive for human immunodeficiency virus. Am J Ophthalmol 109:242, 1990

7. Didier ES, Didier PJ, Friedburg DN, et al: Isolation and characterization of a new human microsporidian, Encephalitozoon hellem, from three AIDS patients with keratoconjunctivitis. J Infect Dis 163:617, 1991

8. Friedberg DN, Stenson SM, Orenstein JM, et al: Microsporidial keratoconjunctivitis in acquired immunodeficiency syndrome. Arch Ophthalmol 108:504, 1990

9. Mietz H, Franzen C, Hoppe T, et al: Microsporidia-induced sclerouveitis with retinal detachment. Arch Ophthalmol 120:864, 2002

10. Keeling PJ, Fast NM: Microsporidia: Biology and evolution of highly reduced intracellular parasites. Annu Rev Microbiol 56:93, 2002

11. Katinka MD, Duprat S, Cornillot E, et al: Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 414:450, 2001

12. Hirt RP, Logsdon JM, Healy B, et al: Microsporidia are related to fungi: Evidence from the largest subunit of RNA polymerase II and other proteins. Proc Natl Acad Sci 96:580, 1999

13. Williams BAP, Hirt RP, Lucocq JM, et al: A mitochondrial remnant in the microsporidian Trachipleistophora hominis. Nature 418:865, 2002

14. Cotte L, Rabodonirina M, Chapuis F, et al: Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. J Infect Dis 180:2003, 1999

15. Dowd SE, Gerba CP, Pepper IL: Confirmation of human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water. Appl Environ Microbiol 64:3332, 1998

16. Enriquez FJ, Taren D, Cruz-Lopez A, et al: Prevalence of intestinal Encephalitozoonosis in Mexico. Clin Infect Dis 26:1227, 1998

17. Dowd SE, John D, Eliopolus J, et al: Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis, and Cryptosporidium parvum in water used for drinking. J Water Health 1:117, 2003

18. Weiss LM: …And now microsporidiosis. Ann Intern Med 123:955, 1995

19. Silverstein BE, Cunningham ET, Margolis TP, et al: Microsporidial keratoconjunctivitis in a patient without human immunodeficiency virus infection. Am J Ophthalmol 124:395, 1997

20. Cali A, Meisler DM, Lowder CY, et al: Corneal microsporidioses: characterization and identification. J Protozool 38:215S, 1991

21. Moon SJ, Mann PM, Matoba AY: Microsporidial keratoconjunctivitis in a healthy patient with a history of LASIK surgery. Cornea 22:271, 2003

22. Bigliardi E, Sacchi L: Cell biology and invasion of the microsporidia. Microbes Infect 3:373, 2001

23. Franzen C: Microsporidia: How can they invade other cells? Trends Parasitol. 20:275, 2004

24. Didier ES, Snowden KF, Shadduck JA: Biology of microsporidian species infecting mammals. Adv Parasitol 40:283, 1998

25. Snowden KF, Didier ES, Orenstein JM, et al: Animal models of human microsporidial infections. Lab Anim Sci. 48:589, 1998

26. Braunfuchsova P, Salat J, Kopecky J: Comparison of the significance of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon cuniculi infection. J Parasitol 88:797, 2002

27. Orenstein JM: Diagnostic pathology of microsporidiosis. Ultrastruct Pathol 27:141, 2003

28. Garcia LS: Laboratory identification of the microsporidia. J Clin Microbiol 40:1892, 2002

29. Conners MS, Gibler TS, Van Gelder RN: Diagnosis of microsporidia keratitis by polymerase chain reaction. Arch Ophthalmol 122:283, 2004

30. Diesenhouse MC, Wilson LA, Corrent GF, et al: Treatment of microsporidial keratoconjunctivitis with topical fumagillin. Am J Ophthalmol 115:293, 1993

31. Rosberger DF, Serdarevic ON, Erlandson RA, et al: Successful treatment of microsporidial keratoconjunctivitis with topical fumagillin in a patient with AIDS. Cornea 12:261, 1993

32. Gross U: Treatment of microsporidiosis including albendazole. Parasitol Res 90:S14, 2003

33. Molina JM, Goguel J, Sarfati C, et al: Trial of oral fumagillin for the treatment of intestinal microsporidiosis in patients with HIV infection. AIDS 14:1341, 2000

34. Rossi P, Urbani C, Donelli G, et al: Resolution of microsporidial sinusitis and keratoconjunctivitis by itraconazole treatment. Am J Ophthalmol 127:210, 1999

35. Visvesvara GS, Belloso M, Moura H, et al: Isolation of Nosema algerae from the cornea of an immunocompetent patient. J Eukaryot Microbiol 46:10S, 1999

36. Canning EU, Curry A, Vavra J, et al: Some ultrastructural data on Microsporidium ceylonensis, a cause of corneal microsporidiosis. Parasite 5:247, 1998

37. Rauz S, Tuft S, Dart JK, et al: Ultrastructural examination of two cases of stromal microsporidial keratitis. J Med Microbiol 53:775, 2004

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