Microsporidia are obligate intracellular, spore-forming parasites infecting every major animal group, especially insects, fish, and mammals [1, 2, 4]. They are sufficiently unique to be classified in a separate phylum, Microspora [1, 2]. Within the phylum are dozens of genera and hundreds of species [4].

Microsporidia have a life cycle consisting of three phases: a proliferative phase, the spore production phase (sporogonial phase), and the spore or infective phase [1, 2]. The spore is characteristic for the phylum (Figure 1) in that it is unicellular, with a resistant spore wall, one nucleus or two abutted nuclei (diplokaryon), sporoplasm, an anchoring disk, and an extrusion apparatus consisting of a single polar tube with an anterior attachment complex [1, 2, 4]. Although spores are environmentally resistant, they may be killed by exposure for 30 minutes to 70% ethanol, 1% formaldehyde, or 1% hydrogen peroxide or by placing them in an autoclave at 120 °C for 10 minutes. In general, spores range in size from 1 to 12 µm.

View larger version (69K): [in this window] [in a new window]   Figure 1. Structure of a microsporidian spore. Depending on the species, the size of the spore can vary from 1 to 10 µm and the number of polar tubule coils can vary from a few to 30 or more. A equals the anchoring disk (the extrusion apparatus consists of the polar tube [Pt], tubular polaroplast [Tp], lamellar polarplast [Lp], and the anchoring disk and is used to identify microsporidia); D equals the nucleus, which may be single (such as in Encephalitozoonae and Enterocytozoonae) or a pair of abutted nuclei called a diplokaryon (such as in Nosema); En equals the endospore, an inner thicker electron-lucent region; Ex equals the exospore, an outer electron-dense region; P equals the plasma membrane separating the spore coat from the sporoplasm (Sp); Pv equals posterior vacuole, a membrane-bound structure; R equals ribosomes, present in a coiled helical array. Reproduced with permission from Cali A, Owen RL. Microsporidiosis. In: Ballows A, Hausler W Jr, Lennette EH, eds. The Laboratory Diagnosis of Infectious Diseases: Principles and Practice. New York: Springer-Verlag; 1988:928-49.

The spore coat consists of an electron-dense, proteinaceous exospore, an electron-lucent endospore composed of chitin and protein, and an inner membrane or plasmalemma [1, 2, 4]. The extrusion apparatus consists of a long polar tube that is attached to the inside of the anterior end of the spore by an anchoring disc, and forms from 4 to approximately 30 coils around the sporoplasm in the spore, depending on the species. While inside the spore, the core of the polar tube contains a fine, particulate, electron-dense material; consequently, the polar tube is sometimes referred to as a polar filament before discharge. The polar tube is a unique vehicle for transmission of infection—by piercing an adjacent host cell, thereby inoculating the sporoplasm directly into that cell, the tube essentially functions as a hypodermic needle.

The classification of microsporidia is currently based on spore structure and developmental life cycle as shown by electron microscopy [4]; molecular taxonomy based on ribosomal RNA (rRNA) is now also being used [5-7]. Within their hosts, microsporidia have been reported from every tissue and organ type.

Six microsporidian genera have been associated with human disease [1, 2]: Nosema (N. corneum has been recently renamed Vittaforma corneae [8]), generally found in insects; Pleistophora, a pathogen of fish and insects; Encephalitozoon, found in many mammals; Enterocytozoon, found in patients with AIDS [9] and several species of fish [10]; and Septata (Encephalitozoon [7]), also found in patients with AIDS [11]. A seventh genus, Microsporidium, has been used to designate microsporidia of uncertain taxonomic status [1]. Pleistophora has been associated with myositis [1, 2]. Encephalitozoon hellem has been associated with superficial keratoconjunctivitis [12] in patients with HIV infection, and these infections reportedly respond to topical fumagillin [12]. Encephalitozoon hellem has also been associated with sinusitis, respiratory disease, prostatic abscesses, and disseminated infection [1, 2], and these infections may respond to albendazole. Nosema, Vittaforma, and Microsporidium have been associated with stromal keratitis, which is seen with trauma in immunocompetent hosts [12]. Enterocytozoon bieneusi, first described in 1985 [9], is associated with malabsorption and diarrhea and has been described only in humans [1, 2]. Encephalitozoon (Septata) intestinalis causes enteric and disseminated infections in patients with AIDS [11] and is found in enterocytes and cells of the lamina propria and urinary tract. On morphologic analysis, Septata intestinalis was most closely aligned with the family Encephalitozoonidae, but its unique structure led to the establishment of a new genus for this organism [11]. Because recent studies on small subunit rRNA have shown that Septata intestinalis is closely related to Encephalitozoon hellem and Encephalitozoon cuniculi, its status as a separate genus has been challenged, and it has been renamed Encephalitozoon intestinalis [7, 13].

Antibodies to Encephalitozoon cuniculi have been found in humans, but it is uncertain whether these represent infection, cross-reactivity to other microsporidian species, or nonspecific reactions [1, 2]. Because Enterocytozoon bieneusi has not been propagated in cell culture or laboratory animals, specific serologic assays remain unavailable. Serologic testing has not proved useful in diagnosing human microsporidial infections [1, 2]. Definitive (that is, species) diagnosis of microsporidiosis currently requires electron microscopic visualization of spore ultrastructure and developmental stages; however, species-specific polymerase chain reaction rRNA primers have been described [6, 14].

In patients with enteric infection, the small intestine has provided the highest diagnostic yield, but organisms have been seen in colon biopsy specimens. In paraffin-embedded sections, microsporidia are discernible with hematoxylin-eosin, tissue Gram, or chromotrope 2R stains [1]. Giemsa-stained touch preparations and semi-thin plastic sections are also useful [1, 2]. Polyclonal serum prepared to Encephalitozoon cuniculi and monoclonal antibodies to Encephalitozoon hellem have been reported to react with Enterocytozoon bieneusi [1, 2, 15]. Reactions of the polymerase chain reaction assay for Encephalitozoon cuniculi, Enterocytozoon bieneusi, and Encephalitozoon (Septata) intestinalis based on cloned rRNA genes from these organisms have been described [1, 6, 14].

Microsporidia have also been seen in stool specimens by electron microscope, modified trichrome stain (chromotrope 2R), Uvitex 2B, and calcofluor white [1]. The sensitivity and specificity of these techniques are being evaluated. The use of a two-step screening process on stool with calcofluor white or Uvitex 2B to identify microsporidia, followed by confirmation with a modified trichrome stain [1], appears to be useful for the noninvasive diagnosis of microsporidiosis.

The major syndrome associated with microsporidiosis is diarrhea and wasting. This condition is usually caused by Enterocytozoon bieneusi (> 90% of cases in the United States) and occasionally by S. intestinalis (although in Europe this organism may be a more frequent cause of diarrhea than is Enterocytozoon bieneusi) [1, 2]. Several studies have been done on the association between microsporidiosis and diarrhea. In HIV-infected patients evaluated for diarrhea, the prevalence of microsporidiosis has ranged from 10% to 40% [1, 2]. In a prospective study, Rabeneck and colleagues [16] found microsporidia in 29% of patients seen at an HIV primary care clinic; however, they saw no association with the presence of diarrhea (18 of 55 patients with diarrhea and 13 of 51 patients without diarrhea had microsporidia on examination of duodenal biopsy specimens). Patients with microsporidiosis in this study had a mean CD4 count of 113 cells/mm³ [16]. During a 15-month follow-up, diarrhea developed in 2 of the 13 asymptomatic patients and continued in the 18 symptomatic patients [17]. In a study of patients presenting to a gastroenterology clinic, Kotler and Orenstein [18] found that 39% of patients with HIV and diarrhea (55 of 141 patients) had microsporidiosis and that the presence of microsporidia was associated with wasting, a mean CD4 count of 28 cells/mm³, and an abnormal D-xylose test result. In contrast, only 1 of 38 HIV-infected patients without diarrhea (2.6%) had microsporidiosis, and this patient subsequently developed diarrhea on follow-up [18].

Using a polymerase chain reaction with primers to an rRNA gene of Enterocytozoon bieneusi [6, 14], Coyle and colleagues (19; manuscript in preparation) found that 3% (1 of 35) of HIV-infected patients without diarrhea and 32% (18 of 56) of HIV-infected patients with diarrhea had microsporidiosis. The one asymptomatic patient had an abnormal D-xylose test result.

These studies suggest that microsporidia are associated with gastrointestinal disease. Recent information on the utility of albendazole for treating microsporidial infection provides further evidence of the association between microsporidia and diarrhea. Therapy with albendazole (400 mg twice daily) eliminated Encephalitzoon (Septata) intestinalis infection and its symptoms [20]. Furthermore, diarrhea was markedly reduced in 50% of patients with Enterocytozoon bieneusi infection who received albendazole treatment [21]. In such treated patients, however, the organism can still be seen in biopsy specimens [21], and relapse is common when treatment is stopped. Although Enterocytozoon bieneusi cannot be cultured in vitro, the other microsporidia that infect humans have now been cultured in vitro and screening for new active agents is in progress [1, 2].

Since microsporidial infection was first described in HIV-infected patients in 1985, our knowledge of the number of microsporidia infecting humans and our ability to treat and diagnose infection with these pathogens has greatly increased. Thus, Enterocytozoon bieneusi has been reported as an important cause of diarrhea and wasting syndrome, as well as biliary system disease, in patients with AIDS and has now been found in immunocompetent patients. Microsporidia of the family Encephalitozoonae have been associated with disseminated disease, diarrhea, sinusitis, and ocular infections. Our ability to culture Encephalitozoonae in vitro and to treat and diagnose infections with these organisms has dramatically increased. However, the most common microsporidian in humans, Enterocytozoon bieneusi, has not been cultured in vitro and both noninvasive diagnosis and better therapeutic agents are needed.

The epidemiology of microsporidiosis is poorly understood. Most likely, these are waterborne pathogens and the environmental reservoirs need to be identified. The importance and prevalence of these organisms in our water supplies is an open question. It is clear, from recent case reports, that these organisms are found in immunocompetent as well as immunocompromised hosts, and, as our diagnostic acumen and testing improve, their importance in other disease syndromes will become clear. As is true for many emerging pathogens, we have just scratched the surface of a complex and evolving relation between the phylum Microspora and ourselves.