INTRODUCTION

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Microsporidia are obligate intracellular protozoan parasites that infect a broad range of vertebrates and invertebrates. In 1857 these parasites were first recognized as pathogens in silkworms (254), and long before they were described as human pathogens, they were recognized as a cause of disease in many nonhuman hosts including insects, mammals, and fish (39, 50, 51, 56). Therefore, they are responsible for considerable infectious disease problems in industries such as fisheries and silk production (39, 50, 56). The first human case of microsporidial infection was reported in 1959 (237), and only 10 well-documented human infections with microsporidia were described until 1985, when a new species, Enterocytozoon bieneusi, was found in a human immunodeficiency virus (HIV)-infected patient in France (99, 245). Since then, many infections with microsporidia have been reported from all over the world, and these parasites are now recognized as one of the most common pathogens in HIV-infected patients (7, 36-38, 53, 55, 56, 60, 66, 72, 87, 88, 96, 124, 130, 131, 136, 142-144, 162, 195, 204-208, 223, 225, 241, 246, 247, 253, 263, 265, 311, 325, 338, 344, 371, 372, 375).

The term "microsporidia" is a nontaxonomic designation commonly used for organisms belonging to the phylum Microspora. This phylum consists of over 100 genera with almost 1,000 species. So far only six genera (Enterocytozoon, Encephalitozoon [including Septata], Pleistophora, Trachipleistophora, Vittaforma, and Nosema) with at least 12 different species belonging to these six genera as well as unclassified microsporidia have been described as pathogens in humans.

For most patients with infectious diseases, microbiological isolation and identification techniques offer the most rapid and specific determination of the etiologic agent (378). This is not a suitable procedure for microsporidia, which are obligate intracellular parasites requiring cell culture systems for growth. Visualization of organisms in cytologic smears, tissue sections, or both is commonly used for diagnosis of infections with microsporidia. However, ultrastructural analysis by transmission electron microscopy is usually necessary for exact species differentiation. This technique may lack sensitivity, and species differentiation can be missed.

During the last 10 years, the detection of infectious disease agents has begun to use nucleic acid-based technologies. Diagnosis of infection caused by parasitic organisms is the last field of clinical microbiology to incorporate these techniques (378). In this paper, we review human microsporidial infections and newly developed molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia with special emphasis on species that infect humans.

All microsporidia are obligate intracellular parasites and have no active stages outside their host cells. They are considered to be ancient organisms, evolutionarily placed as an early branch leading from prokaryotes to eukaryotes (64, 65, 285, 362, 363). Microsporidia lack some typical eukaryotic characteristics. The ribosomes (70S), ribosomal subunits (30S and 50S), and rRNAs (16S and 23S) are of prokaryotic size, and the rRNA has no separate 5.8S rRNA (362). Although mitochondria, peroxisomes, and a classical stacked Golgi apparatus are missing, they are true eukaryotes with a nucleus, an intracytoplasmatic membrane system, and chromosome separation by mitotic spindles (362, 363); polyadenylation occurs on mRNA in microsporidia as in every other eukaryotic organism studied to date (381a).

Spore. Microsporidian spores are between 1 and 20 µm long. Species that infect mammals are usually small, with diameters of 1 to 3 µm. The spores have a thick wall, composed of three layers: (i) an electron-dense outer layer called the exospore, which is proteinaceous; (ii) an electron-lucent inner layer called the endospore, which is chitinous; and (iii) a plasma membrane enclosing the cytoplasm, the nucleus (sometimes two nuclei), a posterior vacuole, the polaroplast membranes, and the unique extrusion apparatus. The extrusion apparatus consists of a coiled polar filament and its anchoring disc, which is characteristic of all microsporidia (Fig. 1). The number and arrangement of coils of the polar filament vary among genera and species. Under appropriate conditions inside a suitable host, the polar filament is discharged through the thin anterior end of the spore, thereby penetrating a new host cell and inoculating the infective sporoplasm into the host cell (Fig. 1 and 2) (41, 43, 50, 58).

View larger version (42K): [in this window] [in a new window]   FIG. 1.   Diagram of a microsporidian spore and representative life cycle (merogonic and sporogonic stages vary among different genera).

View larger version (98K): [in this window] [in a new window]   FIG. 2.   Giemsa stain of Nosema algerae spore with an extruded polar tube and with the sporoplasm at the end of the tube. Giemsa stain. Magnification, ×640.

Merogony. In suitable host cells, the sporoplasms that are released from the spores become meronts. Meronts are rounded, irregular, or elongated simple cells with little differentiated cytoplasm, enclosed by a plasma membrane. Meronts may have isolated or diplokaryon nuclei. Inside the host cell, there is a phase of repeated divisions by binary or multiple fissions called merogony. Nuclear division may occur without cell division, resulting in multinucleated plasmodial forms (41, 43, 50, 58).

Sporogony. Meronts develop into sporonts, which are characterized by a dense surface coat. This surface coat later develops into the exospore layer of the spore wall. Sporonts multiply by binary or multiple fission and divide into sporoblasts that will finally develop into mature spores. Sporonts may have isolated or diplokaryon nuclei. Some sporonts divide directly into sporoblasts by binary fission, whereas others become multinucleated plasmodial stages. Sporoblasts are ovoid bodies that will mature to spores by synthesis of spore organelles (Fig. 1) (41, 43, 50, 58).

The term "microsporidia" is a nontaxonomic designation commonly used for organisms belonging to the phylum Microspora, which is contained within the subkingdom Protozoa (50, 337). In 1882 Balbiani classified these parasites as a separate group, "Microsporidies" (12). Before the middle of this century, since knowledge of this group of organisms was fragmentary, classifications of microsporidia were necessarily simple and artificial. Subsequently, the taxonomy of microsporidia has been subjected to several modifications. Major published microsporidian classifications differ considerably in the characteristics used to produce the major divisions within the microsporidia (337). Larsson (214) considered that many features traditionally used for taxonomic systems (for example, the diplokaryon, sporophorous vesicle, meiosis) have evolved independently in several lineages, and therefore seemed not to be useful for phylogenetic analysis. In his classification system, based on differences in ultrastructural morphology, several characters were subdivided into well-defined categories, thereby creating a tree representing the phylogeny of the microsporidia (214). Weiser (376) based his classification only on the nuclear condition of the spores (one nucleus in Pleistophoridida or two nuclei in Nosematidida), whereas Issi (183) used the spore morphology and developmental stages. Until recently, the classification system of Sprague, proposed in 1977 and updated in 1982, was the most widely used (335, 336). In this scheme the microsporidia were divided into two groups, based on the presence or absence of a membrane surrounding the parasites: the Pansporoblastina (membrane present) and the Apansporoblastina (membrane absent). In systems developed during the last decade, it seems that differences in chromosome cycles constitute the most fundamental basis for distinguishing taxa at the highest level (52, 337). Based on this concept, Sprague et al. (337) proposed a comprehensive revision of the classification system in which differences in the nuclear state and their implications for the chromosome cycle were treated as the most fundamental taxonomic characters (Fig. 3). The microsporidia were separated into the Dihaplophasea, which have a diplokaryon in some phase of their life cycle, and the Haplophasea, which have unpaired nuclei in all stages of their life cycle. Phylogenetic trees constructed on the basis of DNA sequence data now show clearly that this and other classification systems do not reflect the true relationships among microsporidia and that the classification of microsporidia should be completely revised.

View larger version (10K): [in this window] [in a new window]   FIG. 3.   Taxonomy of microsporidia infecting humans, using the revised taxonomy of Sprague et al. from 1992 (337), modified in light of the new taxonomic classification of Vittaformae corneae (324), Encephalitozoon intestinalis (10, 166), Trachipleistophora hominis (180), and T. antropophtera (356a). Comparing this taxonomic system with phylogenetic trees generated on the basis of DNA sequence data (see Fig. 13), it seems clear that the system of Sprague et al. (337) is flawed.

Nucleotide sequence data of the small-subunit (SSU) rRNA of a microsporidian from insects, Vairimorpha necatrix, suggested that microsporidia are very ancient organisms and that the evolutionary developments leading to microsporidia branched very early from those leading to eukaryotes (363). DNA data for protein-encoding genes supported this thesis (35, 64, 65, 186, 187, 387), but phylogenetic trees constructed on the basis of - and -tubulin sequences suggested that the microsporidia are close relatives of fungi, which may be evolved degeneratively from higher forms (125, 126, 221). Recently, genes encoding Hsp70 (a heat shock protein or chaperonin) have been identified in the microsporidia Nosema locustae, V. necatrix, Encephalitozoon hellem, and Encephalitozoon cuniculi, and phylogenetic analyses have shown unequivocally that these genes are most closely related to those encoding Hsp70 proteins from the mitochondria of other eukaryotes, suggesting that microsporidia may be evolved degeneratively from higher forms (159, 173, 193, 251a, 275a). This possible degenerative evolution is discussed in more detail below.

SSU and large-subunit (LSU) rRNA and protein-encoding DNA sequences are now available for several microsporidian species, including six species infecting humans. The taxonomy of microsporidia will be amended significantly in the near future when these and newly generated nucleotide sequence data are considered for future classification systems. For example, molecular analyses have led to the reclassification of Septata intestinalis into Encephalitozoon intestinalis (10, 166). However, this reclassification is still controversial on the basis of ultrastructural data and rules of taxonomy (49). Several phylogenetic trees based on DNA sequence data have been suggested recently (9-11, 125, 233, 280, 364, 365, 381, 396) and are discussed below.

GENUS- AND SPECIES-SPECIFIC CHARACTERISTICS

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Enterocytozoon sp.

To date, there is only one member in the genus Enterocytozoon, Enterocytozoon bieneusi. This organism develops in direct contact with the host cell cytoplasm. Meronts often have electron-lucent inclusions which are present throughout the life cycle. Sporonts form electron-dense precursors of the polar tube and the anchoring disk, which develop before sporogonial plasmodia divide into sporoblasts. Multiple sporoblasts are formed by invagination of the plasma membrane of one large sporogonial plasmodium. Spores are oval and small, measuring only 1.1 to 1.6 by 0.7 to 1.0 µm, with five to seven coils of the polar tubule, arranged in two rows (Fig. 4) (42, 53, 99, 100, 318).

View larger version (155K): [in this window] [in a new window]   FIG. 4.   Transmission electron micrograph of duodenal epithelium from an HIV-infected patient heavily parasitized with Enterocytozoon bieneusi. Several cells are infected with merogonic and sporogonic stages, and one cell is infected with darkly staining spores. Magnification, ×6,600.

E. bieneusi was first detected by Modigliani et al. (245) and described in detail by Desportes et al. (99) in 1985 following examination of a 29-year-old Haitian AIDS patient with chronic diarrhea who lived in France. A similar case was described in the United States in the same year (119). Since then, the number of reported cases has steadily increased in Europe, North and South America, Africa, and Australia (7, 36, 53, 55, 56, 60, 72, 83, 87, 96, 103, 124, 127, 130, 131, 136, 142, 244, 246, 263, 325, 389). The parasite usually infects intestinal enterocytes of HIV-infected patients but has been also detected in lamina propria cells of small-bowel biopsy specimens, biliary tree, gallbladder, liver cells, pancreatic duct, and tracheal, bronchial, and nasal epithelia (93, 129, 165, 250, 278, 279, 310, 367, 368).

The second and last member of the family Enterocytozooidae is Nucleospora salmonis. This microsporidium was originally described by Hedrick et al. in 1991 (169), but shortly thereafter Chilmonczyk et al. (67) described it as Enterocytozoon salmonis. Ultrastructural examinations showed morphological similarities between E. bieneusi and N. salmonis, with Nucleospora exhibiting most of the distinguishing morphological characteristics of the family Enterocytozoonidae. However, in contrast to E. bieneusi, N. salmonis grows in the nucleus rather than in the cytoplasm of cells and parasitizes fish rather than humans (120, 197, 386). Desportes-Livage et al. (101) further described several ultrastructural differences in the development of these two genera. Based on rRNA sequence data first generated by Barlough et al. (13), rules of taxonomy, and the morphology and intranuclear location of the organism, it has been suggested that in the absence of significant reasons for the suppression of the generic name Nucleospora, the original name N. salmonis rather than E. salmonis is valid (120).

All Encephalitozoon species develop within parasitophorous vacuoles. Meronts divide by binary fission and usually remain in the vacular membrane. Sporonts develop a thick surface coat which becomes the exospore of spores, and the sporonts divide into sporoblasts which will develop into spores (Fig. 5). Spores measure 2.0 to 2.5 by 1.0 to 1.5 µm, and the polar tubule has five to seven coils in a single row (Fig. 6) (50, 57, 58, 318, 333).

View larger version (160K): [in this window] [in a new window]   FIG. 5.   Transmission electron micrograph of duodenal epithelium from an HIV-infected patient infected with Encephalitozoon cuniculi. Two meronts with single nuclei, four sporonts with a thickened plasma membrane and highly developed endoplasmatic reticulum, and four spores inside a parasitophorous vacuole are visible. Magnification, ×17,500.

View larger version (125K): [in this window] [in a new window]   FIG. 6.   Transmission electron micrograph of an Encephalitozoon cuniculi spore in nasal discharge from a patient with AIDS and chronic rhinosinusitis. The spore contains a polar tubule with six coils lying in a single row and is coated with an electron-dense exospore. Magnification, ×55,125.

E. cuniculi was the first microsporidium to be recognized as a parasite of mammals. First found in rabbits in 1922 (388), this microsporidium was named by Levaditi et al. in 1923 (220). Subsequently, it has been detected in many mammalian hosts, including humans (50, 51). To date, E. cuniculi is the best studied of the microsporidian species, and much of what is known about the pathogenesis of microsporidial disease has been derived from studies of this organism.

Two pathogenic species of Encephalitozoon which infect humans, E. cuniculi and E. hellem, are morphologically similar by light and electron microscopy and can be distinguished only by antigenic, biochemical, or nucleic acid analysis (104, 112). Several cases of Encephalitozoon infection were reported to occur in patients with and without AIDS prior to 1991. Light and/or electron microscopic analysis indicated that these infections appeared to be due to E. cuniculi. However, in 1991 Didier et al. (104) used biochemical and antigenic methods to describe a new species of Encephalitozoon, E. hellem, which had been found in three patients with AIDS. Since all subsequently published cases of Encephalitozoon infections in humans appeared to be caused by E. hellem (113, 162, 178, 211, 304-306, 369), there was some doubt whether E. cuniculi did in fact cause human infection (293). However, in 1995 De Groote et al. (95) and Franzen et al. (144) described two homosexual men with AIDS and disseminated E. cuniculi infection; identification was confirmed by an immunofluorescence assay and by DNA identification. Recently, E. cuniculi has been detected in several HIV-infected patients (97, 177, 179, 242, 271, 374).

A third Encephalitozoon species, E. intestinalis, infecting HIV-infected patients, was first described in 1992 by Orenstein et al. (266, 268) as a microsporidium with ultrastructural similarities with the genus Encephalitozoon. It was later classified as a new genus and species, Septata intestinalis by Cali et al. (47) on the basis of ultrastructural differences. Based on rRNA sequence data, it has been suggested that this organism be placed in the genus Encephalitozoon and renamed Encephalitozoon intestinalis (10, 166). This reclassification is still controversial (49), as discussed below. E. intestinalis shows a unique parasite-secreted fibrillar network surrounding the developing parasites, so that the parasitophorous vacuole appears septate (Fig. 7) (46, 47, 59, 266).

View larger version (184K): [in this window] [in a new window]   FIG. 7.   Transmission electron micrograph of duodenal epithelium of an HIV-infected patient infected with Encephalitozoon intestinalis. One meront with two nuclei, four sporonts with thickened plasma membrane, and four spores are separated by amorphous material which leads to septation of the parasitophorous vacuole. Magnification, ×17,500.

Most Nosema species are parasitic in invertebrates (41, 58). Their development takes place in direct contact with the host cell cytoplasm, and nuclei are paired throughout the entire life cycle (41, 58).

Although microsporidia of the genus Nosema are widespread parasites, only a few human infections with Nosema spp. have been reported. A case of systemic infection occurred in a 4-month-old thymus-deficient infant (235). At autopsy, numerous mature and immature microsporidian spores measuring 4.0 to 4.5 by 2.0 to 2.5 µm with nuclei in diplokaryon arrangement and 10 to 12 coils of the polar tubule were found. No other developmental stages were documented, but the features of the spores supported its assignment to the genus Nosema as a new species, Nosema connori (235, 334). A microsporidium species infecting the corneal stroma of a 39-year old man from Ohio was named Nosema ocularum (36, 39, 44). Spores were lying freely in direct contact with the host cell cytoplasm and measured 3.0 by 5.0 µm with 9 to 12 coils of the polar tubule (36, 44, 45).

Another microsporidium infecting muscle cells of a 31-year-old HIV-infected patient was described by Cali et al. (48). Development took place in direct contact with the muscle cell cytoplasm, and the organisms contained one or two diplocaryotic pairs of nuclei. The spores measured about 2.5 to 2.9 by 1.9 to 2.0 µm, with 7 to 10 turns of the polar tubule. These features are most closely aligned with the genus Nosema, and this organism is currently named "Nosema-like microsporidian" (48).

Another Nosema-like microsporidium was identified in fecal material of a patient with AIDS (239). Because all the parasites were located in partially digested striated muscle cells, it was concluded that this did not represent a true infection (239).

In 1990, Davis et al. (92) described an otherwise healthy 45-year-old man with an 18-month history of unilateral progressive central keratitis. Microsporidian spores measuring 3.7 by 1.0 µm were identified in deep corneal stroma and were isolated in cell cultures (317). The spores contained polar tubules with six coils and had nuclei in diplokaryotic arrangements. In cell culture, all the observed stages were detected individually in the host cell cytoplasm. This organism was originally assigned to the genus Nosema and named Nosema corneum (317), even though the diplokaryotic arrangement of the nuclei was the only character that conformed with the description of the genus Nosema. Based on the ultrastructure of developmental stages in liver cells of experimentally infected athymic mice (tetrasporoblastic sporogony, band-like sporonts, all stages surrounded by a cisterna of host endoplasmatic reticulum), this organism was later transferred to a new genus and named Vittaforma corneae (323, 324). The reclassification on ultrastructural grounds was later supported by SSU rRNA gene sequence data, which placed Vittaforma distant from Nosema (9, 10). A case of disseminated V. corneae infection recently occurred in Switzerland (375).

Pleistophora spp. are common parasites of fish, and only a few infections have been reported in humans. Three cases of Pleistophora-like microsporidian infection involving skeletal muscles have been described in two HIV-infected patients and in a non-HIV-infected patient (69, 161, 216). The parasites develop within a vesicle, bounded by a thick parasite-formed coat named the sporophorous vesicle. The spores measured 2.0 to 2.8 by 3.0 to 4.0 µm with 10 to 12 coils of the polar tube.

The genus Trachipleistophora was established for a microsporidium responsible for a fourth case of myositis, this time in a patient with AIDS; organisms were found in corneal scrapings, skeletal muscle, and nasal discharge (138). These parasites were cultivated in vitro and in athymic mice (180). Meronts had two to four nuclei and divided by binary fission. In sporogony, the surface coat became separated from the plasma membrane and formed a sporophorous vesicle. The parasite differed from the genus Pleistophora, because no multinucleate sporogonial plasmodium was formed at any stage. Thus, this organism was placed in a new genus and named Trachipleistophora hominis (180).

Recently, two cases of infection with a Pleistophora-like microsporidian, which also seems to be a species of Trachipleistophora, have been reported (271, 390). Sporogony distinguishes this parasite from T. hominis since two different types of sporophorous vesicles and spores are formed (390), and the parasite has recently been classified as a new species T. antropophtera (356a).

One of the Pleistophora-like microsporidia involving skeletal muscles (69), which was described before T. hominis was described as a new species, resembles T. hominis, whereas other Pleistophora-like microsporidia (216) may be different (180, 181).

The collective group Microsporidium is an assemblage of identifiable species for which the generic positions are uncertain because details of their life cycle are missing (50).

Microsporidium ceylonensis was identified in a corneal ulcer of an 11-year-old Tamil boy from Sri Lanka. The spores measured 1.5 by 3.5 µm, and no meronts or sporonts were seen (6, 50). Microsporidium africanum was detected in corneal stroma of a 26-year-old woman from Botswana suffering from a perforated corneal ulcer (50, 277). Spores with 15 to 16 turns of the polar tubule measured 4.5 by 1.5 µm, and no developmental stages of the parasite were seen.

Many other genera in several invertebrate phyla and in all five classes of vertebrates have been described (39, 50, 58). The number of named and unnamed species, now approaching 1,000 and belonging to nearly 100 genera, certainly represents only a small fraction of the total diversity. Examination of new hosts will continue to increase the number of microsporidian genera and species (58).

EPIDEMIOLOGY

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Prevalence and Geographic Distribution

Human infections with microsporidia have been reported from all over the world, and the majority of cases have involved HIV-infected patients (1, 14, 36, 37, 53, 56, 68, 72, 83, 87, 88, 92, 95, 99, 102, 103, 109, 110, 113, 118, 121, 122, 136, 142-144, 177, 179, 195, 204-208, 215, 238, 243, 244, 246, 247, 263, 266, 277-279, 291, 297-299, 304-306, 315, 316, 326, 328, 339, 344, 367-370, 389-391). Among persons without HIV infection, only 35 cases of microsporidiosis have been documented (Tables 1 and 2) (40, 371). Several early reports of suspected cases could not be confirmed because the original material had been lost or reexamination showed that the responsible organism was not a microsporidium (79, 341, 377). Many of the 35 affected patients lived in or had traveled to tropical or subtropical areas (96, 295, 329, 371). Intestinal E. bieneusi infection was also reported in 8 of 990 African children who lived in an area of low HIV prevalence, but the HIV serostatus of these children was unknown (34). Encephalitozoon spores were detected in 20 of 255 stool samples from persons with unknown HIV serostatus living in two rural highland villages in Mexico (133). Although microsporidia seem to be common pathogens in HIV-infected patients in Africa (34, 124, 195, 351), it is uncertain whether they are more common in tropical and subtropical areas than in Europe or North America. Little is known about the epidemiology of microsporidia, but the discovery of self-limiting infections with E. bieneusi and E. intestinalis in immunocompetent persons suggests that microsporidia may be common human pathogens (56). The wide geographical distribution and the high prevalence among HIV-infected patients suggest that microsporidia may be natural parasites of humans, causing disease only in immunosuppressed hosts (56). Recently, microsporidia have been emerging as opportunistic pathogens in organ transplant recipients being treated with immunosuppressive drugs (194, 284, 296).

More than 1,000 cases of microsporidiosis have been documented, the majority with E. bieneusi, in HIV-infected patients (14, 30, 53, 72, 87, 99, 103, 109, 110, 118, 136, 142, 207, 244, 246, 263, 278, 279, 326, 344). Between 2 and 50% of HIV-infected patients with severe immunodeficiency and CD4 cell counts below 100/µl and otherwise unexplained diarrhea are infected, depending on the study group and method of diagnosis (72, 130, 131, 136, 142, 207, 212, 246, 263). When patients do not suffer from diarrhea, E. bieneusi is only rarely reported (127, 282, 283). Rabeneck et al. (282, 283) observed no significant difference in the occurrence of microsporidiosis in patients with (18 of 55 [33%]) and without (13 of 51 [25%]) chronic diarrhea. However, these findings were not duplicated by other investigators, and support for a pathogenic role for microsporidia is based on its identification, often as the sole pathogen, in several hundred patients worldwide. It seems likely that, as with other parasites, a relationship exists between the intensity of infection and clinical illness. Because intestinal microsporidiosis may be a common infection in humans that can exist latently (148, 350, 353), microsporidia are most likely to cause disease if the immune status of a host is such that the infection cannot be controlled. However, quantitation of E. bieneusi spores in stool specimens is not correlated with intensity of diarrhea (71).

Infections with other microsporidian species have been reported less frequently, but more than 100 cases of human infections with Encephalitozoon spp. have been documented (68, 95, 102, 113, 121, 122, 136, 143, 144, 195, 215, 238, 243, 247, 266, 291, 297-299, 304-306, 328, 369, 373, 374). Most of these cases were due to E. intestinalis or E. hellem (68, 113, 121, 122, 136, 143, 195, 238, 243, 247, 266, 291, 297, 298, 304-306, 328, 369, 373), but recently E. cuniculi was detected in several HIV-infected patients as well (1, 95, 144, 177, 179, 242, 374).

Human infections with other species (N. connori, N. ocularum, V. corneae, Pleistophora spp., T. hominis, T. antropophtera, M. ceylonensis, and M. africanum) have occurred only in a few patients so far (6, 44, 48, 50, 69, 92, 138, 161, 216, 235, 277, 375) and these infections may represent only random opportunistic events.

Routes of transmission and sources of human microsporidial infections have been difficult to ascertain. Based on the distribution of lesions, oral, respiratory, and ocular routes of infection are possible and are supported by evidence obtained from experimentally infected rabbits (80, 153, 313), mice (106, 156, 209, 300, 342), and monkeys (106, 343). There is considerable serologic evidence that humans without clinical signs of infection have been exposed to microsporidia (174-176, 327, 353). Whether these persons are chronically or actively infected is unknown.

Microsporidia are released into the environment via stool, urine, and respiratory secretions. Persons or animals infected with microsporidia are possible sources of infection. Experimental Encephalitozoon infections of several animals by the oral, tracheal, and rectal routes have been reported (80, 153, 209, 313). Person-to-person transmission of microsporidia may be significant. In a case-control study, intestinal microsporidiosis was associated with male homosexuality, thereby suggesting sexual routes of transmission (181a). Person-to-person transmission was suspected in an HIV-seronegative partner of an HIV-infected man with intestinal microsporidiosis due to E. intestinalis (139). Another male patient with microsporidial urethritis had a sexual partner with diarrhea due to intestinal microsporidiosis (27).

Whether microsporidiosis in humans is a zoonosis is unknown, and no direct proof of transmission from animals to humans has been documented, with the exception of one case where a 10-year-old girl seroconverted after close contact with a dog infected with E. cuniculi (239a). Animal reservoirs of microsporidia infecting humans have been confirmed recently (Table 3). E. cuniculi is commonly found in several mammals (50, 51), and Encephalitozoon spp. have occasionally been found in lovebirds (50, 196). E. hellem was recently detected in birds (parrots) (28), E. intestinalis has been found in different mammalian animals (donkey, dog, pig, cow, and goat) in Mexico (32a), and Enterocytozoon bieneusi has been found in stool samples of pigs and dogs in Switzerland (98) and in simian immunodeficiency virus-infected macaques (234). E. hellem infection of birds, E. bieneusi infections of pigs, dogs, and monkeys, and E. intestinalis infection of different mammals were confirmed by molecular techniques with rRNA data (32a, 98, 234). Molecular analysis of different human, rabbit, dog, mouse, and blue fox E. cuniculi isolates showed that all isolates from humans were of the same subtype as isolates from dogs and rabbits (97, 98, 108, 111, 181, 236). This fact supports the hypothesis that human infections with E. cuniculi may be a zoonosis (97, 98, 111, 181, 236).

Arthropods are the most common hosts of microsporidia, and experimental infections of mice by a mosquito microsporidium (Nosema algerae) have been accomplished (342, 345). Whether insect microsporidia might infect humans is unknown.

Several microsporidia have been found in surface water samples (8), but whether human microsporidiosis is a waterborne disease is unknown. Results from recent studies involving molecular techniques seemed to indicate the presence of E. intestinalis, E. bieneusi and V. corneae in raw sewage, tertiary effluents, surface water, and groundwater in France and the United States (123a, 332a); there is one report of a presumably waterborne outbreak in Lyon (France) during summer 1995 (78a). Risk factors for intestinal microsporidiosis also suggest water as the source of infection (133, 181a). In a case-control study, the only two factors associated with intestinal microsporidiosis were swimming in a pool and male homosexuality, both suggesting that the mode of transmission is fecal-oral (181a). However, no seasonal variation in the prevalence of microsporidiosis in HIV-infected patients was seen over 4 years in southern California, suggesting a constant presence of microsporidia in the environment rather than a seasonal association with recreational water use or seasonal contamination of the water supply (75a).

Microsporidiosis is truly an emerging infectious disease with a rapidly broadening clinical spectrum of diseases. The spectrum of diseases includes gastrointestinal, pulmonary, nasal, ocular, muscular, cerebral, and systemic infections. Microsporidiosis should be considered in the differential diagnosis of HIV-related symptomatic disease of virtually all organ systems (Table 4) (271).

Enterocytozoon bieneusi. Intestinal infections with microsporidia have been found mainly in HIV-infected patients, and most infections have been due to E. bieneusi. It is most common in patients with severe immunodeficiency and a CD4 cell count below 100/µl (60, 127, 130, 131, 142, 246, 332). The parasites cause a severe, nonbloody, nonmucoid diarrhea with up to 10 or even more bowel movements per day, slowly progressive weight loss, and malabsorption of fat, D-xylose, and vitamin B₁₂ (60, 78, 127, 130, 131, 204, 205, 212). Intestinal infection is associated with lactase deficiency and a reduced activity of alkaline phosphatase and -glucosidase at the basal part of the vilus and with reduced villus height and a vilus surface reduction (301). Diarrhea appears gradually and may continue for months. Patients are often reluctant to eat and may complain of nausea (60, 78, 204, 205 212). Some patients have intermittent diarrhea, but only a few excrete microsporidial spores without having diarrhea (282, 283, 326, 330). In groups of patients with chronic diarrhea who were negative for other enteric pathogens, the prevalence of E. bieneusi was between 7 and 50% (127, 130, 131, 207, 246, 263). Some patients have coinfections with other pathogens (30, 171, 370); cryptosporidia are the most common pathogens coinfecting patients with intestinal microsporidiosis (157, 171, 370).

Encephalitozoon spp. Similar to Enterocytozoon bieneusi, E. intestinalis causes an enteritis with diarrhea, weight loss, and malabsorption (68, 70, 78, 121, 136, 143, 207, 212, 247, 266). Besides intestinal infections, these parasites may infect the biliary tract and gallbladder, resulting in cholangitis and cholecystitis (385). Disseminated infections occur regularly and involve heavy infections of the urinary tract including the kidneys (68, 121, 143, 150, 247, 268). Left untreated, small bowel infection with E. intestinalis can lead to perforation and peritonitis (331). E. cuniculi only occasionally infects the gastrointestinal tract, and its pathogenicity in humans is unknown. Franzen et al. (144) described an AIDS patient with a widely disseminated E. cuniculi infection including the gastrointestinal tract but with no accompanying gastrointestinal symptoms. Weber et al. (374) described a second patient with disseminated infection due to E. cuniculi who had no gastrointestinal symptoms but who had microsporidian spores in the stool samples.

Other species. A Nosema-like microsporidium has been identified in fecal material of a patient with AIDS (239). The parasites were located in partially digested striated muscle cells, suggesting that infected animal musculature had been ingested. It was concluded that this represents an incidental finding rather than a true infection (239).

Hepatitis caused by an Encephalitozoon spp. that was classified as E. cuniculi on an ultrastructural basis was reported in a 35-year-old HIV-infected patient from southern Florida with a CD4 cell count of 48/µl (340). He presented with fatigue, diarrhea, and weight loss. He subsequently developed fever and died of hepatocellular necrosis. Autopsy confirmed the diagnosis of microsporidian hepatitis.

Peritonitis due to E. cuniculi was described in a 45-year-old HIV-infected man with a CD4 cell count of 57/µl (392). The patient presented with a 13-kg weight loss over the course of a year and was treated with trimethoprim-sulfamethoxazole because of Pneumocystis carinii pneumonia. After the end of therapy, he developed renal failure and a tumorlike mass was recognized in the abdomen. He died, and at limited autopsy microsporidia consistent in ultrastructure with E. cuniculi were discovered within areas of mixed nongranulomatous inflammation in sections of the omentum magnum (392). The reports of these two cases were published before E. hellem was described as a new species. In both instances, diagnosis was made only on an ultrastructural basis, so that the exact species identification is uncertain.

A second case of fulminant hepatic failure caused by microsporidial infection with an Encephalitozoon sp. was reported in a 43-year-old homosexual man with AIDS (322). He suffered from microsporidial diarrhea 2 months prior to development of fulminant hepatitis. The patient died before albendazole became available. The autopsy revealed disseminated microsporidial infection involving the liver, gallbladder wall, and a mediastinal lymph node.

Both E. bieneusi and E. intestinalis have been detected in nonparenchymal liver cells of several HIV-infected patients, but the patients did not show any signs of hepatitis (14, 268, 278, 279).

Disseminated Trachipleistophora antropophtera infection involving several organ systems including the liver and the pancreas was reported in an 8-year-old HIV-infected girl with seizures and cerebral lesions. This patient died after empirical antitoxoplasma therapy (271, 390).

Beside gastrointestinal infection, ocular microsporidiosis is the most common manifestation of microsporidiosis in humans (225).

Encephalitozoon spp. In HIV-infected patients, keratoconjunctivitis may be caused by all three Encephalitozoon spp. (E. hellem, E. cuniculi, and E. intestinalis) (44, 45, 102, 104, 105, 113, 144, 152, 211, 224-226, 238, 243, 291, 306, 319, 369). Most patients present with bilateral conjunctival inflammation and also exhibit bilateral punctate epithelial keratopathy, leading to decreased visual acuity. The keratoconjunctivitis is often asymptomatic or moderate but can be severe; it rarely leads to corneal ulcers (225).

Other species. Keratitis with corneal stroma infection was described in an otherwise healthy 45-year-old man from South Carolina who developed decreased vision in his left eye during an 18-month history of unilateral progressive central keratitis (92). There was no history of prior trauma. Corneal biopsy revealed microsporidia invading deep into the corneal stroma. This organism was successfully propagated in vitro and was named Nosema corneum (317). On the basis of ultrastructural data, it is now in a new genus and has been renamed Vittaforma corneae (324).

Sinusitis is a common manifestation of human microsporidiosis. All three Encephalitozoon spp. have caused rhinosinusitis in several HIV-infected patients (129, 144, 147, 211, 250, 274, 292). E. bieneusi and T. hominis have also been detected in sinus biopsy specimens from HIV-infected patients (129, 138, 165, 184); the patients suffered from severe rhinitis, and nasal polyps were often present.

Pulmonary infections with microsporidia have been reported less frequently than other manifestations (150, 213, 287, 297-299, 304, 305, 328, 369). Infection of the lower respiratory tract may be asymptomatic or associated with bronchiolitis; it is rarely associated with pneumonia or progressive respiratory failure in HIV-infected patients (150, 287, 297-299, 304, 305, 369). All three Encephalitozoon spp. have been detected in bronchial epithelial cells of HIV-infected patients with disseminated Encephalitozoon infection, whereas pulmonary involvement with E. bieneusi has been reported only in two patients (93, 367).

Pulmonary microsporidial infection was also found in a 27-year-old woman from India with chronic myeloid leukemia undergoing allogenic bone marrow transplantation (194). The patient died of a fungal infection, and the diagnosis of pulmonary microsporidiosis was reached only postmortem. Ultrastructural examinations confirmed the organism to be a microsporidium, but taxonomic classification could not be done because the organism could not be identified as any of the known pathogenic species of microsporidia (194).

Infections of the urinary tract are a common finding in HIV-infected patients with disseminated Encephalitozoon infections. The clinical presentation and consequences of the presence of microsporidia in the urinary system can vary remarkably; patients may be asymptomatic with or without microhematuria, they may have cystitis and intestinal nephritis with dysuria and gross hematuria, or they may experience progressive renal failure (1, 121, 144, 150, 242, 268, 304, 305, 369).

Myositis caused by Pleistophora-like microsporidia has been described in four immunocompromised patients. Ledford et al. (216) reported a 20-year-old HIV-seronegative man who had a severe immunodeficiency of unknown origin (CD4 cells, 66/µl) with progressive generalized muscle weakness and contractures for 7 months, fever, generalized lymphadenopathy, and an 18-kg weight loss. Pleistophora-like microsporidian spores were seen in muscle biopsy specimens from the quadriceps and deltoid (216). Four years after his initial clinical presentation, the patient was still immunodeficient but remained seronegative for HIV (229, 230).

Chupp et al. (69) reported a 33-year-old Haitian man with AIDS who was admitted to the hospital with fever, cough, and diffuse myalgias and weakness (69, 281). A Pleistophora-like microsporidium was detected in muscle cells in a biopsy specimen from the right quadriceps. A similar case was reported by Grau et al. (161) in a 35-year old HIV-infected Spanish man who originated from The Gambia. The patient suffered from myositis with fever, myalgia, and progressive weakness. Microsporidian spores were detected in a muscle biopsy specimen.

In an Australian patient who presented with a severe, progressive myositis associated with fever and weight loss, Pleistophora-like microsporidia were demonstrated in corneal scrapings, skeletal muscle, and nasal discharge (138). The organisms were cultivated in vitro as well as in athymic mice. Since these parasites differed from Pleistophora, the new genus and species Trachipleistophora hominis was established (180).

A Nosema-like microsporidium was detected by Cali et al. in a biopsy specimen from the left quadriceps of a 31-year-old patient with AIDS and myositis (48).

Encephalitozoon spp. Two cases of disseminated Encephalitozoon infection with cerebral involvement were reported in a 9-year-old Japanese boy and in a 2-year-old Columbian boy. Both patients suffered from cerebral symptoms such as headache, vomiting, spastic convulsions, and convulsive seizures. Encephalitozoon-like organisms were found in urine from both patients and in cerebrospinal fluid from one patient. The exact species differentiation of these two parasites is uncertain (21, 237) (see "Systemic infections" below).

Other species. Cerebral involvement with Trachipleistophora antropophtera was reported in two AIDS patients, a 33-year-old man and an 8-year-old girl, with seizures and cerebral lesions, who died after empirical anti-toxoplasma therapy (20, 271, 390). At autopsy, a pansporoblastic microsporidium was seen in several organ systems including the brain (271, 390).

Urethritis. Two cases of urethritis associated with microsporidia were found in patients with AIDS who suffered from urethritis, sinusitis, and diarrhea (27, 77). Encephalitozoon-like spores were detected in a smear of expressed urethral pus as well as in stool samples, nasal discharge, sputum, and urine of one patient (77) and in stool samples of the second patient (27). Both patients were treated with albendazole, and the symptoms disappeared.

Prostatic abscess. A prostatic abscess due to E. hellem was found in an AIDS patient with disseminated E. hellem infection (308). The prostate was of normal size with a 1.5- by 1.8-cm central periurethral abscess containing necrotic prostatic tissue. Tissue Gram stain revealed gram-positive microsporidian spores, which were identified as E. hellem by an indirect fluorescence assay.

Tongue ulcer. A shallow 1-cm ulceration on the dorsum of the tongue was observed in an HIV-infected patient with severe immunodeficiency (15 CD4 cells/µl) and disseminated infection due to E. cuniculi (95). Spores were identified in several samples and in soft tissue beneath the tongue ulcer. The microsporidian was identified as E. cuniculi by immunofluorescent staining, in vitro cultivation, and molecular analysis of the SSU rRNA gene by PCR. The patient was treated with albendazole, and the symptoms resolved within 2 weeks (95).

Skeletal involvement. Only two cases of skeletal involvement with microsporidia have been found in patients with AIDS (19). Both patients suffered from disseminated microsporidial infections. In one patient the mandible and associated soft tissues were involved. Species identification was not done.

Cutaneous microsporidiosis. One case of nodular cutaneous microsporidiosis that resolved with oral clindamycin therapy was found in an HIV-infected patient (200a). Underlying osteomyelitis that also resolved after therapy was not proven to be caused by the microsporidia. Species differentiation by PCR techniques was not successful.

Encephalitozoon spp. The first case of documented human microsporidial infection was a case of disseminated Encephalitozoon infection in a 9-year-old Japanese boy who suffered from recurrent fever, headache, vomiting, and spastic convulsions reported in 1959. Encephalitozoon-like organisms were found in cerebrospinal fluid and urine. He was treated with sulfisoxazole and penicillin and recovered (237).

Other species. In 1973, Nosema infection and Pneumocystis carinii pneumonia were diagnosed at autopsy in a 4-month-old athymic male infant (235). Shortly after birth, the child developed diarrhea, vomiting, fever, dyspnea, weight loss, and mechanical ileus. Laparatomy and several antibiotics failed to alter the clinical course, and at autopsy sporoblasts with mature and immature spores of a Nosema sp. were seen in almost all tissues examined except the spleen (235). The parasite was named Nosema connori (334).

Successful treatment of microsporidiosis in immunodeficient patients is limited. Several in vitro culture systems and animal models have been used to identify potential antimicrobial agents for treatment of microsporidiosis. Different drugs control the levels of microsporidial infection in invertebrate hosts; these include fumagillin, an antibiotic produced by Aspergillus fumigatus, and itraconazole for control of Nosema apis in honey bees and other microsporidia in weevils (54). However, in vitro investigations with Nosema bombycis showed no effect of itraconazole and metronidazole on the number of cells infected or on the spore harvest (54). On the other hand, albendazole had marked effects on these parameters, and several ultrastructural changes in the parasites were noted (54, 163).

Other in vitro models used to evaluate drug efficacy included E. cuniculi, E. hellem, and E. intestinalis (16, 117, 141, 168, 218, 219, 380). These studies showed that albendazole, fumagillin, 5-fluorouracil, sparfloxacin, oxibendazole, and propamidine isethionate inhibited E. cuniculi growth in cell cultures. Chloroquine, pefloxacin, azithromycin, rifabutin, and thiabendazole were partially effective at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since the concentrations that inhibited microsporidia were also toxic for the cells in the cell culture. Pyrimethamine, piritrexim, sulfonamides, paronomycin, roxithromycin, atovaquone, flucytosine, toltrazuril, ronidazole, and ganciclovir were ineffective (16). Spore germination of E. hellem and E. intestinalis was inhibited by nifedipine, metronidazole, and nitric oxide donors (168), and E. hellem spore germination was also inhibited by cytochalasin D, demecolcine, and itraconazole (218). TNP-470, a semisynthetic analogue of fumagillin, was highly effective against all three Encephalitozoon spp. and V. corneae in cell cultures (82, 111a). A fluorescent probe, designated calcein, and confocal microscopy have been used to demonstrate drug-induced effects in Encephalitozoon-infected green monkey kidney cells, and both albendazole and fumagillin caused different types of parasite changes (219). In vivo efficacy of albendazole, fumagillin, and TNP-470 against E. cuniculi has been demonstrated in experimentally infected SCID mice, athymic mice, and rabbits (82, 210, 314). Unfortunately, long-term in vitro cultivation of Enterocytozoon bieneusi has not been feasible so far; therefore, a direct assay of the effects of agents on this parasite is not yet practicable.

Based on these in vitro studies, several drugs have been used to treat microsporidial infections in humans. Until recently, blinded, placebo-controlled comparative trials were lacking. Therefore, most clinical experience in the therapy of human microsporidiosis consists of only anecdotal observations. Several case reports and small case series have shown that albendazole was highly effective for treatment of Encephalitozoon infection in HIV-infected patients and led to impressive clinical improvement and eradication of the parasites (1, 77, 95, 109, 121, 143, 144, 150, 162, 185, 211, 215, 247, 248a, 269, 328, 373). However, since some patients relapsed after therapy, maintenance therapy may be necessary for these patients (248a, 373). Symptomatic improvement with reduction of clinical findings was also achieved with topical fumagillin (113, 158, 314) in several HIV-infected patients with microsporidial keratoconjunctivitis due to Encephalitozoon species. Resolution of E. hellem infection of the corneal epithelium of an AIDS patient with itraconazole was also reported (391), ^</