Molecular Techniques for Detection, Species Differentiation, and Phylogenetic Analysis of Microsporidia

Transcription

1 CLINICAL MICROBIOLOGY REVIEWS, Apr. 1999, p Vol. 12, No /99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. Molecular Techniques for Detection, Species Differentiation, and Phylogenetic Analysis of Microsporidia CASPAR FRANZEN* AND ANDREAS MÜLLER Department of Internal Medicine I, University of Cologne, Cologne, Germany INTRODUCTION MORPHOLOGY OF MICROSPORIDIA Morphology and Life Cycle Spore Merogony Sporogony TAXONOMY GENUS- AND SPECIES-SPECIFIC CHARACTERISTICS Enterocytozoon sp Encephalitozoon spp Nosema spp Vittaforma sp Pleistophora spp. and Trachipleistophora spp Other Genera EPIDEMIOLOGY Prevalence and Geographic Distribution Sources of Infection and Transmission CLINICAL MANIFESTATIONS Gastrointestinal and Biliary Tract Infections Enterocytozoon bieneusi Encephalitozoon spp Other species Hepatitis, Pancreatitis, and Peritonitis Ocular Infections Encephalitozoon spp Other species Sinusitis Pulmonary Infections Urinary Tract Infections Myositis Cerebral Infections Encephalitozoon spp Other species Rare Manifestations Urethritis Prostatic abscess Tongue ulcer Skeletal involvement Cutaneous microsporidiosis Systemic Infections Encephalitozoon spp Other species THERAPY DIAGNOSTIC METHODS Transmission Electron Microscopy Light Microscopy Cytologic diagnosis and stool examination Histologic diagnosis Cell Culture Animal Models * Corresponding author. Mailing address: Department of Internal Medicine I, University of Cologne, Joseph-Stelzmann Str. 9, D Cologne, Germany. Phone: 49-(0) Fax: 49-(0) Caspar.Franzen@Uni-Koeln.de. 243

2 244 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. Antigen-Based Methods Serologic Testing MOLECULAR METHODS Small- and Large-Subunit rrna Genes of Microsporidia and -Tubulin Genes of Microsporidia Other Genes of Microsporidia DNA Isolation Techniques Molecular Techniques for Diagnosis and Species Differentiation Primer pairs and hybridization probes for E. bieneusi Primer pairs and hybridization probes for E. intestinalis Primer pairs for E. hellem and E. cuniculi General primer pairs and hybridization probes for several microsporidian species Strain differentiation of Encephalitozoon spp. and E. bieneusi Comparison of molecular techniques with light microscopy Molecular Techniques for Phylogenetic Analysis Molecular Techniques for Susceptibility Testing FUTURE TRENDS CONCLUDING REMARKS ACKNOWLEDGMENTS REFERENCES INTRODUCTION 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, , 162, 195, , 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. MORPHOLOGY OF MICROSPORIDIA 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). Morphology and Life Cycle Spore. Microsporidian spores are between 1 and 20 m long. Species that infect mammals are usually small, with diameters of1to3 m. 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). 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 differ-

3 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 245 Downloaded from FIG. 1. Diagram of a microsporidian spore and representative life cycle (merogonic and sporogonic stages vary among different genera). on September 25, 2018 by guest entiated 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). TAXONOMY The term microsporidia is a nontaxonomic designation commonly used for organisms belonging to the phylum Microspora, which is contained within the subkingdom Protozoa

4 246 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. 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. (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. 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 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.

5 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 247 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 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 mm, with five to seven coils of the polar tubule, arranged in two rows (Fig. 4) (42, 53, 99, 100, 318). 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 entero- 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, 36,600.

6 248 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. Downloaded from 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. cytes 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). Encephalitozoon spp. 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 m, and the polar tubule has five to seven coils in a single row (Fig. 6) (50, 57, 58, 318, 333). 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, on September 25, 2018 by guest

7 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 249 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 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, , 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). Nosema spp. 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 mm 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 mm with 9 to 12 coils of the polar tubule (36, 44, 45). Another microsporidium infecting muscle cells of a 31-yearold HIV-infected patient was described by Cali et al. (48). Development took place in direct contact with the muscle cell 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, 355,125.

8 250 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. Downloaded from 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. 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 m, 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). Vittaforma sp. 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 m 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. and Trachipleistophora spp. 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 on September 25, 2018 by guest

9 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 251 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 m 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). Other Genera 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 m, 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 m, 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 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, , 177, 179, 195, , 215, 238, 243, 244, 246, 247, 263, 266, , 291, , , 315, 316, 326, 328, 339, 344, , ). 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 HIVinfected 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 HIVinfected patients with severe immunodeficiency and CD4 cell counts below 100/ l 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, , , 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, , 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. Sources of Infection and Transmission Routes of transmission and sources of human microsporidial infections have been difficult to ascertain. Based on the distri-

10 TABLE 1. Case reports of microsporidiosis in patients not infected with HIV with normal or unknown immune status Microsporidial species a Location Age (yr) b Sex c Clinical history Immune status Diagnostic method Yr of study Reference(s) E. cuniculi Japan 9 M Seizure disorder NK d Cytology of CSF e and urine E. intestinalis Swizerland 36 M Diarrhea (self-limited) Normal Examination of stool specimens E. intestinalis France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens E. intestinalis France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens E. bieneusi Germany (travel to Egypt and Jordan) 26 M Diarrhea (self-limited) Normal Examination of stool specimens E. bieneusi France (returning from tropical country) NK M Diarrhea NK, Crohn s disease Examination of stool specimens E. bieneusi France (returning from tropical country) NK M Diarrhea NK, cardiac-valve graft Examination of stool specimens E. bieneusi France (travel to tropical country) NK M Diarrhea NK Examination of stool specimens E. bieneusi France (travel to tropical country) NK M Diarrhea NK Examination of stool specimens E. bieneusi France (returning from Africa) NK F Diarrhea NK Examination of stool specimens E. bieneusi France NK M Diarrhea NK Examination of stool specimens E. bieneusi Germany (travel to Turkey) 3 F Diarrhea (self-limited) Normal Examination of stool specimens E. bieneusi Swizerland 26 F Diarrhea (self-limited) Normal Examination of stool specimens E. bieneusi Zambia 7 mo M No symptoms Normal Examination of stool specimens E. bieneusi Spain 24 F Diarrhea (chronic) Normal Examination of stool and a duodenal biopsy specimens M. ceylonensis Sri Lanka 11 M Keratitis NK Histologic examination M. africanum Botswana 26 F Corneal ulceration NK Histologic examination N. ocularum USA (Ohio) 39 M Keratitis Normal Histologic examination , 44 V. corneae USA (South Carolina) (travel to 45 M Keratitis/iritis Normal Histologic examination Caribbean and Central America) Not determined France 44 F Fever, myalgia Normal Examination of stool specimens Not determined France 72 M No symptoms Liver cirrhosis after Examination of stool specimens hepatitis B Not determined France 63 M No symptoms Chronic alcoholism Examination of stool specimens Not determined France 68 M No symptoms Chronic alcoholism Examination of stool specimens Not determined France 64 M No symptoms Chronic alcoholism Examination of stool specimens Not determined France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens Not determined France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens a Classification is based only on morphology. b Age in years unless otherwise indicated. c M, male; F, female. d NK, not known. e CSF, cerebrospinal fluid. 252 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. Downloaded from on September 25, 2018 by guest

11 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 253 a Classification is based only on morphology. b Age in years unless otherwise indicated. c M, male; F, female. Not determined France 52 M Diarrhea (chronic), malabsorption Immunosuppression after heart-lung transplantation Examination of stool specimens and histologic examination (gastrointestinal biopsy) Not determined India 27 F Pneumonia Immunosuppression after bone marrow transplantation Histologic examination (autopsy) Pleistophora sp. USA (Florida) 20 M Myositis T-cell immunodeficiency Histologic examination N. connori USA (Washington D.C.) 4 mo M Disseminated infection Thymic aplasia Histologic examination (autopsy) E. bieneusi Spain 66 M Diarrhea (chronic) Lymphocytopenia and hypogammaglobulinemia Examination of stool and duodenal biopsy specimens a E. bieneusi France 48 M Diarrhea Immunosuppression after heart-lung transplantation Examination of stool specimens E. cuniculi Sweden (born in Colombia) 2 M Seizure disorder Low CD4/CD8 cell ratio Cytology of urine, serum antibody E. bieneusi USA (Boston) 48 F Diarrhea (self-limited) Immunosuppression after Examination of stool specimens liver transplantation E. bieneusi USA (Boston) 42 M Diarrhea (chronic) Decreased CD4 cell count Examination of stool specimens a Microsporidial species a Location Age (yr) b Sex c Clinical history Immune status Diagnostic method Yr of study Reference TABLE 2. Case reports of microsporidiosis in patients not infected with HIV but with immunodeficiency TABLE 3. Animal hosts of human microsporidia Species Nonhuman host Reference(s) E. bieneusi Pigs, dogs, macaque monkeys 234 E. intestinalis Dogs, donkeys, pigs, cows, goats 32a E. hellem Birds (parrots) 28 E. cuniculi Several mammals 50, 97, 98, 111, 118, 181, 236a, 365 Strain I Rabbits, mice Strain II Strain III Mice, blue foxes Domestic dogs, rodents, goats, sheep, swine, horses, foxes, cats N. connori None known N. ocularum None known Nosema-like sp. None known V. corneae None known Pleistophora spp. Fish 50 T. hominis None known T. antropophtera None known M. africanum None known M. ceylonensis None known bution 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 ( , 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

12 254 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. Species TABLE 4. Clinical manifestations of human microsporidial infections Clinical manifestation Reference(s) (first reports) E. bieneusi Enteritis 99, 119, 245 Cholangitis, cholecystitis 240, 278 Pneumonia, bronchitis 367 Sinusitis, rhinitis 129, 165 E. intestinalis Enteritis 266 Cholangitis, cholecystitis 268, 385 Nephritis, urinary tract infection 47, 268 Sinusitis, rhinitis 121, 147 Bronchitis 121, 150 Keratoconjunctivitis 226 Disseminated infection 47, 268 E. hellem Keratoconjunctivitis 104, 306 Sinusitis, rhinitis 178, 211 Pneumonia, bronchiolitis 304, 305 Nephritis, urinary tract infection 304 Prostatic abscess 308 Disseminated infection 304 E. cuniculi Hepatitis, peritonitis a 340, 392 Encephalitis 237, 242, 374 Intestinal infection 144 Urinary tract infection 95, 144 Keratoconjunctivitis 95, 144 Sinusitis, rhinitis 95, 144 Disseminated infection 95, 144 T. hominis Myositis 138, 180 Keratoconjunctivitis 138, 180 Sinusitis, rhinitis 138, 180 T. antropophtera Encephalitis 271, 390 Myositis 271, 390 Disseminated infection 271, 390 Pleistophora spp. Myositis 216, 229, 230 V. corneae Keratitis 92, 317, 324 N. ocularum Keratoconjunctivitis 36, 44 N. connori Disseminated infection 235, 334 Nosema-like sp. Myositis 239 M. africanum Corneal ulcer 50, 277 M. ceylonensis Corneal ulcer 6, 50 a Species not confirmed because classification was based only on ultrastructure morphology. 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). CLINICAL MANIFESTATIONS 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). Gastrointestinal and Biliary Tract Infections 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/ l (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 B 12 (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, ). 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). E. bieneusi infection of the biliary tract, with or without cholecystitis, is responsible for some of AIDS-related cholangiopathies which are not explained by Cryptosporidium spp. or cytomegalovirus infection (33, 201, 240, 278, 279). Dissemination of E. bieneusi is very uncommon, but it has been detected in duodenal lamina propria cells (310), in bronchoalveolar lavage fluid and transbronchial biopsy specimens (93, 367), and in nasal sinuses of HIV-infected patients (129, 165, 184). Intestinal infections with E. bieneusi in non-hiv-infected patients have been reported in only 15 patients in Germany, Switzerland, France, Spain, Zambia, and the United States (96,

13 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA a, 167, 249, 284, 295, 296, 329): one patient had Crohn s disease (96), an African woman had a cardiac-valve graft (96), one patient had a congenital disorder of the lymphatic system (154a), another had a low CD4 cell count of unknown origin (365a), and two patients were immunosuppressed due to treatment associated with liver and heart-lung transplantation (284, 296). The other nine patients were otherwise healthy (Tables 1 and 2). Although patients usually presented with self-limiting diarrhea, some patients were treated with albendazole or metronidazole (284, 296). 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. Among persons not infected with HIV, only three cases of intestinal infection with an Encephalitozoon spp. have been reported (139). A 36-year-old HIV-seronegative homosexual man was asked to provide stool for examination after E. intestinalis was demonstrated in stool samples of his HIV-infected partner. E. intestinalis was detected in two of seven stool samples from the non-hiv-infected man and again 4 months later, together with Isospora belli, when he became mildly symptomatic after a trip to Brazil (139). Two other patients were travelers presenting with chronic diarrhea, and microsporidian spores were detected in their stools (286). Molecular identification of microsporidian species as E. intestinalis was based on PCR amplification of an SSU rrna sequence. Albendazole treatment led to the elimination of spores in the stool, but the clinical signs persisted. 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, Pancreatitis, and Peritonitis 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/ l (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/ l (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). Ocular Infections 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, , 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). A microsporidium was found to be responsible for the symptoms in a 39-year old man from Ohio who developed blurred vision and irritation in his left eye. His visual symptoms persisted despite the discovery and surgical removal of a foreign body (36, 39). A subsequent biopsy of the persistent corneal ulcer revealed organisms with typical microsporidian ultrastructure; the species was named Nosema ocularum (44). Trachipleistophora hominis was found in the corneal scrapings of an HIV-infected patient with disseminated infection who suffered from myositis and keratoconjunctivitis (138). In 1973 and 1981, two cases with corneal involvement were documented in a 11-year-old Tamil boy from Sri Lanka with a corneal ulcer and a 26-year-old woman from Botswana suffering from a perforated corneal ulcer (6, 277). Both otherwise healthy patients did not have HIV infection. The genera could not be determined, and the organisms were named Microspo-

14 256 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. ridium ceylonensis and Microsporidium africanum, respectively (50). Sinusitis 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 Pulmonary infections with microsporidia have been reported less frequently than other manifestations (150, 213, 287, , 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, , 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). Urinary Tract Infections 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 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/ l) 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). Cerebral Infections 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). Cerebral microsporidiosis due to E. cuniculi was recently described by Weber et al. (374) in a 29-year-old HIV-infected man with a CD4 cell count of 0 cells/ l. The patient was hospitalized because of headache, visual and cognitive impairment, nausea, and vomiting. Magnetic resonance imaging scans showed right maxillary sinusitis and multiple small, contrast-enhanced lesions in the hippocampal, mesencephalitic, and intracortical regions. Examination of cerebrospinal fluid showed microsporidial spores, which were also detected in sputum, urine, and stool specimens. The microsporidium was cultivated in vitro and was classified as E. cuniculi by Western blot analysis, ribotyping, and sequencing of the rrna intergenic spacer region (374). A similar case was reported by Mertens et al. (242) in a 25-year-old HIV-infected woman. Microsporidian spores, classified as E. cuniculi by immunohistochemistry and PCR, were detected in the brain, heart, kidneys, urinary bladder, spleen, lymph nodes, adrenals, and trachea at autopsy. Other species. Cerebral involvement with Trachipleistophora antropophtera was reported in two AIDS patients, a 33-yearold 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). Rare Manifestations 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.

15 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 257 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/ l) 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. Systemic Infections 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 Encephalitozoon-like organisms were found in cerebrospinal fluid and urine. He was treated with sulfisoxazole and penicillin and recovered (237). A similar case occurred in 1984 in a 2-year-old Columbian boy who lived in Sweden. He had convulsive seizures, and gram-positive organisms consistent with an Encephalitozoon sp. were found in urine. Anti-E. cuniculi antibodies (immunoglobulin G [IgG] and IgM) were detected in serum samples (21). Disseminated infections with all three Encephalitozoon spp. are now increasingly recognized in severely immunosuppressed HIV-infected patients, usually in those with CD4 cell counts below 100/ l (95, 121, 144, 150, 162, 215, 247, 268, 297, 304, 305, 328, 369). The spectrum of disease has expanded to include keratoconjunctivitis, bronchiolitis and pneumonia, sinusitis, nephritis, urethritis, cystitis, prostatitis, hepatitis, peritonitis, gastroenteritis, and cholangitis, but there are clear differences in the typical distribution pattern for each microsporidian species: E. hellem parasitizes mainly the keratoconjunctiva, urinary tract, nasal sinuses, and bronchial system; on the other hand, E. intestinalis appears to be confined mainly to the gastrointestinal and biliary tract with dissemination to the kidneys, eyes, nasal sinuses, and sometimes the respiratory tract; finally, E. cuniculi causes widely disseminated infections involving nearly all organ systems, but the clinical manifestations vary substantially, ranging from no symptoms to severe disease (144, 150, 162, 215, 247, 268, 304, 305, 328, 369). 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). T. hominis was reported as the cause of myositis in a 34- year-old HIV-infected man; parasites were recognized in corneal scrapings, skeletal muscle, and nasal discharge (138). The newly recognized pansporoblastic microsporidium, T. antropophtera caused disseminated infection involving the brain, heart, kidneys, pancreas, thyroid, parathyroid, liver, bone marrow, lymph nodes, and spleen in an HIV-infected 8-year-old child (271, 390). T. antropophtera infection of the brain was also seen in a 33-year-old HIV-infected male in whom autopsy was limited to the brain (20, 271, 390). THERAPY 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 clin-

16 258 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. ical 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), but this finding could not be duplicated (113). Itraconazole seems to be ineffective in preventing Enterocytozoon bieneusi infection in HIV-infected patients (2). Keratoconjunctivitis due to an Encephalitozoon species in another AIDS patient responded to topical dibromopropamidine isethionate (238). Infections due to E. bieneusi are much more difficult to treat, and currently there is no acceptable treatment. Pneumocystis carinii prophylaxis with co-trimoxazole seems to have no influence on the prevalence of intestinal microsporidiosis in HIVinfected patients, suggesting that this drug may be ineffective (3). Improvement or disappearance of diarrhea caused by E. bieneusi has been reported after treatment with metronidazole, but repeated biopsies showed that microsporidia persisted (127, 128), and other investigators did not observe any response after treatment with metronidazole (114, 136). The efficacy of albendazole for E. bieneusi infections is controversial (257). Diarrhea may improve in 50 to 60% of patients (29, 103, 114), but persistence of organisms was seen in posttreatment biopsy specimens despite several ultrastructural changes in the parasite (29, 31, 114). Other trials with albendazole showed much lower response rates (142, 172, 246). Doubleblind placebo-controlled trials with albendazole are in progress. Despite a remarkable clinical response with atovaquone in symptomatic AIDS patients with intestinal E. bieneusi infection, there was no apparent decrease in the parasite burden in either stools or biopsy specimens (5). A double-blind placebo-controlled study with atovaquone is also under way. Similarly, azithromycin treatment showed only partial effect on diarrhea in E. bieneusi-infected patients, and microsporidial infection persisted on repeat biopsy and stool examinations (172). Improvement of diarrhea with clearance of microsporidian shedding in stool was observed in three E. bieneusiinfected patients treated with furazolidone (116). Purified fumagillin was also able to clear E. bieneusi infection from stool as well as intestinal biopsy specimens in three patients, but the drug is toxic and causes thrombocytopenia in nearly all patients (248). These observations must be confirmed by treatment of more patients with these two drugs. As mentioned above, TNP-470, a synthetic analogue of fumagillin that is less toxic, is as effective as fumagillin against several microsporidian species in vitro and in athymic mice and holds promise as a new antimicrosporidial compound (111a). Of note, elevated tumor necrosis factor alpha levels in stool samples have been demonstrated in patients with microsporidial diarrhea. Therefore, the anti-tumor necrosis factor alpha agent thalidomide has been used to treat diarrhea due to E. bieneusi. Some patients responded to this therapy, but again this observation must be confirmed in controlled trials (320, 321). Symptomatic improvement has been achieved with octreotide, but this drug has no effect on the parasites (325). Combination antiretroviral therapy that includes a protease inhibitor can restore immunity to microsporidia. The use of potent antiretroviral therapy in patients with advanced HIV infection can improve symptoms due to microsporidiosis and in some cases leads to disappearance of the parasites (61, 75b, 140, 160a). However, the rapid time to release in patients with declining CD4 lymphocyte counts suggest that the microsporidial infections are not eradicated. Since no effective therapy is available for E. bieneusi infection whereas infections with Encephalitozoon spp. respond very well to albendazole therapy, exact species differentiation of microsporidia infecting humans is absolutely necessary. DIAGNOSTIC METHODS Diagnosis of human microsporidiosis is dependent on the identification of spores in clinical samples, e.g., stool specimens, duodenal or bile juice, urine, conjunctival smears, bronchoalveolar lavage fluid, sputum, nasal discharge, or biopsy tissues. The detection of spores in clinical samples, however, is a laborious, challenging, and time-consuming task because the tiny organisms can easily be missed. Originally, definitive diagnosis of microsporidiosis required transmission electron microscopy, but during the last few years new staining methods, suitable for light microscopy, have been developed. Microsporidia have now been found in virtually every tissue and body fluid in humans. Although the diagnosis and identification of microsporidia by light microscopy have greatly improved during the last few years, species differentiation is usually impossible by these techniques. Immunofluorescent staining techniques have been developed for species differentiation of microsporidia, but antibodies used in these procedures are available only at research laboratories so far. Similarly, cell culture systems can be used for in vitro cultivation of microsporidia, but this is not a suitable technique for routine use because it is laborious and time-consuming. A variety of serological tests has been developed to detect antibodies to microsporidia, but the sensitivity and specificity of these tests are unknown. Also, these tests are not suitable methods to diagnose infections in immunosuppressed persons. Recent success in nucleotide sequencing of various microsporidia has now led to the application of new molecular techniques for the diagnosis of human microsporidiosis. Transmission Electron Microscopy Originally, definitive diagnosis of microsporidiosis required ultrastructural examination of biopsy tissues, body fluid specimens (urine, nasal discharge, sinus aspirates, sputum, bronchoalveolar lavage fluid, duodenal or bile juice, cerebrospinal fluid), or stool samples by transmission electron microscopy, because of the small size of the organisms and their poor and variable staining characteristics (83, 88, 241, 346). Visualization of the unique ultrastructure of the spores with their characteristic coiled polar tube is diagnostic. Microsporidia can be identified to the genus or even species level based on the fine-structure features of the spores and proliferative forms, method of division, and nature of the host cell-parasite interface. In tissue, all stages of the life cycle can often be observed, whereas in body fluids or stool samples only spores are visible. The ultrastructural characteristics of microsporidian species found in humans are summarized in Table 5. Detection of microsporidia by transmission electron microscopy is highly specific, but the technique may lack sensitivity, especially when performed on body fluids and stool samples. However, large studies to evaluate the sensitivity of transmission electron microscopy are lacking (62, 73). Likewise, sample preparation and examination are laborious and time-consuming (62, 73). Light Microscopy Histologic examination of biopsy specimens or cytologic examination of body fluids by light microscopy allows diagnosis of microsporidial infection, but genus or species differentiation is uncertain (76, 227). The size of the spores and the distribu-

17 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 259 Special features Electron-lucent inclusion, development of the polar tubule beginning in the sporonts Diplosporoblastic sporogony Tetrasporoblastic sporogony, band-like sporonts, all stages are surrounded by a cisterna of host endoplasmatic reticulum Multinucleate sporogonial plasmodia No multinucleate sporogonial plasmodia, two different types of sporophorous vesicles and spores are formed during development of T. antropophtera Vacuole No vacuole, in direct contact with the host cell cytoplasma Septated parasitophorous vacuole in E. intestinalis No vacuole, in direct contact with the host cell cytoplasma No vacuole, in direct contact with the host cell cytoplasma Sporophorous vesicle Sporophorous vesicle Nucleus Unikaryotic Unikaryotic Diplokaryotic Diplokaryotic Unikaryotic Unikaryotic Arrangement of polar tube Two rows One row One row One row Two rows One or two rows No. of coils of polar tubule Size of spores ( m) by by by by by by 2.4 Characteristic E. bieneusi Encephalitozoon spp. Nosema spp. V. corneae Pleistophora spp. Trachipleistophora spp. TABLE 5. Morphological characteristics of microsporidia infecting humans FIG. 8. Encephalitozoon cuniculi spores in conjunctival swab from an HIVinfected patient with disseminated infection. Modified chromotrope-based stain. Magnification, 870. tion pattern of the infection may be of limited use, but exact species differentiation is impossible. Cytologic diagnosis and stool examination. Microsporidian spores have been detected in several body fluids and stool samples (264, 347). The outer layer of the spore wall (exospore) is proteinaceous, and the inner layer (endospore) is chitinous (26, 50) so that Gram, Giemsa, and trichrome stains, as well as fluorescent dyes, have been advocated for staining microsporidian spores (74). Gram and Giemsa stains are not suitable for cytologic diagnosis because they do not differentiate between microsporidia and other elements present in body fluids or stool specimens that can be confused with microsporidian spores (75). Microscopic examination of body fluids to diagnose microsporidia did not become routine until special chromotrope-based and fluorescent stains were developed. Nowadays these chromotrope- and/or fluorochromebased stains are used in several modifications. The chromotrope-based stain developed by Weber et al. (366) has markedly improved spore detection in stool samples and body fluids. In this technique, involving a modification of the trichrome stain with a concentration of chromotrope 2R that is 10 times higher than that used in the trichrome stain, microsporidian spores stain characteristic pinkish red. Usually the spores have a characteristic appearance when examined under high-power magnification ( 1,000). The spore wall stains intense red, and some spores show a distinct beltlike stripe that grids the spores diagonally or equatorially (Fig. 8) (366). This staining technique is lengthy, and spores are difficult to detect if only a few are present in the sample (94). Several modifications (changes in temperature and staining time [203], decrease in the phosphotungstic acid level, and substitution of a color-fast counterstain [294]) of the chromotrope-based stain have been suggested for speeding the process and for better contrast between the spores and the background. The improved hot Gram-chromotrope technique provides some real advantages in staining microsporidia for light microscopy. In this procedure, samples are stained in solutions of crystal violet and iodine used in Gram s stain and then in a

18 260 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. FIG. 9. Encephalitozoon intestinalis spores derived from in vitro culture. Fluorescence microscopy after Uvitex 2B stain. Magnification, 1,000. modified chromotrope solution heated to 50 to 55 C. With this stain, microsporidian spores are stained dark violet against a pale green background and the total staining time is shortened to 5 min (251). The epifluorescence of microsporidian spores stained with optical brighteners was a second major breakthrough for detecting spores in stool samples and body fluids (74, 75, 228, 348, 349). Of these fluorescent stains, Uvitex 2B, Calcofluor white, and Fungifluor (a formulation of Calcofluor white in 10% KOH) are the stains of first choice, although Calcofluor white produces somewhat greater background staining (74). The staining procedure is easy and quick, but examination requires a fluorescent microscope with a 350- to 380-nm excitation filter and a high-magnification objective lens (magnification, 1,000). Fluorochromes bind to the chitin of the endospore, and when excited under UV light, the bound dye fluoresces brightly in the visible spectrum (74). Spores are identified by their size, shape, and fluorescent staining properties (Fig. 9) (74, 348). Several modifications of the fluorescent stain with Uvitex 2B, originally described by van Gool et al. (348), were introduced to use on touch preparations (74, 356), brush cytologic specimens (270), and smears of several body fluids (74, 107) and for staining of paraffin-embedded material (74, 146). Uvitex 2B was used a number of years ago for detecting nonhuman microsporidia in tissue sections (355). Although fluorescent stains seem to be more sensitive than chromotrope-based trichrome stains, they may lead to some false-positive results due to the similarity in staining of small yeast cells (74, 75, 94, 228). In addition, other studies have not shown superior sensitivity of the fluorescent stains over the chromotrope-based stains (107, 182). Most authors conclude that the two techniques should be used simultaneously to enhance performance and to provide greater accuracy, especially for patients with light infections (94, 107, 182). Because the number of microsporidia in clinical samples can be very small, centrifugation of body fluids may be necessary. FIG. 10. Paraffin-embedded duodenal biopsy specimen from a patient with AIDS with intestinal Enterocytozoon bieneusi infection. The microsporidial spores are easily visualized within the enterocytes. Fluorescence microscopy after Uvitex 2B stain. Magnification, 1,000. Whether concentration techniques are useful for detection of microsporidian spores in stool specimens remains controversial. Some authors reported that concentration techniques such as the formalin-ethyl acetate concentration procedure or different flotation methods led to a substantial loss of microsporidial spores and thus to false-negative results (62, 366). In contrast, others found that concentration techniques such as the water-ether sedimentation or centrifugation of KOHtreated stool samples increased the yield of microsporidian spores (62, 349). Therefore, one of these concentration techniques should be used for stool samples. Histologic diagnosis. Because of the small size of the spores, reliable visualization of microsporidia by light microscopy depends on a distinct contrast between spores and other cellular contents. Routine hematoxylin-and-eosin stained parasites in tissue are easily overlooked even by experienced pathologists (241). Tissue Gram stains such as Brown-Brenn or Brown- Hopps seem to be very useful for reliable identification of microsporidia in paraffin-embedded tissue sections (206, 267). Microsporidian spores are Gram variable, but with these stains they stain dark blue or red against a faint brown-yellow background (206). Fluorescent staining techniques with optical brighteners are easy and quick to perform on tissues, and the sensitivity of these stains seems to be very high (Fig. 10) (74, 146). A major advantage of these stains is that they can be combined with other staining techniques (74). Silver stains such as the Warthin-Starry stain (136, 137) or a modified chromotrope-based trichrome stain (160) are preferred by some, but both techniques are time-consuming and interpretation of sections may be difficult. Giemsa, chromotrope 2R, or fluorochrome staining of touch preparations of intestinal tissue and of endoscopic brush cytologic specimens is useful in the diagnosis of intestinal microsporidiosis, but these techniques require fresh material (15, 74, 270, 288, 309). Semithin sections of resin-embedded biopsy material, stained with a variety of different stains (hematoxylin and eosin, Giemsa, toluidine blue, methylene blue-azure, and basic fuchsin), are useful methods for visualization of spores and tissue stages of microsporidia. However, resin embedding of biopsy specimens is not routinely used in most laboratories (Fig. 11) (263, 267, 273, 309).

19 VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 261 Animal Models FIG. 11. Resin-embedded semithin (1- m) section of duodenal mucosa from a patient with AIDS and intestinal Enterocytozoon bieneusi infection. Epithelial cells contain spores of Enterocytozoon bieneusi. Toluidine blue stain. Magnification, 800. Cell Culture The in vitro cultivation of several microsporidian species that infect humans has been of enormous benefit, both for our understanding of the biologic aspects of the host cell-parasite relationship and for the development of immunologic reagents for diagnosis and species differentiation. In vitro cultures have been also used to assess the effects of antimicrobial agents on several microsporidian species including E. cuniculi, E. hellem, and E. intestinalis (16, 54, 117, 141, 168, 218, 219). In vitro cultures combined with ultrastructural, biochemical, antigen, or molecular analyses have been used to confirm infections with existing species of microsporidia (95, , ), as well as to define new species (104, 180, 317). However, their use in routine clinical diagnosis is not practical because they are time-consuming and laborious and only specialized laboratories maintain cell cultures with microsporidia. Microsporidia have been successfully cultivated in a number of mammalian cell lines including monkey and rabbit kidney cells (Vero and RK13), human fetal lung fibroblasts (MRC-5), MDCK cells, and several other cell lines (50, 102, , 275, 350, 354). Species that have been cultivated in vitro from a variety of human specimens include E. hellem (102, 178, 359), E. cuniculi (95, 177, 179, 312), E. intestinalis (122, 151, 350, 360), V. corneae (317), and T. hominis (180). Attempts to culture Enterocytozoon bieneusi from small intestinal biopsy specimens laden with merogonic stages and spores by using a range of cell lines and pretreatments have had limited success (54); to date, E. bieneusi has been propagated only in short term cultures (6 months) (361). In the culture systems (human lung fibroblasts and Vero monkey kidney cells) used, E. bieneusi seems to exert a greater cytotoxic effect than has been observed with cell cultures of Encephalitozoon spp. The inability to grow E. bieneusi in a continuous-culture system may reflect a need of this organism for some specific nutritional requirements that are not provided by the cell cultures used so far (361). Animal models provide a basis for studying immune responses and for evaluating diagnostic methods, vaccine candidates, therapeutic strategies, and routes of transmission (106). Furthermore, they are essential for producing poly- and monoclonal antibodies (307, 309, 359, 360). Several animal models have been established to study microsporidial infections (50, 80, 106, 153, 156, 209, 236, 313, 343, 384). Most of these models used E. cuniculi as pathogen. This organism had long been recognized as an important cause of latent infections in laboratory rodents, sometimes complicating the interpretation of experimental results obtained with these animals (50, 51, 80). BALB/c and C57Bl/6 athymic mice have been used as animal models and have been infected intraperitoneally with E. cuniculi, E. hellem, or V. corneae (106, 156). SCID mice have also been infected by oral inoculation of E. cuniculi spores. This animal model was used to study the in vivo efficacy of albendazole against E. cuniculi (209). Experimental E. cuniculi infections in immunocompetent mice produced only chronic asymptomatic infection. Successful transmission of E. cuniculi to rabbits by administration of spores orally and rectally has been reported (80, 153). Simian immunodeficiency virus-infected rhesus macaque monkeys have been also infected with E. cuniculi, E. hellem, and V. corneae per os (106). Animal models for E. bieneusi infection are difficult to establish. Attempts to infect immunosuppressed gnotobiotic piglets, gamma interferon knockout mice, and SCID mice treated with anti-gamma interferon monoclonal antibodies with E. bieneusi have been unsuccessful. Recently, experimental oral transmission of E. bieneusi to simian immunodeficiency virusinfected rhesus monkeys has been reported (343). Antigen-Based Methods Microsporidium-specific antibodies in immunofluorescence tests have been used for the diagnosis and species differentiation of microsporidia. Poly- and monoclonal antibodies were also used for Western blot analysis of several microsporidian species (4, 84, 85, 132, , 256, 259, 261, , , 379, 397). Immunofluorescent antibody tests involving polyclonal antisera against E. hellem, E. cuniculi, and E. intestinalis produced in mice or rabbits showed that several species of microsporidia demonstrated immunological cross-reactivity (4, 259, 359, 360, 397). This cross-reactivity of the polyclonal antisera against Encephalitozoon spp. was used for the detection of several microsporidian species including E. bieneusi in various clinical samples by using different immunofluorescence tests (4, 259, 359, 360, 397). However, the cross-reactivity of the antisera limit their use as diagnostic tools because species differentiation is not possible with these reagents. Several monoclonal antibodies which recognized E. hellem (4, 359), E. cuniculi, or E. intestinalis (17) were developed. Most of these monoclonal antibodies are specific to spore antigens (4, 227a, 359), whereas other researchers used polar tube protein-reactive monoclonal antibodies in combination with monoclonal antibodies that recognize the surfaces of spores (17). Some of these monoclonal antibodies are species specific, whereas others react against spore walls or the polar tubes of several microsporidian species (227a). Mono- and polyclonal antibodies are useful tools for species differentiation of microsporidia in different clinical samples (307), but antibody-staining techniques may be less sensitive than other techniques. Didier et al. (107) compared a chromo-

20 262 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV. A variety of serological tests (carbon immunoassay, indirect immunofluorescence test, enzyme-linked immunosorbent assay, counterimmunoelectrophoresis, and Western blotting) have been developed to detect IgG and IgM antibodies to microsporidia, especially to E. cuniculi (18, 21, 22, , 327, 352, 353, 379, 383). Some of these tests are commonly used to detect antibodies in several animal species (18, 32, 170, 175). Of these assays, the indirect immunofluorescence test and enzyme-linked immunosorbent assay are probably the most useful because they are easy to perform, but the sensitivity and specificity of all these tests are unknown ( ). Antibodies to E. cuniculi and E. intestinalis have been found in humans with and without HIV infection (174, 176, 327, 352, 353), but it is uncertain whether these represent true infection, cross-reactivity with other species, or nonspecific reaction. Serologic surveys for antibodies to E. cuniculi have suggested a possible link between exposure to a tropical environment and infection with microsporidia. In patients with malaria and schistosomiasis, the microsporidial seropositivity rate was 4.7 and 9.1%, respectively (176). A study of homosexual men in Sweden reported that 10 of 30 persons (33%) were seropositive for antibodies to E. cuniculi; all the seropositive patients had at some time visited a tropical area (22, 176). Explanations for this apparent relationship remain speculative, and clinicopathologic correlations have not been reported for any of these serologic surveys. Of interest, a high seroprevalence of antibodies against E. intestinalis were observed among Dutch blood donors (24 of 300 [8%]), pregnant French women (13 of 276 [5%]), and patients with various infectious and noninfectious diseases (6 of 150 [4%]) (353). For E. bieneusi, which has not been propagated in long-term cell cultures so far, specific serologic assays remain unavailable. Whether antigens of Encephalitozoon spp. cross-react with those of E. bieneusi has not been determined exactly so far (259, 302, 379, 397). However, serologic methods are not useful as diagnostic tools for microsporidiosis because at least half of the serum samples from persons without a history of microsporidial infection may have positive titers and immunosuppressed persons may have a poor response to antigen challenge. FIG. 12. Indirect immunofluorescent staining of Encephalitozoon cuniculi spores in nasal discharge of an HIV-infected patient with disseminated infection, using polyclonal anti-encephalitozoon cuniculi antiserum. Magnification, 400. trope-based stain, a fluorescent stain containing Calcofluor white, and a fluorescent polyclonal antibody stain. The fluorescent polyclonal antibody stain was the least sensitive method for detecting microsporidial spores in stool samples, urine, and duodenal fluid (107). Therefore, antibodies should be used for species differentiation in samples only when the initial diagnosis of microsporidiosis by using fluorescent stains with optical brighteners and/or chromotrope-based stains has been made (Fig. 12) (107). E. bieneusi-specific antibodies have not been developed so far. Serologic Testing MOLECULAR METHODS Molecular studies of microsporidia are in their infancy. A limited number of genes have been located, and only a few DNA sequences have been reported in a few microsporidian species so far. Compared with other eukaryotes, microsporidia have extremely small genomes, often in the size range of bacterial genomes. Pulsed-field gel electrophoresis studies of the karyotypes of several microsporidian species showed that the haploid genome usually ranges from only 5.3 to 19.5 Mb, which represents the largest microsporidian genome measured to date in Glugea atherinae (23, 24, 252). However, the haploid genome of E. cuniculi was estimated to be only 2.9 Mb, which is smaller than all the other microsporidian genomes. This size is about half that of the previously smallest known microsporidian genome, 5.3 Mb in Nosema locustae (23). Eleven chromosomal DNA bands obtained by pulsed-field gel electrophoresis with DNA isolated from E. cuniculi spores ranged only from 217 to 315 kb. However, the chromosome number of E. cuniculi was larger than that of a Vairimorpha sp. and Nosema costelytrae, both of which have only eight chromosomes (231). The very small genome in E. cuniculi may be related to the early divergence of microsporidia, but it may be also a derived characteristic, perhaps related to the highly adapted parasitic lifestyle of this organism. Chromosomal localization of genes in microsporidia were done for only a few genes in a limited number of species. Eight genes in E. cuniculi have been localized so far. Each of the 11 chromosomes of E. cuniculi contains rdna, which strongly contrasts with the location of rdna within a single but rather large chromosome (760 kb) in Nosema bombycis, an insect microsporidium with a genome of 15.3 Mb (25). Further analysis of eluted DNA from each E. cuniculi chromosome by restriction enzyme digestion and rdna hybridization showed identical patterns, suggesting the presence of a single rdna unit copy per chromosome (25). However, most probes were assigned to single chromosomes, indicating a prevalence of low-copy-number nucleotide sequences within the very small genome of E. cuniculi. Both -tubulin and aminopeptidase genes were located on two different chromosomes (the -tubulin gene on chromosomes II and III, and the aminopeptidase gene on chromosomes I and VIII), whereas thymidylate synthase, dihydrofolate reductase, and serine hydroxymethyl transferase genes were located only on chromosome I, cdc2- like and ERCC6-like genes were located only on chromosome VIII (25), and an Hsp70 gene was located only on chromosome XI (275a). Small- and Large-Subunit rrna Genes of Microsporidia Although microsporidia are true eucaryotic organisms with a nucleus, an intracytoplasmatic membrane system, and chromosome separation by mitotic spindles, their rrna genes show features related to prokaryotic sequences (86, 362). They are composed of a 16S SSU rrna and a 23S LSU rrna sepa-