CLINICAL MICROBIOLOGY REVIEWS, Apr. 1999, p. 243–285 Vol. 12, No.

2
0893-8512/99/$04.0010
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, 50924 Cologne, Germany

INTRODUCTION .......................................................................................................................................................244
MORPHOLOGY OF MICROSPORIDIA ...............................................................................................................244
Morphology and Life Cycle ...................................................................................................................................244
Spore.....................................................................................................................................................................244
Merogony..............................................................................................................................................................244
Sporogony.............................................................................................................................................................245

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TAXONOMY ...............................................................................................................................................................245
GENUS- AND SPECIES-SPECIFIC CHARACTERISTICS .................................................................................247
Enterocytozoon sp. ....................................................................................................................................................247
Encephalitozoon spp.................................................................................................................................................248
Nosema spp. .............................................................................................................................................................249
Vittaforma sp. ...........................................................................................................................................................250
Pleistophora spp. and Trachipleistophora spp.......................................................................................................250
Other Genera...........................................................................................................................................................251
EPIDEMIOLOGY .......................................................................................................................................................251
Prevalence and Geographic Distribution ............................................................................................................251
Sources of Infection and Transmission ...............................................................................................................251
CLINICAL MANIFESTATIONS ..............................................................................................................................254
Gastrointestinal and Biliary Tract Infections.....................................................................................................254
Enterocytozoon bieneusi........................................................................................................................................254
Encephalitozoon spp.............................................................................................................................................255
Other species .......................................................................................................................................................255
Hepatitis, Pancreatitis, and Peritonitis ...............................................................................................................255
Ocular Infections ....................................................................................................................................................255
Encephalitozoon spp.............................................................................................................................................255
Other species .......................................................................................................................................................255
Sinusitis....................................................................................................................................................................256
Pulmonary Infections .............................................................................................................................................256
Urinary Tract Infections ........................................................................................................................................256
Myositis ....................................................................................................................................................................256
Cerebral Infections .................................................................................................................................................256
Encephalitozoon spp.............................................................................................................................................256
Other species .......................................................................................................................................................256
Rare Manifestations ...............................................................................................................................................256
Urethritis..............................................................................................................................................................256
Prostatic abscess .................................................................................................................................................256
Tongue ulcer ........................................................................................................................................................257
Skeletal involvement...........................................................................................................................................257
Cutaneous microsporidiosis ..............................................................................................................................257
Systemic Infections .................................................................................................................................................257
Encephalitozoon spp.............................................................................................................................................257
Other species .......................................................................................................................................................257
THERAPY ....................................................................................................................................................................257
DIAGNOSTIC METHODS........................................................................................................................................258
Transmission Electron Microscopy ......................................................................................................................258
Light Microscopy ....................................................................................................................................................258
Cytologic diagnosis and stool examination .....................................................................................................259
Histologic diagnosis............................................................................................................................................260
Cell Culture .............................................................................................................................................................261
Animal Models ........................................................................................................................................................261

* Corresponding author. Mailing address: Department of Internal

Medicine I, University of Cologne, Joseph-Stelzmann Str. 9, D-50924
Cologne, Germany. Phone: 49-(0)221-478-4433. Fax: 49-(0)221-478-
6456. E-mail: Caspar.Franzen@Uni-Koeln.de.

243
244 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV.

Antigen-Based Methods .........................................................................................................................................261

Serologic Testing.....................................................................................................................................................262
MOLECULAR METHODS .......................................................................................................................................262
Small- and Large-Subunit rRNA Genes of Microsporidia ...............................................................................262
a- and b-Tubulin Genes of Microsporidia .........................................................................................................263
Other Genes of Microsporidia ..............................................................................................................................264
DNA Isolation Techniques.....................................................................................................................................265
Molecular Techniques for Diagnosis and Species Differentiation ...................................................................265
Primer pairs and hybridization probes for E. bieneusi..................................................................................265
Primer pairs and hybridization probes for E. intestinalis .............................................................................268
Primer pairs for E. hellem and E. cuniculi ......................................................................................................269
General primer pairs and hybridization probes for several microsporidian species ...............................269
Strain differentiation of Encephalitozoon spp. and E. bieneusi......................................................................270
Comparison of molecular techniques with light microscopy........................................................................271
Molecular Techniques for Phylogenetic Analysis...............................................................................................272
Molecular Techniques for Susceptibility Testing ...............................................................................................275

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FUTURE TRENDS .....................................................................................................................................................275
CONCLUDING REMARKS ......................................................................................................................................275
ACKNOWLEDGMENTS ...........................................................................................................................................276
REFERENCES ............................................................................................................................................................276

INTRODUCTION field of clinical microbiology to incorporate these techniques

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 hu-
man 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 consider-
able infectious disease problems in industries such as fisheries
and silk production (39, 50, 56). The first human case of mi-
crosporidial infection was reported in 1959 (237), and only 10
well-documented human infections with microsporidia were
described until 1985, when a new species, Enterocytozoon bie-
neusi, was found in a human immunodeficiency virus (HIV)-
infected patient in France (99, 245). Since then, many infec-
tions with microsporidia have been reported from all over the
world, and these parasites are now recognized as one of the
most common pathogens in HIV-infected patients (7, 36–38,
53, 55, 56, 60, 66, 72, 87, 88, 96, 124, 130, 131, 136, 142–144,
162, 195, 204–208, 223, 225, 241, 246, 247, 253, 263, 265, 311,
325, 338, 344, 371, 372, 375).
The term “microsporidia” is a nontaxonomic designation
commonly used for organisms belonging to the phylum Micro-
spora. This phylum consists of over 100 genera with almost
1,000 species. So far only six genera (Enterocytozoon, Enceph-
alitozoon [including Septata], Pleistophora, Trachipleistophora,
Vittaforma, and Nosema) with at least 12 different species be-
longing 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 in-
tracellular 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 transmis-
sion 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. Di-
agnosis of infection caused by parasitic organisms is the last
(378). In this paper, we review human microsporidial infec-
tions and newly developed molecular techniques for detection,
species differentiation, and phylogenetic analysis of microspo-
ridia 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 consid-
ered 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 charac-
teristics. 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 mitochon-
dria, peroxisomes, and a classical stacked Golgi apparatus are
missing, they are true eukaryotes with a nucleus, an intracyto-
plasmatic 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 mm long.
Species that infect mammals are usually small, with diameters
of 1 to 3 mm. 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-
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FIG. 1. Diagram of a microsporidian spore and representative life cycle (merogonic and sporogonic stages vary among different genera).

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 char-
acterized by a dense surface coat. This surface coat later de-
velops into the exospore layer of the spore wall. Sporonts
multiply by binary or multiple fission and divide into sporo-
blasts 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 be-
come multinucleated plasmodial stages. Sporoblasts are ovoid
bodies that will mature to spores by synthesis of spore or-
ganelles (Fig. 1) (41, 43, 50, 58).
TAXONOMY
The term “microsporidia” is a nontaxonomic designation
commonly used for organisms belonging to the phylum Mi-
crospora, which is contained within the subkingdom Protozoa
246 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV.

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
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
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,
3640.

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(50, 337). In 1882 Balbiani classified these parasites as a sep-
arate group, “Microsporidies” (12). Before the middle of this
century, since knowledge of this group of organisms was frag-
mentary, classifications of microsporidia were necessarily sim-
ple and artificial. Subsequently, the taxonomy of microsporidia
has been subjected to several modifications. Major published
microsporidian classifications differ considerably in the char-
acteristics used to produce the major divisions within the mi-
crosporidia (337). Larsson (214) considered that many features
traditionally used for taxonomic systems (for example, the dip-
lokaryon, sporophorous vesicle, meiosis) have evolved inde-
pendently 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 mi-
crosporidia (214). Weiser (376) based his classification only on
the nuclear condition of the spores (one nucleus in Pleistopho-
ridida or two nuclei in Nosematidida), whereas Issi (183) used
the spore morphology and developmental stages. Until re-
cently, 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 devel-
oped during the last decade, it seems that differences in chro-
mosome cycles constitute the most fundamental basis for dis-
tinguishing taxa at the highest level (52, 337). Based on this
concept, Sprague et al. (337) proposed a comprehensive revi-
sion 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 Dihaplo-
phasea, which have a diplokaryon in some phase of their life
that the system of Sprague et al. (337) is flawed.

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 microspo-
ridia should be completely revised.
Nucleotide sequence data of the small-subunit (SSU) rRNA
of a microsporidian from insects, Vairimorpha necatrix, sug-
gested 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 a- and b-tubulin sequences suggested that the
microsporidia are close relatives of fungi, which may be
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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.

evolved degeneratively from higher forms (125, 126, 221). Re-
cently, genes encoding Hsp70 (a heat shock protein or chap-
eronin) have been identified in the microsporidia Nosema
locustae, V. necatrix, Encephalitozoon hellem, and Encephalito-
zoon cuniculi, and phylogenetic analyses have shown unequiv-
ocally that these genes are most closely related to those en-
coding Hsp70 proteins from the mitochondria of other
eukaryotes, suggesting that microsporidia may be evolved de-
generatively 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 fu-
ture 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 ultra-
structural data and rules of taxonomy (49). Several phyloge-
netic 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 Enterocyto-
zoon, Enterocytozoon bieneusi. This organism develops in direct
contact with the host cell cytoplasm. Meronts often have elec-
tron-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 de-
scribed 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-
248 FRANZEN AND MÜLLER
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, 317,500.
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 salmo-
nis. Ultrastructural examinations showed morphological simi-
larities between E. bieneusi and N. salmonis, with Nucleospora
exhibiting most of the distinguishing morphological character-
istics 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 sev-
eral ultrastructural differences in the development of these two
genera. Based on rRNA sequence data first generated by Bar-
lough et al. (13), rules of taxonomy, and the morphology and
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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 mm, 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,
VOL. 12, 1999
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.
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 hu-
mans, 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). Sev-
eral cases of Encephalitozoon infection were reported to occur
in patients with and without AIDS prior to 1991. Light and/or
electron microscopic analysis indicated that these infections
appeared to be due to E. cuniculi. However, in 1991 Didier et
al. (104) used biochemical and antigenic methods to describe a
new species of Encephalitozoon, E. hellem, which had been
found in three patients with AIDS. Since all subsequently pub-
lished cases of Encephalitozoon infections in humans appeared
to be caused by E. hellem (113, 162, 178, 211, 304–306, 369),
there was some doubt whether E. cuniculi did in fact cause
human infection (293). However, in 1995 De Groote et al. (95)
and Franzen et al. (144) described two homosexual men with
AIDS and disseminated E. cuniculi infection; identification
was confirmed by an immunofluorescence assay and by DNA
identification. Recently, E. cuniculi has been detected in sev-
eral 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 sim-
ilarities 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
MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 249
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placed in the genus Encephalitozoon and renamed Encephali-
tozoon intestinalis (10, 166). This reclassification is still contro-
versial (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 wide-
spread 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 mea-
suring 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 fea-
tures of the spores supported its assignment to the genus
Nosema as a new species, Nosema connori (235, 334). A mi-
crosporidium 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-year-
old HIV-infected patient was described by Cali et al. (48).
Development took place in direct contact with the muscle cell
250 FRANZEN AND MÜLLER
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, 317,500.
cytoplasm, and the organisms contained one or two diplocary-
otic pairs of nuclei. The spores measured about 2.5 to 2.9 by 1.9
to 2.0 mm, with 7 to 10 turns of the polar tubule. These features
are most closely aligned with the genus Nosema, and this or-
ganism 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 para-
sites 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 pro-
gressive central keratitis. Microsporidian spores measuring 3.7
by 1.0 mm 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 arrange-
ments. In cell culture, all the observed stages were detected
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individually in the host cell cytoplasm. This organism was orig-
inally assigned to the genus Nosema and named Nosema cor-
neum (317), even though the diplokaryotic arrangement of the
nuclei was the only character that conformed with the descrip-
tion of the genus Nosema. Based on the ultrastructure of de-
velopmental stages in liver cells of experimentally infected
athymic mice (tetrasporoblastic sporogony, band-like sporonts,
all stages surrounded by a cisterna of host endoplasmatic re-
ticulum), 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
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
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 mm with 10 to 12 coils of the polar tube.
The genus Trachipleistophora was established for a micro-
sporidium responsible for a fourth case of myositis, this time in
a patient with AIDS; organisms were found in corneal scrap-
ings, skeletal muscle, and nasal discharge (138). These para-
sites were cultivated in vitro and in athymic mice (180). Mer-
onts 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 Trachipleisto-
phora hominis (180).
Recently, two cases of infection with a Pleistophora-like mi-
crosporidian, which also seems to be a species of Trachipleis-
tophora, have been reported (271, 390). Sporogony distin-
guishes 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 de-
scribed 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 uncer-
tain 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 mea-
sured 1.5 by 3.5 mm, 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 per-
forated corneal ulcer (50, 277). Spores with 15 to 16 turns of
the polar tubule measured 4.5 by 1.5 mm, and no developmen-
tal 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, 142–144,
177, 179, 195, 204–208, 215, 238, 243, 244, 246, 247, 263, 266,
277–279, 291, 297–299, 304–306, 315, 316, 326, 328, 339, 344,
367–370, 389–391). Among persons without HIV infection,
only 35 cases of microsporidiosis have been documented (Ta-
bles 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
251
was not a microsporidium (79, 341, 377). Many of the 35
affected patients lived in or had traveled to tropical or sub-
tropical areas (96, 295, 329, 371). Intestinal E. bieneusi infec-
tion 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 de-
tected 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 sub-
tropical areas than in Europe or North America. Little is
known about the epidemiology of microsporidia, but the dis-
covery of self-limiting infections with E. bieneusi and E. intes-
tinalis in immunocompetent persons suggests that microspo-
ridia may be common human pathogens (56). The wide
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geographical distribution and the high prevalence among HIV-
infected patients suggest that microsporidia may be natural
parasites of humans, causing disease only in immunosup-
pressed hosts (56). Recently, microsporidia have been emerg-
ing as opportunistic pathogens in organ transplant recipients
being treated with immunosuppressive drugs (194, 284, 296).
More than 1,000 cases of microsporidiosis have been docu-
mented, the majority with E. bieneusi, in HIV-infected patients
(14, 30, 53, 72, 87, 99, 103, 109, 110, 118, 136, 142, 207, 244,
246, 263, 278, 279, 326, 344). Between 2 and 50% of HIV-
infected patients with severe immunodeficiency and CD4 cell
counts below 100/ml and otherwise unexplained diarrhea are
infected, depending on the study group and method of diag-
nosis (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 signif-
icant difference in the occurrence of microsporidiosis in pa-
tients 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 inten-
sity of diarrhea (71).
Infections with other microsporidian species have been re-
ported less frequently, but more than 100 cases of human
infections with Encephalitozoon spp. have been documented
(68, 95, 102, 113, 121, 122, 136, 143, 144, 195, 215, 238, 243,
247, 266, 291, 297–299, 304–306, 328, 369, 373, 374). Most of
these cases were due to E. intestinalis or E. hellem (68, 113, 121,
122, 136, 143, 195, 238, 243, 247, 266, 291, 297, 298, 304–306,
328, 369, 373), but recently E. cuniculi was detected in several
HIV-infected patients as well (1, 95, 144, 177, 179, 242, 374).
Human infections with other species (N. connori, N. ocula-
rum, 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-
 252
FRANZEN AND MÜLLER
TABLE 1. Case reports of microsporidiosis in patients not infected with HIV with normal or unknown immune status
Microsporidial speciesa Location Age (yr)b Sexc Clinical history Immune status Diagnostic method Yr of study Reference(s)
d e
E. cuniculi Japan 9 M Seizure disorder NK Cytology of CSF and urine 1959 237
E. intestinalis Swizerland 36 M Diarrhea (self-limited) Normal Examination of stool specimens 1995 139
E. intestinalis France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens 1998 286
E. intestinalis France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens 1998 286
E. bieneusi Germany (travel to Egypt and Jordan) 26 M Diarrhea (self-limited) Normal Examination of stool specimens 1994 295
E. bieneusi France (returning from tropical country) NK M Diarrhea NK, Crohn’s disease Examination of stool specimens 1994 89
E. bieneusi France (returning from tropical country) NK M Diarrhea NK, cardiac-valve graft Examination of stool specimens 1994 96
E. bieneusi France (travel to tropical country) NK M Diarrhea NK Examination of stool specimens 1994 96
E. bieneusi France (travel to tropical country) NK M Diarrhea NK Examination of stool specimens 1994 96
E. bieneusi France (returning from Africa) NK F Diarrhea NK Examination of stool specimens 1994 96
E. bieneusi France NK M Diarrhea NK Examination of stool specimens 1994 96
E. bieneusi Germany (travel to Turkey) 3 F Diarrhea (self-limited) Normal Examination of stool specimens 1995 329
E. bieneusi Swizerland 26 F Diarrhea (self-limited) Normal Examination of stool specimens 1995 139
E. bieneusi Zambia 7 mo M No symptoms Normal Examination of stool specimens 1997 167
E. bieneusi Spain 24 F Diarrhea (chronic) Normal Examination of stool and 1998 154a
duodenal biopsy specimens
M. ceylonensis Sri Lanka 11 M Keratitis NK Histologic examination 1973 6
M. africanum Botswana 26 F Corneal ulceration NK Histologic examination 1981 277
N. ocularum USA (Ohio) 39 M Keratitis Normal Histologic examination 1991 36, 44
V. corneae USA (South Carolina) (travel to 45 M Keratitis/iritis Normal Histologic examination 1990 92
Caribbean and Central America)
Not determined France 44 F Fever, myalgia Normal Examination of stool specimens 1995 249
Not determined France 72 M No symptoms Liver cirrhosis after Examination of stool specimens 1995 249
hepatitis B
Not determined France 63 M No symptoms Chronic alcoholism Examination of stool specimens 1995 249
Not determined France 68 M No symptoms Chronic alcoholism Examination of stool specimens 1995 249
Not determined France 64 M No symptoms Chronic alcoholism Examination of stool specimens 1995 249
Not determined France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens 1998 286
Not determined France (travel to tropical country) NK M Diarrhea Normal Examination of stool specimens 1998 286
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.

CLIN. MICROBIOL. REV.

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VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 253

TABLE 3. Animal hosts of human microsporidia

Microsporidial speciesa
c
b
a

Not determined

Not determined
N. connori
Pleistophora sp.

E. bieneusi

E. bieneusi
E. bieneusi

E. bieneusi
E. cuniculi
M, male; F, female.
Age in years unless otherwise indicated.
Classification is based only on morphology.
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
France

India
USA (Washington D.C.)
USA (Florida)

Spain

France
USA (Boston)

USA (Boston)
Sweden (born in Colombia)
Strain I Rabbits, mice
Strain II Mice, blue foxes
Strain III Domestic dogs, rodents, goats,
sheep, swine, horses, foxes,

Location
cats
N. connori None known
N. ocularum None known

TABLE 2. Case reports of microsporidiosis in patients not infected with HIV but with immunodeficiency
Nosema-like sp. None known
V. corneae None known

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Pleistophora spp. Fish 50
T. hominis None known
T. antropophtera None known
Age (yr)b

M. africanum None known

52

27

20

66

48
42

48
4 mo

M. ceylonensis None known

Sexc
M

F
M
M

M
M

F
M

bution of lesions, oral, respiratory, and ocular routes of infec-

tion are possible and are supported by evidence obtained from
Diarrhea (chronic),

Pneumonia
Disseminated infection
Myositis

Diarrhea (chronic)

Diarrhea
Diarrhea (chronic)

Diarrhea (self-limited)
Seizure disorder
malabsorption

experimentally infected rabbits (80, 153, 313), mice (106, 156,

Clinical history

209, 300, 342), and monkeys (106, 343). There is considerable

serologic evidence that humans without clinical signs of infec-
tion have been exposed to microsporidia (174–176, 327, 353).
Whether these persons are chronically or actively infected is
unknown.
Microsporidia are released into the environment via stool,
urine, and respiratory secretions. Persons or animals infected
with microsporidia are possible sources of infection. Experi-
Immunosuppression after

Immunosuppression after
Thymic aplasia
T-cell immunodeficiency

Lymphocytopenia and

Immunosuppression after
Decreased CD4 cell count

Immunosuppression after
Low CD4/CD8 cell ratio

mental Encephalitozoon infections of several animals by the

heart-lung transplantation

transplantation
bone marrow

hypogammaglobulinemia

heart-lung transplantation

liver transplantation

oral, tracheal, and rectal routes have been reported (80, 153,
209, 313). Person-to-person transmission of microsporidia may
Immune status

be significant. In a case-control study, intestinal microsporidi-

osis was associated with male homosexuality, thereby suggest-
ing 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).
Examination of stool specimens

Histologic examination (autopsy)

Histologic examination (autopsy)
Histologic examination

Examination of stool and

Examination of stool specimens

Examination of stool specimens

Examination of stool specimens

Cytology of urine, serum antibody

Whether microsporidiosis in humans is a zoonosis is un-

(gastrointestinal biopsy)
and histologic examination

duodenal biopsy specimens

known, and no direct proof of transmission from animals to

humans has been documented, with the exception of one case
Diagnostic method

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 Mex-
ico (32a), and Enterocytozoon bieneusi has been found in stool
samples of pigs and dogs in Switzerland (98) and in simian
Yr of study

immunodeficiency virus-infected macaques (234). E. hellem in-

1995

1994

1998
1973
1985

1996

1995
1996

1984

fection of birds, E. bieneusi infections of pigs, dogs, and mon-

keys, and E. intestinalis infection of different mammals were
confirmed by molecular techniques with rRNA data (32a, 98,
Reference

234). Molecular analysis of different human, rabbit, dog,

249

194

154a
235
216

284

296
365a

21

mouse, and blue fox E. cuniculi isolates showed that all isolates
from humans were of the same subtype as isolates from dogs
254 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV.

TABLE 4. Clinical manifestations of human disease is unknown. Results from recent studies involving mo-
microsporidial infections lecular techniques seemed to indicate the presence of E. intes-
Reference(s)
tinalis, E. bieneusi and V. corneae in raw sewage, tertiary efflu-
Species Clinical manifestation ents, surface water, and groundwater in France and the United
(first reports)
States (123a, 332a); there is one report of a presumably water-
E. bieneusi Enteritis 99, 119, 245 borne outbreak in Lyon (France) during summer 1995 (78a).
Cholangitis, cholecystitis 240, 278 Risk factors for intestinal microsporidiosis also suggest water
Pneumonia, bronchitis 367
Sinusitis, rhinitis 129, 165
as the source of infection (133, 181a). In a case-control study,
the only two factors associated with intestinal microsporidiosis
E. intestinalis Enteritis 266 were swimming in a pool and male homosexuality, both sug-
Cholangitis, cholecystitis 268, 385 gesting that the mode of transmission is fecal-oral (181a).
Nephritis, urinary tract infection 47, 268 However, no seasonal variation in the prevalence of micros-
Sinusitis, rhinitis 121, 147 poridiosis in HIV-infected patients was seen over 4 years in
Bronchitis 121, 150 southern California, suggesting a constant presence of micro-
Keratoconjunctivitis 226 sporidia in the environment rather than a seasonal association
Disseminated infection 47, 268 with recreational water use or seasonal contamination of the

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E. hellem Keratoconjunctivitis 104, 306
water supply (75a).
Sinusitis, rhinitis 178, 211
Pneumonia, bronchiolitis 304, 305
Nephritis, urinary tract infection 304 CLINICAL MANIFESTATIONS
Prostatic abscess 308
Disseminated infection 304 Microsporidiosis is truly an emerging infectious disease with
a rapidly broadening clinical spectrum of diseases. The spec-
E. cuniculi Hepatitis, peritonitisa 340, 392 trum of diseases includes gastrointestinal, pulmonary, nasal,
Encephalitis 237, 242, ocular, muscular, cerebral, and systemic infections. Microspo-
374 ridiosis should be considered in the differential diagnosis of
Intestinal infection 144 HIV-related symptomatic disease of virtually all organ systems
Urinary tract infection 95, 144 (Table 4) (271).
Keratoconjunctivitis 95, 144
Sinusitis, rhinitis 95, 144
Disseminated infection 95, 144 Gastrointestinal and Biliary Tract Infections
T. hominis Myositis 138, 180 Enterocytozoon bieneusi. Intestinal infections with microspo-
Keratoconjunctivitis 138, 180 ridia have been found mainly in HIV-infected patients, and
Sinusitis, rhinitis 138, 180 most infections have been due to E. bieneusi. It is most com-
T. antropophtera Encephalitis 271, 390
mon in patients with severe immunodeficiency and a CD4 cell
Myositis 271, 390 count below 100/ml (60, 127, 130, 131, 142, 246, 332). The
Disseminated infection 271, 390 parasites cause a severe, nonbloody, nonmucoid diarrhea with
up to 10 or even more bowel movements per day, slowly pro-
Pleistophora spp. Myositis 216, 229, gressive weight loss, and malabsorption of fat, D-xylose, and
230 vitamin B12 (60, 78, 127, 130, 131, 204, 205, 212). Intestinal
infection is associated with lactase deficiency and a reduced
V. corneae Keratitis 92, 317, 324 activity of alkaline phosphatase and a-glucosidase at the basal
part of the vilus and with reduced villus height and a vilus
N. ocularum Keratoconjunctivitis 36, 44
surface reduction (301). Diarrhea appears gradually and may
N. connori Disseminated infection 235, 334 continue for months. Patients are often reluctant to eat and
may complain of nausea (60, 78, 204, 205 212). Some patients
Nosema-like sp. Myositis 239 have intermittent diarrhea, but only a few excrete microspo-
ridial spores without having diarrhea (282, 283, 326, 330). In
M. africanum Corneal ulcer 50, 277 groups of patients with chronic diarrhea who were negative for
other enteric pathogens, the prevalence of E. bieneusi was
M. ceylonensis Corneal ulcer 6, 50 between 7 and 50% (127, 130, 131, 207, 246, 263). Some pa-
a
Species not confirmed because classification was based only on ultrastructure tients have coinfections with other pathogens (30, 171, 370);
morphology. 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 cholan-
and rabbits (97, 98, 108, 111, 181, 236). This fact supports the giopathies which are not explained by Cryptosporidium spp. or
hypothesis that human infections with E. cuniculi may be a cytomegalovirus infection (33, 201, 240, 278, 279).
zoonosis (97, 98, 111, 181, 236). Dissemination of E. bieneusi is very uncommon, but it has
Arthropods are the most common hosts of microsporidia, been detected in duodenal lamina propria cells (310), in bron-
and experimental infections of mice by a mosquito microspo- choalveolar lavage fluid and transbronchial biopsy specimens
ridium (Nosema algerae) have been accomplished (342, 345). (93, 367), and in nasal sinuses of HIV-infected patients (129,
Whether insect microsporidia might infect humans is un- 165, 184).
known. Intestinal infections with E. bieneusi in non-HIV-infected
Several microsporidia have been found in surface water sam- patients have been reported in only 15 patients in Germany,
ples (8), but whether human microsporidiosis is a waterborne Switzerland, France, Spain, Zambia, and the United States (96,
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
154a, 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 treat-
ment 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 met-
ronidazole (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 cho-
lecystitis (385). Disseminated infections occur regularly and
involve heavy infections of the urinary tract including the kid-
neys (68, 121, 143, 150, 247, 268). Left untreated, small bowel
infection with E. intestinalis can lead to perforation and peri-
tonitis (331). E. cuniculi only occasionally infects the gastroin-
testinal 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 gastrointesti-
nal tract but with no accompanying gastrointestinal symptoms.
Weber et al. (374) described a second patient with dissemi-
nated 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. intes-
tinalis was demonstrated in stool samples of his HIV-infected
partner. E. intestinalis was detected in two of seven stool sam-
ples from the non-HIV-infected man and again 4 months later,
together with Isospora belli, when he became mildly symptom-
atic after a trip to Brazil (139). Two other patients were trav-
elers presenting with chronic diarrhea, and microsporidian
spores were detected in their stools (286). Molecular identifi-
cation 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 clas-
sified 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/ml (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/ml (392). The
patient presented with a 13-kg weight loss over the course of a
year and was treated with trimethoprim-sulfamethoxazole be-
cause 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
255
microsporidia consistent in ultrastructure with E. cuniculi were
discovered within areas of mixed nongranulomatous inflamma-
tion 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 micro-
sporidial 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 micros-
poridial infection involving the liver, gallbladder wall, and a
mediastinal lymph node.
Both E. bieneusi and E. intestinalis have been detected in
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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 in-
volving several organ systems including the liver and the pan-
creas 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, keratocon-
junctivitis may be caused by all three Encephalitozoon spp. (E.
hellem, E. cuniculi, and E. intestinalis) (44, 45, 102, 104, 105,
113, 144, 152, 211, 224–226, 238, 243, 291, 306, 319, 369). Most
patients present with bilateral conjunctival inflammation and
also exhibit bilateral punctate epithelial keratopathy, leading
to decreased visual acuity. The keratoconjunctivitis is often
asymptomatic or moderate but can be severe; it rarely leads to
corneal ulcers (225).
Other species. Keratitis with corneal stroma infection was
described in an otherwise healthy 45-year-old man from South
Carolina who developed decreased vision in his left eye during
an 18-month history of unilateral progressive central keratitis
(92). There was no history of prior trauma. Corneal biopsy
revealed microsporidia invading deep into the corneal stroma.
This organism was successfully propagated in vitro and was
named Nosema corneum (317). On the basis of ultrastructural
data, it is now in a new genus and has been renamed Vittaforma
corneae (324).
A microsporidium was found to be responsible for the symp-
toms in a 39-year old man from Ohio who developed blurred
vision and irritation in his left eye. His visual symptoms per-
sisted 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 ultra-
structure; the species was named Nosema ocularum (44).
Trachipleistophora hominis was found in the corneal scrap-
ings 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 suffer-
ing 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-
256 FRANZEN AND MÜLLER
ridium ceylonensis and Microsporidium africanum, respectively
(50).
Sinusitis
Sinusitis is a common manifestation of human microsporidi-
osis. 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, 297–
299, 304, 305, 328, 369). Infection of the lower respiratory tract
may be asymptomatic or associated with bronchiolitis; it is
rarely associated with pneumonia or progressive respiratory
failure in HIV-infected patients (150, 287, 297–299, 304, 305,
369). All three Encephalitozoon spp. have been detected in
bronchial epithelial cells of HIV-infected patients with dissem-
inated Encephalitozoon infection, whereas pulmonary involve-
ment 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 pulmo-
nary microsporidiosis was reached only postmortem. Ultra-
structural examinations confirmed the organism to be a mi-
crosporidium, 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 in-
fections. The clinical presentation and consequences of the
presence of microsporidia in the urinary system can vary re-
markably; patients may be asymptomatic with or without mi-
crohematuria, they may have cystitis and intestinal nephritis
with dysuria and gross hematuria, or they may experience pro-
gressive 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/ml)
with progressive generalized muscle weakness and contrac-
tures 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 seronega-
tive 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 spec-
imen 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
CLIN. MICROBIOL. REV.
myositis with fever, myalgia, and progressive weakness. Mi-
crosporidian spores were detected in a muscle biopsy speci-
men.
In an Australian patient who presented with a severe, pro-
gressive myositis associated with fever and weight loss, Pleis-
tophora-like microsporidia were demonstrated in corneal
scrapings, skeletal muscle, and nasal discharge (138). The or-
ganisms 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 Encephali-
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tozoon 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 head-
ache, 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/ml. The patient was
hospitalized because of headache, visual and cognitive impair-
ment, nausea, and vomiting. Magnetic resonance imaging
scans showed right maxillary sinusitis and multiple small, con-
trast-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 inter-
genic 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 immunohis-
tochemistry and PCR, were detected in the brain, heart, kid-
neys, urinary bladder, spleen, lymph nodes, adrenals, and tra-
chea at autopsy.
Other species. Cerebral involvement with Trachipleistophora
antropophtera was reported in two AIDS patients, a 33-year-
old man and an 8-year-old girl, with seizures and cerebral
lesions, who died after empirical anti-toxoplasma therapy (20,
271, 390). At autopsy, a pansporoblastic microsporidium was
seen in several organ systems including the brain (271, 390).
Rare Manifestations
Urethritis. Two cases of urethritis associated with microspo-
ridia 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 infec-
tion (308). The prostate was of normal size with a 1.5- by
1.8-cm central periurethral abscess containing necrotic pros-
tatic tissue. Tissue Gram stain revealed gram-positive micro-
sporidian spores, which were identified as E. hellem by an
indirect fluorescence assay.
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
Tongue ulcer. A shallow 1-cm ulceration on the dorsum of
the tongue was observed in an HIV-infected patient with se-
vere immunodeficiency (15 CD4 cells/ml) 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 immuno-
fluorescent 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 os-
teomyelitis 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 Encepha-
litozoon infection in a 9-year-old Japanese boy who suffered
from recurrent fever, headache, vomiting, and spastic convul-
sions reported in 1959. 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 (immuno-
globulin 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/ml (95, 121, 144, 150, 162, 215, 247, 268, 297, 304,
305, 328, 369). The spectrum of disease has expanded to in-
clude keratoconjunctivitis, bronchiolitis and pneumonia, sinus-
itis, nephritis, urethritis, cystitis, prostatitis, hepatitis, peritoni-
tis, gastroenteritis, and cholangitis, but there are clear
differences in the typical distribution pattern for each micro-
sporidian species: E. hellem parasitizes mainly the keratocon-
junctiva, 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 manifesta-
tions 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 devel-
oped diarrhea, vomiting, fever, dyspnea, weight loss, and me-
chanical ileus. Laparatomy and several antibiotics failed to
alter the clinical course, and at autopsy sporoblasts with ma-
ture 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 cor-
neal scrapings, skeletal muscle, and nasal discharge (138). The
257
newly recognized pansporoblastic microsporidium, T. antropo-
phtera 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 lim-
ited 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 con-
trol the levels of microsporidial infection in invertebrate hosts;
these include fumagillin, an antibiotic produced by Aspergillus
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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 in-
fected or on the spore harvest (54). On the other hand, al-
bendazole 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 is-
ethionate inhibited E. cuniculi growth in cell cultures. Chloro-
quine, pefloxacin, azithromycin, rifabutin, and thiabendazole
were partially effective at high concentrations. Arprinocid,
metronidazole, minocycline, doxycycline, itraconazole, and di-
fluoromethylornithine were not evaluable, since the concentra-
tions that inhibited microsporidia were also toxic for the cells
in the cell culture. Pyrimethamine, piritrexim, sulfonamides,
paronomycin, roxithromycin, atovaquone, flucytosine, toltra-
zuril, 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 semisyn-
thetic 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 ef-
fects in Encephalitozoon-infected green monkey kidney cells,
and both albendazole and fumagillin caused different types of
parasite changes (219). In vivo efficacy of albendazole, fum-
agillin, and TNP-470 against E. cuniculi has been demon-
strated 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. Sev-
eral case reports and small case series have shown that al-
bendazole was highly effective for treatment of Encephalito-
zoon 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-
258 FRANZEN AND MÜLLER
ical findings was also achieved with topical fumagillin (113,
158, 314) in several HIV-infected patients with microsporidial
keratoconjunctivitis due to Encephalitozoon species. Resolu-
tion 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 infec-
tion in HIV-infected patients (2). Keratoconjunctivitis due to
an Encephalitozoon species in another AIDS patient re-
sponded 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 influ-
ence on the prevalence of intestinal microsporidiosis in HIV-
infected 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 re-
sponse after treatment with metronidazole (114, 136). The
efficacy of albendazole for E. bieneusi infections is controver-
sial (257). Diarrhea may improve in 50 to 60% of patients (29,
103, 114), but persistence of organisms was seen in posttreat-
ment biopsy specimens despite several ultrastructural changes
in the parasite (29, 31, 114). Other trials with albendazole
showed much lower response rates (142, 172, 246). Double-
blind placebo-controlled trials with albendazole are in
progress. Despite a remarkable clinical response with atova-
quone in symptomatic AIDS patients with intestinal E. bieneusi
infection, there was no apparent decrease in the parasite bur-
den 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 microspo-
ridian shedding in stool was observed in three E. bieneusi-
infected patients treated with furazolidone (116). Purified fum-
agillin 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 microspo-
ridial 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 oct-
reotide, 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 microspo-
ridial infections are not eradicated.
Since no effective therapy is available for E. bieneusi infec-
tion whereas infections with Encephalitozoon spp. respond very
CLIN. MICROBIOL. REV.
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 speci-
mens, duodenal or bile juice, urine, conjunctival smears, bron-
choalveolar 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 di-
agnosis of microsporidiosis required transmission electron mi-
croscopy, but during the last few years new staining methods,
suitable for light microscopy, have been developed. Microspo-
ridia have now been found in virtually every tissue and body
fluid in humans.
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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 an-
tibodies 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 suit-
able 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 micros-
poridia has now led to the application of new molecular tech-
niques for the diagnosis of human microsporidiosis.
Transmission Electron Microscopy
Originally, definitive diagnosis of microsporidiosis required
ultrastructural examination of biopsy tissues, body fluid spec-
imens (urine, nasal discharge, sinus aspirates, sputum, bron-
choalveolar 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). Visualiza-
tion of the unique ultrastructure of the spores with their char-
acteristic 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 inter-
face. 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 micros-
copy 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 transmis-
sion electron microscopy are lacking (62, 73). Likewise, sample
preparation and examination are laborious and time-consum-
ing (62, 73).
Light Microscopy
Histologic examination of biopsy specimens or cytologic ex-
amination 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-
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 259

Special features

Vacuole

Nucleus

Arrangement of polar tube

No. of coils of polar tubule

Size of spores (mm)

Characteristic
Electron-lucent

No vacuole, in direct

Unikaryotic

Two rows

5–7

1.1–1.6 by 0.7–1.0
sporonts
beginning in the
polar tubule
development of the
inclusion,

cell cytoplasma
contact with the host

E. bieneusi

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TABLE 5. Morphological characteristics of microsporidia infecting humans
Septated parasitophorous

Unikaryotic

One row

5–7

2.0–2.5 by 1.0–1.5
intestinalis
vacuole in E.

Encephalitozoon spp.

FIG. 8. Encephalitozoon cuniculi spores in conjunctival swab from an HIV-

infected patient with disseminated infection. Modified chromotrope-based stain.
Magnification, 3870.

tion pattern of the infection may be of limited use, but exact

species differentiation is impossible.
Cytologic diagnosis and stool examination. Microsporidian
Diplosporoblastic

No vacuole, in direct

Diplokaryotic

One row

7–12

2.5–5.0 by 2.0–2.5
sporogony

cell cytoplasma
contact with the host

spores have been detected in several body fluids and stool

samples (264, 347). The outer layer of the spore wall (exo-
Nosema spp.

spore) 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 differen-
tiate between microsporidia and other elements present in
body fluids or stool specimens that can be confused with mi-
Tetrasporoblastic

No vacuole, in direct

Diplokaryotic

One row

5–7

3.05–4.55 by 0.77–1.27

crosporidian spores (75). Microscopic examination of body

reticulum
endoplasmatic
cisterna of host
surrounded by a
sporonts, all stages are
sporogony, band-like

cell cytoplasma
contact with the host

fluids to diagnose microsporidia did not become routine until

special chromotrope-based and fluorescent stains were devel-
V. corneae

oped. Nowadays these chromotrope- and/or fluorochrome-

based 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,
Multinucleate

Sporophorous vesicle

Unikaryotic

Two rows

9–12

3.2–3.4 by 2.8

microsporidian spores stain characteristic pinkish red. Usually

plasmodia
sporogonial

Pleistophora spp.

the spores have a characteristic appearance when examined

under high-power magnification (31,000). 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 diffi-
cult 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
No multinucleate

Unikaryotic
Sporophorous vesicle

One or two rows

11

4.0 by 2.4
Trachipleistophora spp.

substitution of a color-fast counterstain [294]) of the chromo-

antropophtera
development of T.
are formed during
vesicles and spores
sporophorous
different types of
plasmodia, two
sporogonial

trope-based stain have been suggested for speeding the process

and for better contrast between the spores and the back-
ground. The improved hot Gram-chromotrope technique pro-
vides 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
260 FRANZEN AND MÜLLER
FIG. 9. Encephalitozoon intestinalis spores derived from in vitro culture. Flu-
orescence microscopy after Uvitex 2B stain. Magnification, 31,000.
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 de-
tecting 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 exci-
tation filter and a high-magnification objective lens (magnifi-
cation, 31,000). Fluorochromes bind to the chitin of the en-
dospore, 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 prop-
erties (Fig. 9) (74, 348). Several modifications of the fluores-
cent 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 en-
hance 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.
CLIN. MICROBIOL. REV.
FIG. 10. Paraffin-embedded duodenal biopsy specimen from a patient with
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AIDS with intestinal Enterocytozoon bieneusi infection. The microsporidial
spores are easily visualized within the enterocytes. Fluorescence microscopy after
Uvitex 2B stain. Magnification, 31,000.
Whether concentration techniques are useful for detection of
microsporidian spores in stool specimens remains controver-
sial. Some authors reported that concentration techniques such
as the formalin-ethyl acetate concentration procedure or dif-
ferent flotation methods led to a substantial loss of microspo-
ridial 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 KOH-
treated stool samples increased the yield of microsporidian
spores (62, 349). Therefore, one of these concentration tech-
niques should be used for stool samples.
Histologic diagnosis. Because of the small size of the spores,
reliable visualization of microsporidia by light microscopy de-
pends 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 back-
ground (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 chro-
motrope-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 cyto-
logic specimens is useful in the diagnosis of intestinal micros-
poridiosis, 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 eo-
sin, 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).
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
FIG. 11. Resin-embedded semithin (1-mm) section of duodenal mucosa from
a patient with AIDS and intestinal Enterocytozoon bieneusi infection. Epithelial
cells contain spores of Enterocytozoon bieneusi. Toluidine blue stain. Magnifica-
tion, 3800.
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, 177–179, 358–360),
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 labo-
ratories 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, 177–179,
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. bie-
neusi seems to exert a greater cytotoxic effect than has been
observed with cell cultures of Encephalitozoon spp. The inabil-
ity 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).
261
Animal Models
Animal models provide a basis for studying immune re-
sponses and for evaluating diagnostic methods, vaccine candi-
dates, therapeutic strategies, and routes of transmission (106).
Furthermore, they are essential for producing poly- and mono-
clonal 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 labo-
ratory 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. cunic-
uli, E. hellem, or V. corneae (106, 156). SCID mice have also
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been infected by oral inoculation of E. cuniculi spores. This
animal model was used to study the in vivo efficacy of albenda-
zole against E. cuniculi (209). Experimental E. cuniculi infec-
tions in immunocompetent mice produced only chronic asymp-
tomatic 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. cu-
niculi, E. hellem, and V. corneae per os (106).
Animal models for E. bieneusi infection are difficult to es-
tablish. Attempts to infect immunosuppressed gnotobiotic pig-
lets, gamma interferon knockout mice, and SCID mice treated
with anti-gamma interferon monoclonal antibodies with E. bie-
neusi have been unsuccessful. Recently, experimental oral
transmission of E. bieneusi to simian immunodeficiency virus-
infected rhesus monkeys has been reported (343).
Antigen-Based Methods
Microsporidium-specific antibodies in immunofluorescence
tests have been used for the diagnosis and species differentia-
tion of microsporidia. Poly- and monoclonal antibodies were
also used for Western blot analysis of several microsporidian
species (4, 84, 85, 132, 198–200, 256, 259, 261, 304–309, 358–
360, 379, 397).
Immunofluorescent antibody tests involving polyclonal anti-
sera 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 differentia-
tion 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-
262 FRANZEN AND MÜLLER
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, 3400.
trope-based stain, a fluorescent stain containing Calcofluor
white, and a fluorescent polyclonal antibody stain. The fluo-
rescent 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
A variety of serological tests (carbon immunoassay, indirect
immunofluorescence test, enzyme-linked immunosorbent as-
say, counterimmunoelectrophoresis, and Western blotting)
have been developed to detect IgG and IgM antibodies to
microsporidia, especially to E. cuniculi (18, 21, 22, 174–176,
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 sensi-
tivity and specificity of all these tests are unknown (174–176).
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 sug-
gested a possible link between exposure to a tropical environ-
ment 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 seroposi-
tive 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 clinico-
pathologic correlations have not been reported for any of these
serologic surveys. Of interest, a high seroprevalence of anti-
bodies 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 noninfec-
tious 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 unavail-
CLIN. MICROBIOL. REV.
able. 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 in-
fection may have positive titers and immunosuppressed per-
sons may have a poor response to antigen challenge.
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 ex-
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tremely small genomes, often in the size range of bacterial
genomes. Pulsed-field gel electrophoresis studies of the karyo-
types of several microsporidian species showed that the hap-
loid 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 microspo-
ridian genome, 5.3 Mb in Nosema locustae (23). Eleven chro-
mosomal DNA bands obtained by pulsed-field gel electro-
phoresis 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 chromo-
somes (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 anal-
ysis 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 b-tubulin and aminopeptidase
genes were located on two different chromosomes (the b-tu-
bulin gene on chromosomes II and III, and the aminopeptidase
gene on chromosomes I and VIII), whereas thymidylate syn-
thase, 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 chromo-
some 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-
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
rated by an intergenic nontranscribed spacer (363). In contrast
to true eukaryotic organisms, microsporidia lack a separate
5.8S rRNA, which is believed to be a universal eukaryotic
characteristic, and also a second intergenic spacer (363–365).
As in the prokaryotes, microsporidia have LSU rRNA whose
59 region corresponds to the 5.8S rRNA of eukaryotes (363–
365). The SSU and LSU rRNA genes are shorter than typical
eukaryotic SSU and LSU rRNA genes and lack several uni-
versal sequences (158a, 275b, 363–365).
The first sequence data of SSU-rRNA of a microsporidium,
V. necatrix, were reported by Vossbrinck et al. in 1987 (363)
(accession no. M24612). Subsequently, PCR amplification with
primers complementary to conserved sequences of the V. ne-
catrix SSU rRNA gene has been used to amplify and sequence
DNA fragments of the SSU rRNA gene from several micro-
sporidia that infect humans (164–166, 359, 364, 365, 393–396).
Several nucleotide sequences of SSU and LSU rRNA genes
including the intergenic spacer regions of different microspo-
ridian species, including six species infecting humans (E. bie-
neusi, E. cuniculi, E. hellem, E. intestinalis, T. hominis, and V.
corneae), have been published and are accessible via the Gen-
Bank and EMBL databases (October 1998) (Table 6).
The rRNA sequences of E. bieneusi were first reported by
Zhu et al. and deposited in the GenBank database under
accession no. L07123 and L20290 (395). Gene amplification
was performed with DNA extracted from human intestinal
biopsy specimens from HIV-infected patients with electron
microscopy-confirmed E. bieneusi infection. One primer pair
was in a conserved region of the SSU rRNA (V1 and 1492),
and a second was in a conserved region of the SSU rRNA
(530f) and LSU rRNA (580r). Primer V1 is based on the
sequence of the SSU rRNA gene of V. necatrix at the 59 end,
and primer 1492 is based on rRNA sequences highly conserved
across many organisms at the 39 end. An amplification product
of about 1,300 bp was obtained, cloned, and sequenced (395).
Resequencing of the SSU and LSU rRNA regions of different
E. bieneusi isolates by other researchers (89, 165, 289, 361)
(accession no. L16868, and AF024657) showed a 2.3 to 2.7%
dissimilarity even in conserved regions with respect to the
sequences reported by Zhu et al. Some of these differences
may be caused by polymorphisms, but many are probably due
to unresolved sequencing artifacts in the sequence reported by
Zhu et al. (395).
E. intestinalis SSU rRNA sequences were reported by sev-
eral researchers (10, 166, 360, 394, 396). Zhu et al. used primer
pair V1 and 1492 to amplify a 1,300-bp DNA fragment from
intestinal biopsy specimens with electron microscopy-con-
firmed E. intestinalis infection (394, 396). The DNA fragment
was cloned and sequenced, and the obtained sequence was
deposited in the GenBank database under accession no.
L19567 (394, 396). Resequencing of the SSU rRNA region of
different E. intestinalis isolates by other investigators (10, 166,
360) (accession no. U09929, L39113, L16866, and L16867)
showed several dissimilarities to the sequence reported by Zhu
et al. Although some may be caused by polymorphisms, dis-
similarities are also reported in conserved regions of the SSU-
rRNA, thus indicating that the E. intestinalis sequence of Zhu
et al. may contain several sequencing errors.
The E. cuniculi SSU rRNA sequence was also first reported
by Zhu et al. and was deposited in the GenBank database
under accession no. Z19563 (393). They amplified the SSU-
rRNA gene with primer pair V1 and 1492 from rabbit kidney
cells (RK-13) infected with E. cuniculi in vitro. A 1,200-bp
DNA fragment was amplified, cloned, and sequenced (393).
Several further SSU and LSU rRNA sequences of different E.
cuniculi strains have been deposited in the GenBank database,
263
and there are significant differences between the SSU rRNA
sequence determined by Zhu et al. and that obtained by others
(10, 164, 359, 364) (accession no. L07255, L17072, L39107,
X98469, X98470, and X98467) which includes also bases in
conserved regions of the rRNA gene. Again, some dissimilar-
ities may be caused by polymorphisms, but dissimilarities in
conserved regions indicate that the sequence reported by Zhu
et al. contained several sequencing errors.
The complete E. hellem SSU rRNA sequence data were first
described by Visvesvara et al. (359). They used a primer pair
based on nucleotides 1 to 21 (Micro-F [the nucleotide se-
quence of this primer is identical to that of primer V1]) and
1244 to 1218 (Micro-R) of the V. necatrix SSU rRNA gene to
amplify, clone, and sequence the SSU rRNA gene of E. hellem,
E. cuniculi, and E. intestinalis (359, 360). The obtained se-
quences are deposited in the GenBank database under acces-
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sion no. L19070, L17072, and U09929.
SSU rRNA sequences of V. corneae (accession no. L39112
and U11046) and T. hominis (accession no. AJ002605) are
available via the GenBank database as well.
Vossbrinck et al. (364) used highly conserved primers 530f in
the SSU rRNA and 580r and 228r in the LSU rRNA to amplify
the 39 end of the SSU rRNA gene, the intergenic spacer, and
the 59 end of the LSU-rRNA gene of E. hellem (accession no.
L13331) and E. cuniculi (accession no. L13332). The same
primers were used by Zhu et al. (396) to amplify the same
region of SSU and LSU rRNA genes including the intergenic
spacer of E. bieneusi (accession no. L20290) and E. intestinalis
(accession no. L20292) (396). The 39 end of the SSU-rRNA
gene, the intergenic spacer, and the 59 end of the LSU-rRNA
gene of several microsporidian species found in humans have
now been sequenced by many researchers and are available via
the GenBank or EMBL database (Table 6).
To date, complete LSU rRNA sequences have been deter-
mined only for E. cuniculi (accession no. AJ005581) and N.
apis (accession no. U97150). The sizes were estimated to be
about 2,487 and 2,480 bp, respectively, which is in the size
range of prokaryotic 23S rRNA; this shortening is essentially
due to truncation of divergent domains, with most of them
being removed (158a, 275b). Most variable stems of the con-
served core are also deleted, reducing the LSU rRNA to only
the structural features preserved in all living cells (275b).
a- and b-Tubulin Genes of Microsporidia
The tubulin gene family consists of three distinct but highly
conserved subfamilies, a-, b-, and g-tubulin. a- and b-tubulins
are the most abundant in eukaryotic cells and have been stud-
ied most extensively. Microtubules are a characteristic feature
of all eukaryotic organisms, since they are major components
of the cytoskeleton and the mitotic spindle. Microtubules form
by polymerization of tubulin, a dimer of a- and b-subunits,
each approximately 440 amino acids long (125, 189). The func-
tion of g-tubulin is less clear, although it is known to be im-
portant in microtubule-organizing centers and has been impli-
cated in several other processes. About 100 b-tubulin
sequences as well as many a-tubulin sequences from a wide
range of eukaryotic cells have been reported. In contrast to
rRNA sequences and many other proteins, deletions or inser-
tions are rare in tubulin genes, so that there are no problems
in alignment. Phylogenetic analysis with a- and b-tubulin se-
quences is consistent in several respects with rRNA-based phy-
logenetics but has generated different results for some amito-
chondrial protozoa including microsporidia (125).
b-Tubulin sequences of E. cuniculi (accession no. L31807)
264 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV.

TABLE 6. Human microsporidian genes in GenBanka

Accession no. Gene Organism Reference(s)

X98470 SSU rRNA E. cuniculi Donovan 181

X98469 SSU rRNA E. cuniculi Stewart (host, Canis familiaris) 181
X98467 SSU rRNA E. cuniculi D. Owen (host, Mus musculus) 181
L39107 SSU rRNA E. cuniculi 10
L17072 SSU rRNA E. cuniculi 359
L13295 SSU rRNA E. cuniculi partial cds 364
Z19563 SSU rRNA E. cuniculi 393
L07255 SSU rRNA E. cuniculi 164
AJ005581 SSU rRNA E. cuniculi 275b
L13393 SSU rRNA E. hellem partial cds 364
L39108 SSU rRNA E. hellem 10
L19070 SSU rRNA E. hellem 359
AF039229, AF039230 SSU rRNA E. hellem (host, Electus roratus) partial cds 281a
U39297 SSU rRNA E. intestinalis partial cds 148
U09929 SSU rRNA E. intestinalis 360

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L39113 SSU rRNA E. intestinalis 10
L19567 SSU rRNA E. intestinalis 394
L16867 SSU rRNA Encephalitozoon sp. 166a
L16866 SSU rRNA Encephalitozoon sp. 164
L16868 SSU rRNA E. bieneusi 165
L07123 SSU rRNA E. bieneusi 395
AF023245 SSU rRNA E. bieneusi (host, Macaca mulatta) 234a
U61180 SSU rRNA E. bieneusi (host, pig) 98
AF024657 SSU rRNA E. bieneusi 89
AJ002605 SSU rRNA T. hominis 66a
L39112 SSU rRNA V. cornea 10
U11046 SSU rRNA V. cornea 276a
X98466 39-end SSU, ITS,b 59-end LSU E. cuniculi Donovan 181
X98468 39-end SSU, ITS, 59-end LSU E. cuniculi D. Owen (host, Mus musculus) 181
L29560 39-end SSU, ITS, 59-end LSU E. cuniculi 189
L13332 39-end SSU, ITS, 59-end LSU E. cuniculi 364
AJ005581 39-end SSU, ITS, 59-end LSU E. cuniculi 275b
AF069064 39-end SSU, ITS, 59-end LSU E. hellem 151a
L13331 39-end SSU, ITS, 59-end LSU E. hellem 364
L29556 39-end SSU, ITS, 59-end LSU E. hellem 189
AF069064 39-end SSU, ITS, 59-end LSU E. hellem 151a
L20292 39-end SSU, ITS, 59-end LSU E. intestinalis 396
Y11611 39-end SSU, ITS, 59-end LSU E. intestinalis 301a
L20290 39-end SSU, ITS, 59-end LSU E. bieneusi 396
U61180 39-end SSU, ITS, 59-end LSU E. bieneusi (host, pig) 98
AF023245 39-end SSU, ITS, 59-end LSU E. bieneusi (host, Macaca mulatta) 234a
AF059610 39-end SSU, ITS, 59-end LSU E. bieneusi (host, Canis familiaris) 236a
AJ005581 LSU rRNA E. cuniculi 275b
U66908 a-Tubulin E. hellem 192
L47271 b-Tubulin E. hellem 221
L31808 b-Tubulin E. hellem partial cds 125
L31807 b-Tubulin E. cuniculi partial cds 125
L47274 b-Tubulin E. intestinalis partial cds 221
AJ005666 Polar tube protein E. cuniculi 95a
AF044915 Polar tube protein 55 precursor E. hellem 200
AF054829 Ribosomal protein L27a gene E. cuniculi 25a
AJ005644 Dihydrofolate reductase E. cuniculi 124a
AJ005644 Serine hydroxymethyltransferase E. cuniculi 124a
AJ005644 Aminopeptidase E. cuniculi 124a
AJ005644 Thymidylate synthase E. cuniculi 124a
AF031701 Actin (ACT1) E. hellem 126a
a
For a more complete list including microsporidian species not infecting humans, see reference 381a.
b
ITS, internal transcribed spacer.

and E. intestinalis (accession no. L47274) and a- and b-tubulin
sequences of E. hellem (accession no. U66908, L47271, and
L31808) were amplified with primers corresponding to con-
served sequences (125, 126, 188, 189, 192, 221). Southern blots
indicate that E. cuniculi, E. hellem, and E. intestinalis possess a
single b-tubulin gene copy and that the three b-tubulin se-
quences are very similar to each other (125, 188, 189).
a-Tubulin sequences of Nosema locustae (accession no.
U66907) and Spraguea lophii (U66906) have been determined
and are available via the GenBank or EMBL databases (192).
Other Genes of Microsporidia
Besides rRNA and a- and b-tubulin genes, only those en-
coding isoleucyl-tRNA synthase (accession no. L37097) and
glutamyl-tRNA synthetase in N. locustae (AF005490 and
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
AF005489) (35), homologues of U2 spliceosomal RNA in V.
necatrix (115) and of U2 and U6 spliceosomal RNA in N.
locustae (AF053588 and AF053589) (133a), reverse transcrip-
tase (AF019229), chitin synthase (AF019228), and DNA-di-
rected RNA polymerase II (AF019227) in S. lophii (172a),
translation elongation factors EF-1a (D32139) and EF-2
(D79220) in Glugea plecoglossi (186, 187), a chaperone protein
Hsp70 in N. locustae (U97520), V. necatrix (AF008215), E.
hellem, and E. cuniculi (159, 173, 275a), actin (ACT1) in E.
hellem (AF031701) and N. locustae (AF031702), a polar tube
protein in E. cuniculi (AJ005666) (95a) and E. hellem
(AF044915) (200), a ribosomal protein (AF054829), dihydro-
folate reductase, serine hydroxymethyltransferase, aminopep-
tidase, and thymidylate synthase (AJ005644) in E. cuniculi
(25a, 124a) have been partially characterized in microsporidia.
DNA Isolation Techniques
Microsporidian DNA can be easily extracted from tissue
samples or in vitro cultures by routine procedures such as
proteinase K digestion followed by phenol-chloroform extrac-
tion and ethanol precipitation or by DNA purification methods
with commercial kits such as Magic Minipreps (Promega
Corp.) or QIAmp tissue kits (Qiagen) (81, 91, 145, 148, 149,
236).
DNA isolation from microsporidian spores is more difficult,
and harsh conditions must be used to destroy the spore wall.
Mechanical disruption of the spores with glass beads in com-
bination with proteinase K digestion is commonly used (109,
110, 134). Some authors recommend additional incubation
with enzymes that resolve chitin (lyticase or chitinase) (134) or
boiling the samples to release DNA from spores (260, 262).
If stool samples are used, treatment of stool with 0.5%
sodium hypochlorite, 10% formalin, or 1 M KOH is recom-
mended to remove PCR inhibitors (134, 190, 222), whereas
others have reported that inhibition can be nullified by simple
dilution of the samples (262).
Molecular Techniques for Diagnosis and
Species Differentiation
Several PCR-based methods have been published to amplify
different regions of the SSU and LSU rRNA gene as well as
the intergenic spacer region for diagnosis and species differ-
entiation of microsporidia infecting humans (135) and animals
(232, 233) (Tables 7 and 8).
Primer pairs and hybridization probes for E. bieneusi. PCR
diagnosis of E. bieneusi was first reported by Zhu et al. (395).
Primer pair V1 and EB450 amplified cloned E. bieneusi SSU
rRNA sequences and DNA from E. bieneusi-infected tissues
but also amplified E. hellem DNA from cell cultures (81, 145,
149, 381, 395). Specificity was tested with DNA prepared from
Toxoplasma gondii, Trypanosoma cruzi, Escherichia coli, Sac-
charomyces cerevisiae, E. intestinalis, V. necatrix, E. cuniculi,
Glugea stephani, N. locustae, N. bombycis, Pleistophora, and E.
hellem and with gastrointestinal biopsy specimens infected with
E. cuniculi, E. intestinalis, Cryptosporidium spp., Giardia lam-
blia, Mycobacterium avium, and Candida albicans (81, 145, 381,
395). Only weak amplification of DNA prepared from E. hel-
lem was seen (395). In other studies, these primers did not
amplify DNA from E. hellem (81). Based on the SSU rRNA
sequence of E. hellem, amplification should not occur. It is
likely that the amplification observed by Zhu et al. (395) was
due either to contamination of the original E. hellem culture
with spores of E. bieneusi (which were being cultivated in the
265
same incubator) or to contamination of the culture with a
plasmid containing the cloned E. bieneusi SSU rRNA gene
(81). The V1-EB450 primer pair reliably amplifies E. bieneusi
DNA from gastrointestinal biopsy specimens from patients
with light and electron microscopy-confirmed E. bieneusi in-
fection (81, 145, 149, 381, 395) and from stool specimens from
patients with light microscopy-confirmed intestinal microspo-
ridiosis (262, 339a). Further evaluation of this primer pair gave
negative results with one bile sample that was confirmed to
contain E. bieneusi by electron microscopy and with E. bieneusi
derived from a short-term culture (89). Resequencing of the
SSU rRNA region of different E. bieneusi isolates by other
researchers showed a 2.3 to 2.7% dissimilarity to the sequence
reported by Zhu et al. (395), resulting in a base mismatch of
primer EB450 (position 9 of the EB450 primer has G in place
of C) (89). The upstream primer V1 is not specific for E.
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bieneusi but is directed against a conserved sequence for many
microsporidian species (381). The base mismatch of primer
EB450 combined with this lack of specificity may lead to neg-
ative results with some samples. An internal 30-mer oligonu-
cleotide, EB150, complementary to a region of the amplicon
produced by V1 and EB450 has been used as a probe for
Southern blot hybridization analysis of the PCR products (81,
145, 149, 381, 395) by isotopic and nonisotopic procedures
(145, 149, 395). Probe EB150 hybridized with E. hellem DNA
when amplified by V1 and EB450 (149, 395); again, it is likely
that this was due either to contamination of the original E.
hellem culture with spores of E. bieneusi or to contamination of
the culture with a plasmid containing the cloned E. bieneusi
SSU rRNA gene (81).
The primer pair V1-EB450 and a second pair, V1-Mic3
(Mic3 is located at positions 445 to 427 of the SSU rRNA gene
of E. bieneusi), have been used to confirm E. bieneusi infection
in simian immunodeficiency virus-infected rhesus monkeys.
The specificity of the PCR products was confirmed by DNA
sequencing and restriction fragment length polymorphism
analysis with MnlI and DdeI restriction endonucleases (343).
The primer pair V1-Mic3 has also been used to amplify a
446-bp fragment from the E. bieneusi SSU rRNA gene of
HIV-infected patients with intestinal microsporidiosis due to
E. bieneusi (63).
The primer pair EBIEF1 and EBIER1 described by Da Silva
et al. (89) amplified cloned E. bieneusi SSU rRNA sequences
and DNA from bile fluid, a duodenal aspirate from an AIDS
patient with electron microscopy-confirmed E. bieneusi infec-
tion, and a short-term culture of an E. bieneusi isolate. Speci-
ficity was tested with human DNA as well as with cloned
microsporidian SSU rRNA coding regions of 13 species of
microsporidia, and positive amplification was shown only with
the E. bieneusi cloned template. PCR products generated with
this primer pair were analyzed only by ethidium bromide-
stained gel analysis, and the identity of the PCR products was
not confirmed by Southern blot hybridization, DNA sequenc-
ing, or restriction enzyme digestion (89). Estimating the size of
the PCR-generated fragment by gel electrophoresis alone is
usually not sufficient to confirm the specificity of a PCR. Every
primer that is nonspecifically elongated at its 39 end because of
unwanted annealing to partially complementary sequences not
only is lost for specific priming but also enhances further non-
specific amplifications. This may lead to false-positive results,
which should be ruled out by the confirmation techniques
mentioned above.
An E. bieneusi-specific primer pair and hybridization probe
were designed by Schuitema et al. (303) and further evaluated
by David et al. (91) and Liguory et al. (222). The primer pair
and probe were used for species differentiation for samples
266 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV.

TABLE 7. Primer pairs and hybridization probes used for diagnosis and species differentiation of human microsporidia
Primer pair sequence
Primer designation Hybridization probe

59-CACCAGGTTGATTCTGCCTGAC-39 V1 59-TGTTGCGGTAATTTGGTCTCTGTGTGTAAA-39
59-ACTCAGGTGTTATACTCACGTC-39 EB450

59-GCCTGACGTAGATGCTAGTC-39 59-GTTCAATAGCGATGAGTTTGCTAATGTT-39
59-ATGGTTCTCCAACTGAAACC-39

59-GAAACTTGTCCACTCCTTACG-39 EBIEF1
59-CCATGCACCACTCCTGCCATT-39 EBIER1

59-CACCAGGTTGATTCTGCCTGAC-39 V1
59-CAGCATCCACCATAGACAC-39 Mic3

59-TCAGTTTTGGGTGTGGTATCGG-39 Eb.gc
59-GCTACCCATACACACATCATTC-39 Eb.gt

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59-CACCAGGTTGATTCTGCCTGAC-39 V1 59-TGTTGATGAACCTTGTGG-39
59-CTCGCTCCTTTACACTCGAA-39 SI500 59-TGGTAGTTTAGGGTAATGGCCTAACTAGGCG-39

59-GGGGGCTAGGAGTGTTTTTG-39 59-TTTGGGGGATTATGTCCTGATGTGGAT-39
59-CAGCAGGCTCCCTCGCCATC-39

59-TTTCGAGTGTAAAGGAGTCGA-39 SINTF1
59-CCGTCCTCGTTCTCCTGC-39 SINTR

59-CACCAGGTTGATTCTGCCTGAC-39 V1
59-CCTGCCCGCTTCAGAACC-39 Sep1

59-TGAGAAGTAAGATGTTTAGCA-39 EHEL-F
59-GTAAAAACACTCTCACACTCA-39 EHEL-R

59-ATGAGAAGTGATGTGTGTGTGCG-39 ECUN-F
59-TGCCATGCACTCACAGGCATC-39 ECUN-R

59-CACCAGGTTGATTCTGCCTGAC-39 PMP1 (V1)

59-CCTCTCCGGAACCAAACCTG-39 PMP2

59-CACCAGGTTGATTCTGCC-39 C1
59-GTGACGGGCGGTGTGTAC-39 C2

59-TGAATG(G/T)GTCCCTGT-39 MSP-1 Nested PCR (first PCR)

59-GGAATTCACACCGCCCGTC(A/G)(C/T)TAT-39 MSP-3
59-GTTCATTCGCACTACT-39 MSP-2B Nested PCR (second PCR)
59-CCAAGCTTATGCTTAAGTCCAGGGAG-39 MSP-4B

59-TGAATG(G/T)GTCCCTGT-39 MSP-1 Nested PCR (first PCR)

59-GGAATTCACACCGCCCGTC(A/G)(C/T)TAT-39 MSP-3
59-TCACTCGCCGCTACT-39 MSP-2A Nested PCR (second PCR)
59-CCAAGCTTATGCTTAAGT(C/T)(A/C)AA(A/G)GGGT-39 MSP-4A

59-CCAGGUTGATUCTGCCUGACG-39 Mic3U Nested PCR (first PCR)

59-TUACCGCGCCUGCUGGCAC-39 Mic421U

59-AAGGAGCCTGAGAGATGGCT-39 Mic266 Nested PCR (second PCR)

59-CAATTGCTTCACCCTAAGGTC-39 Eb379

59-AAGGAGCCTGAGAGATGGCT-39 Mic266 Nested PCR (second PCR)

59-CACCCCTTTGCACTCGCACAC-39 Ec378

59-AAGGAGCCTGAGAGATGGCT-39 Mic266 Nested PCR (second PCR)

59-TGCCCTCCAGTAAATCACAAC-39 Eh410

59-AAGGAGCCTGAGAGATGGCT-39 Mic266 Nested PCR (second PCR)

59-CCTCCAATCAATCTCGACTC-39 Ei395

59-TGCAGTTAAAATGTCCGTAGT-39 Int530f 59-TAGCGGCTGACGAAGCTGC-39

59-TTTCACTCGCCGCTACTCAG-39 Int580r 59-CGGGCAGGAGAACGAGGACGG-39
59-CGGGCAGGAGAACGAGGACGG-39
a
ISR, intergenic spacer region.
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 267

TABLE 7—Continued
Probe
Species Target gene Amplicon size (bp) Reference(s)
designation

EB150 E. bieneusi SSU rRNA 348 145, 149, 260, 262, 381, 395

E. bieneusi SSU rRNA 1,265 91, 222, 303

E. bieneusi SSU rRNA 607 89

E. bieneusi SSU rRNA 443 343

E. bieneusi SSU rRNA, ISRa, LSU rRNA 210 357

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SI60 E. intestinalis SSU rRNA 370 81, 148, 149, 260, 262

E. intestinalis SSU rRNA 930 91, 222, 303

E. intestinalis SSU rRNA 520 90

E. intestinalis SSU rRNA 874 343

E. hellem SSU rRNA 547 359, 360

E. cuniculi SSU rRNA 549 359, 360

E. bieneusi, E. cuniculi, E. intestinalis, E. hellem SSU rRNA 250, 268, 270, 279 134

E. bieneusi, E. cuniculi, E. intestinalis, E. hellem SSU rRNA 1,200 286

E. bieneusi SSU rRNA, ISR, LSU-rRNA 508 190, 191

E. cuniculi, E. intestinalis, E. hellem SSU rRNA, ISR, LSU rRNA 289–305 190, 191

E. bieneusi, E. cuniculi, E. intestinalis, E. hellem SSU rRNA 410, 419, 421, 433 202

E. bieneusi SSU rRNA 132

E. cuniculi SSU rRNA 113 202

E. hellem SSU rRNA 134 202

E. intestinalis SSU rRNA 128 202

E. cuniculi SSU rRNA, ISR, LSU rRNA ca. 1,000 109

E. hellem
E. intestinalis
268 FRANZEN AND MÜLLER CLIN. MICROBIOL. REV.

TABLE 8. Primer pairs used for phylogenetic analysis of human microsporidia

Approximate
Primer
Primer pair Species amplified Target gene amplicon size Reference(s)
designation
(bp)

59-CACCAGGTTGATTCTGCCTGAC-39 V1 E. bieneusi, E. intestinalis, SSU rRNA 1,200 363, 381, 395,

59-GGTTACCTTGTTACGACTT-39 1492 E. cuniculi, E. hellem, 396
other species

59-CACCAGGTTGATTCTGCC-39 18f E. bieneusi, E. intestinalis, SSU rRNA 1,300 10

59-TTATGATCCTGCTAATGGTTC-39 1537r E. cuniculi, E. hellem,
other species

59-CACCAGGTTGATTCTGCCTGA-39 Micro-F E. intestinalis, E. cuniculi, SSU rRNA 1,300 359, 360

59-TTATGATCCTGCTAATGGTTCTCCAAC-39 Micro-R E. hellem, other
species

59-CACCAGGTTGATTCTGCCTGA-39 Micro-F E. bieneusi SSU rRNA 1,200 361

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59-CCAACTGAAACCTTGTTACGACTT-39 1492N4

59-CACCAGGTTGATTCTGCCTGACG-39 E. bieneusi, E. intestinalis, SSU rRNA 1,300 303

59-TTATGATCCTGCTAATGGTTCTCC-39 E. cuniculi, E. hellem,
other species

59-GTGCCAGC(C/A)GCCGCGG-39 530f E. bieneusi, E. intestinalis, SSU rRNA, 1,550, 1,350, 108, 364, 381,
59-GGTCCGTGTTTCAAGACGG-39 580r E. cuniculi, E. hellem, ISRa, 1,350, 1,350, 396
V. cornae, other LSU rRNA 1,300b
species

59-TGCAGTTAAAATGTCCGTAGT-39 int580f E. intestinalis, E. cuniculi, SSU rRNA, 1,000, 1,000, 108, 303
59-TTTCACTCGCCGCTACTCAG-39 int580r E. hellem, other ISR, 1,000b
species LSU rRNA
a
ISR, intergenic spacer region.
b
Sizes given for listed species respectively.

that were found to be PCR positive by using another primer
pair, which amplifies different microsporidian species (see be-
low) (91), and for diagnosis of intestinal infection with E.
bieneusi by using DNA from stool samples (222). Examination
of 31 stool specimens from 26 patients with intestinal E. bie-
neusi infection, 6 stool specimens from 3 patients with E. in-
testinalis infection, and 61 stool specimens from 45 patients
without intestinal microsporidiosis showed a sensitivity and
specificity of 100% (222). When this primer pair was used with
target DNA from 25 gastrointestinal biopsy specimens from
patients with confirmed E. bieneusi infection, 23 samples were
found to be positive; no amplification was seen with DNA from
three biopsy specimens from patients with confirmed E. intes-
tinalis infection (91).
The unique rRNA intergenic spacer sequence of E. bieneusi
was used to design a primer pair, Eb.gc and Eb.gt, which
amplified a 210-bp DNA fragment (357). This primer pair was
used to amplify DNA from gastrointestinal biopsy specimens,
stool samples, and duodenal aspirates of patients with E. bie-
neusi infection but not from material infected with E. intesti-
nalis, Isospora belli, Cryptosporidium spp., or G. lamblia. The
identity of the amplified PCR products was not confirmed by
Southern blot hybridization, DNA sequencing, or restriction
enzyme digestion. Again, false-positive results which may oc-
cur during unwanted annealing of primers to partially comple-
mentary sequences should be ruled out by the confirmation
techniques mentioned above.
To date, in situ hybridization has only occasionally been
used to diagnose infections with microsporidia. A 446-bp PCR-
product, amplified with the E. bieneusi-specific primer pair
V1-Mic3, was labeled with digoxigenin–11-dUTP by a random-
priming reaction and used for in situ hybridization of paraffin-
embedded jejunal biopsy specimens from patients with intes-
tinal E. bieneusi infection (63). The same technique was used
with necropsy-derived tissue samples from simian immunode-
ficiency virus-infected monkeys after experimental E. bieneusi
infection (343). Histologic examination of tissue sections failed
to identify microsporidial infection, but in situ hybridization
with the E. bieneusi probe revealed characteristic supranuclear
staining of scattered villous-tip epithelial cells within the jeju-
nal mucosa, which was also confirmed by electron microscopy
(343).
Primer pairs and hybridization probes for E. intestinalis. A
primer pair, V1 and SI500, described by Weiss et al. (381)
amplified cloned E. intestinalis SSU rRNA sequences as well as
DNA from E. intestinalis-infected tissues, body fluids, stool
samples, and cell cultures (81, 148–150, 260, 262, 381). These
primers did not amplify cloned E. bieneusi SSU rRNA se-
quences or DNA from E. bieneusi- or E. cuniculi-infected tis-
sue samples (81, 148). The specificity of this primer pair was
further tested with DNA prepared from a variety of microspo-
ridian species, S. cerevisiae, and E. coli, and no amplification
was observed (81). To confirm the identity of the PCR prod-
ucts, an internal 18-mer oligonucleotide (SI60), that was used
by Weiss et al. as upstream primer in combination with the
reverse primer SI500 (381), was used for isotopic and noniso-
topic Southern blot hybridization by others (81, 148–150). An-
other internal 30-mer oligonucleotide was used as a hybridiza-
tion probe by others (262). Although the primer pair V1-SI500
reliably amplified DNA products of the correct size from tis-
sues from patients with electron microscopy-confirmed E. in-
testinalis infection, Franzen et al. (148) detected a PCR prod-
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
uct in biopsy specimens from patients with E. bieneusi infection
and in a biopsy specimen from a patient with E. cuniculi in-
fection. The PCR products hybridized with the E. intestinalis-
specific probe (SI60), and partial sequencing of the amplified
DNA fragments showed high homology (96 to 100%) to pub-
lished E. intestinalis sequences. This confirmed that the ampli-
fied PCR products were derived from the SSU rRNA gene of
E. intestinalis and provided genetic evidence for latent E. in-
testinalis infection in these patients (148). These results were
reinforced in another study which used primer pair V1-SI500
in combination with the hybridization probe SI60 (149). A total
of 46 HIV-infected patients with diarrhea were studied, and
PCR gave positive results for E. intestinalis in 10 patients and
indicated five cases of double infection with E. intestinalis and
E. bieneusi. Histologic examination by light microscopy showed
microsporidian spores in all cases, but light microscopy was
unable to distinguish between species in almost all cases; un-
fortunately, electron microscopy results were available only for
3 of the 10 patients (149). Primer pair V1-SI500 in combina-
tion with the hybridization probe SI60 was also used to detect
E. intestinalis DNA in blood from an HIV-infected patient with
disseminated infection (150).
Primer pair SINTF1 and SINTR, described by Da Silva et al.
(90), amplified cloned E. intestinalis SSU rRNA sequences and
DNA from a duodenal-jejunal segment from a patient with
AIDS and suspected intestinal microsporidiosis and from eight
different cultured samples of E. intestinalis. Specificity was
tested with cloned microsporidian SSU rRNA coding regions
of E. cuniculi, E. hellem, E. intestinalis, E. bieneusi, and V.
corneae; positive amplification was shown only with the E.
intestinalis SSU rRNA as the template. This primer pair was
also used for species differentiation of microsporidia in stool
samples from several animals (32a). PCR products generated
by this primer pair were analyzed only by ethidium bromide-
stained gel analysis, and the identity of the PCR products was
not confirmed by Southern blot hybridization, DNA sequenc-
ing, or restriction enzyme digestion. Again, false-positive re-
sults cannot be ruled out without confirmation as mentioned
above.
An E. intestinalis-specific primer pair and hybridization
probe were designed by Schuitema et al. (303) and were fur-
ther evaluated by David et al. (91) and Liguory et al. (222). The
primer pair and probe were used for species differentiation of
samples found to be PCR positive with a primer pair that
amplified different microsporidian species in a previous PCR
(see below) (91) and for diagnosis of intestinal infection with
E. intestinalis by using DNA from stool samples (222). Exam-
ination of 6 stool specimens from 3 patients with E. intestinalis
infection, 31 stool specimens from 26 patients with intestinal E.
bieneusi infection, and 61 stool specimens from 45 patients
without intestinal microsporidiosis showed a sensitivity and
specificity of 100%, but the number of stool samples from
patients with E. intestinalis infection was very small in this study
(222). When this primer pair was used with target DNA from
three gastrointestinal biopsy specimens from patients with con-
firmed E. intestinalis infection, all samples were found to be
PCR-positive and no amplification was seen with DNA from 25
biopsy specimens from patients with confirmed E. bieneusi
infection. Again, the number of biopsy samples from patients
with E. intestinalis infection was very small in this study (91).
Another E. intestinalis-specific primer pair, V1 and Sep1, has
been used to exclude E. intestinalis infection in patients with E.
bieneusi-infected stool samples (63) and in simian immunode-
ficiency virus-infected rhesus monkeys with experimental E.
bieneusi infection (343).
269
Primer pairs for E. hellem and E. cuniculi. Two primer pairs
specific for either E. cuniculi (positions 344 to 364 and 872 to
892 of the E. cuniculi SSU rRNA sequence [ECUN-F and
ECUN-R]) or E. hellem (positions 358 to 378 and 884 to 904 of
the E. hellem SSU rRNA sequence [EHEL-F and EHEL-R])
were designed for species-specific amplification of DNA frag-
ments by Visvesvara et al. (359, 360). These two primer pairs
were used for species differentiation of cultured organisms and
organisms present in various clinical samples (95, 297, 298, 359,
360).
Two primer pairs, originally designed for genus- and species-
specific detection of Echinococcus multilocularis, surprisingly
also amplified DNA of the SSU rRNA gene of E. cuniculi (154,
255). The PCR products amplified with these two primer pairs
were sequenced, and it was shown that the DNA of E. cuniculi
carried segments that are characteristic for the genus Echino-
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coccus and the species Echinococcus multilocularis. Perhaps E.
cuniculi infected host animals of E. multilocularis or E. mul-
tilocularis larvae, so that the DNA of E. cuniculi may unknow-
ingly have been cloned and sequenced in a former analysis, or
some genome sequences may be shared between E. cuniculi
and E. multilocularis (154, 255).
General primer pairs and hybridization probes for several
microsporidian species. Primer pair PMP1 (the nucleotide se-
quence of this primer is identical to that of primer V1) and
PMP2 amplified four microsporidian pathogens that infect hu-
mans (E. bieneusi, E. hellem, E. cuniculi, and E. intestinalis)
(134). This primer pair was useful for the detection of micro-
sporidian DNA from cultured organisms and stool samples as
well as from several other clinical samples including gastroin-
testinal biopsy specimens (123, 134). These primers also am-
plified V. corneae from culture (134). To confirm the identity of
PCR amplicons, restriction enzyme digestion of amplified PCR
products with PstI and HaeIII was used. Since E. bieneusi does
not have a PstI restriction site in the amplified region but PstI
cuts the Encephalitozoon amplicons into two fragments, E.
bieneusi could be easily differentiated from Encephalitozoon
spp. However, E. intestinalis could not be differentiated from E.
cuniculi (134), thus limiting the use of this primer pair for
species differentiation. The same primer pair, PMP1-PMP2,
was used by Dowd et al. (123), and the detection sensitivity was
two spores in vitro culture-derived spores. Restriction frag-
ment length polymorphism analysis after digestion of the PCR
product with PstI was used to differentiate E. bieneusi from
Encephalitozoon spp., whereas species differentiation of En-
cephalitozoon spp. was done by DNA sequencing (123). The
same authors used this primer pair for the detection of E.
intestinalis, E. bieneusi, and V. corneae in environmental water
samples as well (123a).
A similar approach was chosen by Raynaud et al. (286), who
used the primer pair C1 (partial sequence of primer V1) and
C2 to amplify a conserved region of the SSU rRNA gene of
four microsporidia found in humans (E. bieneusi, E. hellem, E.
cuniculi, and E. intestinalis). To confirm the identity of the
amplified 1,200-bp PCR products, restriction enzyme digestion
with HindIII and HinfI was used. The amplified PCR products
displays one HindIII restriction site in E. bieneusi and none in
any Encephalitozoon spp. On the other hand, Encephalitozoon
spp. could be differentiated on the basis of the number of HinfI
restriction sites: one for E. cuniculi, two for E. hellem, and
three for E. intestinalis. The technique was used to confirm E.
intestinalis infection diagnosed by light microscopy in two non-
HIV-infected travelers (286).
Another primer pair that amplified DNA from four known
human microsporidian species was designed by Schuitema et
al. (303) and further evaluated by David et al. (91). For species
270 FRANZEN AND MÜLLER
differentiation, digoxigenin-labeled E. bieneusi- and E. intesti-
nalis-specific probes were used in a nonisotopic hybridization
assay. Two species-specific primer sets for E. bieneusi and E.
intestinalis were used in a second PCR (91, 222, 303). About 10
copies of recombinant plasmids carrying amplified SSU rRNA
genes from E. bieneusi and E. intestinalis were detected by
using the primer pairs and the hybridization assay. Of 28 in-
testinal biopsy specimens from patients with electron micros-
copy-confirmed infection due to E. bieneusi or E. intestinalis, 26
were considered PCR positive, and species differentiation was
correctly done in all cases. No false-positive results were seen
in 23 intestinal biopsy specimens from patients without intes-
tinal microsporidiosis (91).
A set of pan-Encephalitozoon primers, int530f and int580r,
selected from sequences conserved between E. cuniculi and E.
hellem, was first described by Schuitema et al. (303) and was
further used by Didier et al. (108–111). This primer pair am-
plified a product of approximately 1,000 bp that includes a
large portion of the SSU rRNA gene, the intergenic spacer,
and a small portion of the LSU rRNA gene of E. hellem, E.
cuniculi, and E. intestinalis, but did not amplify DNA from
culture-derived V. corneae or intestinal biopsy-derived E. bie-
neusi as the target (108). Species differentiation was done by
Southern blot hybridization with a nonradioactive, chemilumi-
nescent 39-oligolabeling and detection system involving spe-
cies-specific hybridization probes specific for E. cuniculi, E.
hellem, and E. intestinalis (109). The specificity of the three
hybridization probes was determined by using culture-derived
E. cuniculi, E. hellem, E. intestinalis, and V. corneae isolates
(109), but the sensitivity was not determined, and the clinical
utility is limited because the primer pair did not amplify E.
bieneusi DNA.
A nested PCR assay which detects four microsporidian spe-
cies that infect humans (E. bieneusi, E. hellem, E. cuniculi, and
E. intestinalis) and an additional four species not known to be
pathogenic to humans (V. necatrix, V. lymantriae, Ameson
[Nosema] michaelis, and Ichthyosporidium giganteum) was de-
scribed by Katzwinkel-Wladarsch et al. (190, 191). Stool sam-
ples spiked with E. cuniculi spores and stool samples, nasal
discharge, urine, sputum, and cerebrospinal fluid from patients
with light microscopy-confirmed E. bieneusi, E. hellem, E. cu-
niculi, or E. intestinalis infection were assayed. Two upstream
primers, MSP-1 and MSP-3, complementary to the 39 region of
the SSU rRNA gene of the microsporidian species mentioned
above and four downstream primers, MSP-2B and MSP-4B for
E. bieneusi and MSP-2A and MSP-4A for the other mentioned
microsporidian species, complementary to the 59 region of the
LSU rRNA gene were used (190, 191). E. bieneusi was distin-
guished from Encephalitozoon spp. because the PCR products
differed in size, and E. cuniculi could be distinguished from E.
intestinalis by restriction endonuclease digestion with MnlI,
which resulted in distinct restriction fragment patterns. The
results were confirmed by DNA sequence analysis, and the
generated sequences were homologous to previously published
DNA sequences. The limit of detection varied between 3 and
100 spores per 0.1 g of stool (190). Comparison of this PCR
assay with light microscopy on 34 specimens from 31 HIV-
infected patients produced identical results in 82% of speci-
mens (28 of 34) (191). Of the discrepant results, four samples
were microscopy negative and PCR positive and two were
microscopy positive and PCR negative. Species differentiation
was 100% accurate. These primer pairs were also used for
amplification of E. bieneusi DNA sequences from fecal samples
of pigs with suspected E. bieneusi infection confirmed by light
microscopy (191). The intergenic spacer region of the ampli-
fied PCR products was sequenced and compared with pub-
CLIN. MICROBIOL. REV.
lished sequences of E. bieneusi, and the DNA sequence of the
amplified intergenic spacer region (accession no. U61180)
showed 97% identity to the corresponding sequence of E.
bieneusi (191).
Another nested PCR assay which detects four microsporid-
ian species that infect humans (E. bieneusi, E. hellem, E. cu-
niculi, and E. intestinalis) was developed by Kock et al. (202). A
PCR with upstream primer Mic3U and downstream primer
Mic421U was used to amplify 410- to 433-bp DNA fragments
of the SSU rRNA gene of all the above mentioned microspo-
ridia. A nested PCR with upstream primer Mic266 and down-
stream primers Eb379, Ec378, Eh410, and Ei395 was used to
amplify species-specific, 113- to 134-bp DNA fragments. The
assay was tested with culture-derived spores of E. cuniculi and
E. hellem and with stool samples spiked with culture-derived
spores. Stool samples from HIV-infected patients with elec-
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tron microscopy-confirmed E. bieneusi (n 5 8), Encephalito-
zoon spp. (n 5 2), and E. intestinalis (n 5 1) infection and
intestinal biopsy specimens from three patients with electron
microscopy-confirmed E. bieneusi (n 5 2) and E. intestinalis
(n 5 1) infection were also examined (202). Without exception,
the PCR assay verified electron microscopy-confirmed infec-
tions in stool samples and biopsy specimens of all patients as
well as in spiked stool samples and tissue cultures (202).
Strain differentiation of Encephalitozoon spp. and E. bie-
neusi. Molecular techniques have confirmed the existence of
different strains of E. cuniculi. Didier et al. (108) first demon-
strated differences in the intergenic spacer region of eight E.
cuniculi isolates from mice, rabbits, and dogs. Two primer
pairs, int530f-int580r and 530f-580r, were used to amplify the
39 end of the SSU rRNA gene, the intergenic spacer, and the
59 end of the LSU rRNA gene of E. cuniculi (108). Using a
double-stranded DNA heteroduplex mobility assay and restric-
tion fragment length polymorphism after digestion with the
restriction endonuclease FokI, they found differences in the
isolates from mice, rabbits, and dogs. After DNA sequencing
of the intergenic spacer region, the isolates were shown to
differ by a small repetitive sequence of 59-GTTT-39. This se-
quence was repeated twice in two isolates from mice, three
times in three isolates from rabbits and in one isolate from a
mouse, and four times in the two isolates from domestic dogs.
These differences were used to characterize a rabbit strain
(strain I), a mouse strain (strain II), and a dog strain (strain
III). The three strains also differed antigenically by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and West-
ern blotting.
Hollister et al. (181) also studied the intergenic spacer re-
gion of three isolates of E. cuniculi: a putative mouse isolate
(strain D. Owen [accession no. X98467 and X98468]), a dog
isolate (strain Stewart [accession no. X98469]), and a human
isolate (strain Donovan [accession no. X98466 and X98470])
established in vitro from the urine of a patient with AIDS (1,
177, 179). In the intergenic spacer region, four tetranucleotide
repeats 59-GTTT-39 were found for the dog and human iso-
lates and three were found for the mouse isolate (181). An-
other human E. cuniculi isolate from a patient with AIDS (95)
also corresponded to dog strain III, with four tetranucleotide
repeats (111, 189).
Deplazes et al. (97) used primers that corresponded to po-
sitions 1 to 19 (which are the first 19 bases of primer V1) and
1277 to 1299 of the SSU rRNA gene sequence of E. cuniculi to
amplify SSU rRNA gene fragments from organisms cultured
from six HIV-infected patients infected with E. cuniculi, E.
hellem, and E. intestinalis and from nine rabbits infected with
E. cuniculi. PCR products were cleaved with restriction en-
zymes (MboI and HpaII) and analyzed on ethidium bromide-
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
TABLE 9. Number of 59-GTTT-39 repeats in the intergenic spacer
region of different E. cuniculi isolates from different hosts and
geographical areas
No. of No. of Geographic
Source Reference(s)
repeats isolates area
2 Blue fox 4 Norway 236
Mouse 2 Czech Republic, UK 108
3 Rabbit 11 Swizerland 236
Rabbit 3 USA 108
Human 6 Swizerland 97, 98
4 Human 3 Mexico, USA 97, 98, 111,
189
Dog 2 USA 108
stained gels. Restriction fragment length polymorphisms of all
E. cuniculi isolates from humans did not differ from the rabbit
isolates (97). Further analysis of the intergenic spacer region of
these six E. cuniculi isolates from humans and of different E.
cuniculi isolates of animal origin was done by Mathis et al.
(236). Five E. cuniculi isolates from Swiss patients were found
to be homologous to isolates from rabbits, whereas the sixth
isolate, from a patient from Mexico, corresponded to dog
strain III (236).
In the light of these data (summarized in Table 9), dogs or
rabbits might be the source of human infection. However, it is
difficult to understand how host-related differences can be
maintained in microsporidial isolates when strains can be
transmitted among different types of hosts, at least experimen-
tally. Too few isolates of E. cuniculi have been obtained and
analyzed so far to assess host specificity or to determine if
humans are at risk of infection by exposure to dogs or rabbits.
More data on strains from many different hosts including hu-
mans are needed, before the epidemiology of E. cuniculi is
clarified.
E. hellem has been found once in a bird, and E. intestinalis
has recently been detected in several mammals. It remains
unclear if these animals are relevant reservoirs of the parasites.
Double-stranded DNA heteroduplex mobility assays, restric-
tion fragment length polymorphisms after digestion with the
restriction endonucleases, and DNA sequencing of the inter-
genic spacer between the SSU rRNA and the LSU rRNA of
different E. hellem and E. intestinalis isolates from different
geographical regions (Europe and America) showed no differ-
ences among these isolates (109, 110, 364). However, Schnitt-
ger et al. recently reported genotypic differences of the SSU
and LSU rRNA coding regions in E. intestinalis (GenBank
accession no. Y11611). Again, too few isolates of E. hellem and
E. intestinalis have been analyzed so far to determine if there
are different strains of these species. Additional isolates must
be analyzed to help define the epidemiology of these micro-
sporidian species.
Three distinct genotypes of E. bieneusi have been identified
by Rinder et al. (289) by amplification of a 484-bp DNA frag-
ment between primers MSP-3 and MSP-4B from 12 stool sam-
ples of eight HIV-infected patients (289). Nine polymorphic
sites were found in the 243-bp intergenic spacer region of the
rRNA gene. The genotypes were stable in samples taken dur-
ing 11 weeks of infection. The intergenic spacer rRNA E.
bieneusi sequence reported by Zhu et al. (accession no.
L20290) (396) was not considered to be a fourth genotype by
the authors. This sequence is only 86.8% identical to those of
the other three genotypes; this may be caused not only by
271
polymorphisms but also by several sequencing errors. Liguory
et al. (222a) identified four genetically unrelated lineages
among 78 different isolates of E. bieneusi from 65 patients with
intestinal microsporidiosis. Strain differentiation was done by
restriction fragment length polymorphism with NlaIII and
Fnu4HI after amplification of the intergenic spacer region with
the primer pair Eb.gc and Eb.gt. Type I strains were found in
the majority of samples (78%), whereas type II, III, and IV
strains were found only in 12, 5, and 5% of the samples,
respectively. The same strains were found in sequential stool
specimens from the same patients. DNA sequences were de-
termined only for strain I, which showed 100% homology to
the E. bieneusi sequence of Zhu et al. (accession no. L20290)
(396), and for strain II, which showed seven mismatches with
this sequence (97% identity) (222a). rRNA sequences of E.
bieneusi isolates from humans, a pig (accession no. U61180),
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and rhesus macaques (accession no. AF023245) exhibit addi-
tional variability over the intergenic spacer region and the 59
end of the LSU rRNA gene (62a). The rRNA sequence of the
pig belongs to the type IV lineage, which has been found in
only 5% of humans (222a).
When rRNA genes are used to discriminate among strains,
several problems must be considered. The presence of differ-
ent genotypes for individual isolates does not automatically
warrant their consideration as different strains, and different
copies of a gene within one genome may not be identical (289).
It is not clear whether different prevalences of strains in hu-
mans reflect different sources of strains that lead to human
infection or are related to the pathogenicity of strains in par-
ticular types of hosts. However, comparisons of different
strains from humans, animals, and environmental sources will
provide a better understanding of the epidemiology of micro-
sporidial infection.
Comparison of molecular techniques with light microscopy.
In-house validations of PCR protocols for the detection of
microsporidia, especially by those who developed them, are
generally satisfactory to excellent. However, until recently
none of these techniques had been validated in a blinded,
externally controlled fashion. In a multicenter evaluation of
the detection of microsporidia in stool specimens by light mi-
croscopy and PCR, quality parameters were determined with
identical sets of 50 stool samples for six laboratories using light
microscopy and six laboratories using their own PCR protocol
(290). A total of 14 clinical samples contained E. bieneusi
spores, and 14 samples were spiked with spores of E. hellem, E.
cuniculi, and E. intestinalis at different concentrations ranging
from 102 to 106 spores/g; 22 samples were negative. The aver-
age overall sensitivity was 67% (36 to 96%) for laboratories
using PCR and 54% (25 to 71%) for laboratories using light
microscopy, and the specifities were 98 and 95%, respectively.
A uniform detection limit of between 104 and 106 spores/g of
stool was apparent for light microscopy, while PCR detected
concentrations as low as 102 spores/g. However, the greatest
differences were found between individual laboratories rather
than between the two techniques used (290). These apparent
differences in quality between laboratories suggest that indi-
vidual laboratories first should improve their established tech-
nique as much as possible and not give up one method in favor
of the other. Nevertheless, it will certainly be helpful to have a
second technique available when confirmation of the first is
desired. Since the threshold of detection for light microscopy
seems to be between 104 and 106 spores/g of stool, epidemio-
logical studies involving only light microscopy may give prev-
alence data which did not reflect the true prevalence of mi-
crosporidia. Application of PCR to stool samples offers an
approach that can be adapted for processing large numbers of
272 FRANZEN AND MÜLLER
specimens to define the true prevalence of intestinal micros-
poridiosis in human populations and to determine whether
microsporidia are present in the general population (272).
Because all the participating laboratories in this trial re-
mained anonymous, no recommendations about DNA isola-
tion techniques, primer pairs, or PCR methods can be given.
Nevertheless, testing all clinical samples with separate species-
specific primer pairs is a time-consuming task. An efficient
approach for PCR detection of microsporidia in clinical sam-
ples would involve using general microsporidian primer pairs
that amplify several species. The species of microsporidia de-
tected could then be determined by PCR with species-specific
primer pairs. Primers V1 and PMP2 amplify microsporidian
DNA of different microsporidian species. Primer V1 is used by
several researchers, and primer PMP2 has also been evaluated
by different groups (123, 134, 149). Other published general
microsporidian primer pairs (190, 202, 286, 303) have not been
evaluated by others so far. The primer pairs V1-EB450 and
V1-SI500, although constructed by using SSU rRNA se-
quences which appear to contain several sequencing errors, are
best evaluated for diagnosis of microsporidiosis due to E. bie-
neusi and E. intestinalis. Several researchers have published
studies with these primer pairs, usually with excellent results
(81, 145, 148–150, 260, 262, 339a, 381, 395).
Molecular Techniques for Phylogenetic Analysis
Molecular techniques are rapidly becoming an important
and integral part of biosystematic studies. Traditional classifi-
cations of microsporidia have been done on the basis of mor-
phological characteristics, but the four major published classi-
fications differ significantly, and the taxonomy of microsporidia
is still controversial (183, 214, 335–337, 376). Microsporidia are
prime candidates for phylogenetic analysis based on DNA se-
quence data because of the relatively small number of useful
morphological characters. Advances in DNA sequencing tech-
nology have resulted in a great increase in the number of useful
characters available for phylogenetic analysis. Moreover, DNA
amplification by PCR has made it possible to obtain sequence
data from organisms which cannot be cultured and for which
material cannot be obtained in sufficient quantities for cloning
and sequencing.
16S rRNA genes are found in all prokaryotic organisms and
accumulate mutations at a low, constant rate over time. Hence,
they may be used as “molecular clocks” (258, 387). Highly
variable portions of the SSU-rRNA genes provide unique
signatures for any organism and useful information about
possible relationships (387). Several groups have been using
molecular approaches to develop taxonomic systems for mi-
crosporidia based on rRNA genes and also on protein-coding
DNA sequences. This analysis has yielded insights into new
relationships among microsporidia, which are being continu-
ously integrated into a revised taxonomy. For example, it has
been shown that Nosema trichoplusiae is a synonym for Nosema
bombycis based on the sequence of the SSU rRNA coding
region (276). Therefore, new molecular techniques have led
not only to the discovery of new organisms but also to the
elimination of an old organism.
The first microsporidian DNA sequence data were reported
by Vossbrinck et al. (363). The SSU rRNA gene of the insect
microsporidian V. necatrix was sequenced, and a phylogenetic
tree was then constructed. The root of the eukaryotic tree lay
between the V. necatrix rRNA sequence and those of all other
eukaryotic lineages for which sequences were available, show-
ing that the V. necatrix sequence is consistent with a very early
branch in the eukaryotic line of descent (363).
CLIN. MICROBIOL. REV.
rRNA sequences for phylogenetic construction of microspo-
ridia pathogenic to humans were also first used by Vossbrinck
et al. (364). They used a highly conserved primer pair, 530f in
the SSU rRNA and 580r in the LSU rRNA, to amplify a DNA
fragment of about 1,350 bp that contains the 39 end of the SSU
rRNA, the intergenic spacer, and the 59 end of the LSU rRNA
of E. hellem, E. cuniculi, and V. corneae obtained from tissue
culture (364). Restriction digests of the amplified DNA frag-
ments with SauIIIA, EcoRI, DraI, and HinfI showed clear
differences among the three species. Three regions of the am-
plified DNA fragment were sequenced with primer 228r (in the
LSU rRNA) in addition to primers 530f and 580r. The se-
quence difference between E. hellem and E. cuniculi was
23.1%, while the sequence difference between the two Enceph-
alitozoon spp. and other studied families (Vairimorpha and
Ichtyosporidium) ranged between 54.2 and 56.2% (364). These
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data indicate that E. hellem and E. cuniculi are different or-
ganisms which are closely related.
The same primers (530f, 580r, and 228r) were used by Zhu
et al. (396) for phylogenetic analysis of E. bieneusi, E. intesti-
nalis, and A. michaelis. E. bieneusi and E. intestinalis DNA was
extracted from intestinal biopsy specimens from patients with
electron microscopy-confirmed microsporidial infection;
spores of A. michaelis were obtained from the muscle of blue
crabs (Callinectus sapidus). Following amplification and se-
quencing of PCR product, sequences were aligned with those
of several other microsporidian species including E. hellem and
E. cuniculi. By using three sequenced regions, Zhu et al. cal-
culated that there was a 16.7% sequence difference between E.
hellem and E. cuniculi and that there were 21.9 and 26.4%
sequence differences between E. intestinalis and E. cuniculi and
between E. intestinalis and E. hellem, respectively (396). These
differences calculated by Zhu et al. (396) differed from those
reported by Vossbrinck et al. (364), perhaps due to differences
in alignment or to the version of PAUP (phylogenetic analysis
using parsimony) used. Based on their data, Zhu et al. (396)
stated that E. cuniculi, E. hellem, and E. intestinalis are closely
related, most likely at the family level, with E. cuniculi and E.
hellem belonging to the same genus and E. intestinalis belong-
ing to the Encephalitozoonidae family but as a separate genus.
Much larger differences were found between the other micro-
sporidia. Since E. bieneusi differed from E. intestinalis by
45.7%, from E. cuniculi by 43.2%, and from E. hellem by
42.2%, it is clear that E. bieneusi is placed in a separate family
(396).
Baker et al. presented a phylogeny of various species of
microsporidia based on sequence data of the whole SSU rRNA
gene (10). DNA sequences either were obtained by PCR with
primer pair 18f (partial sequence of primer V1) and 1537r,
complementary to conserved regions of the SSU rRNA gene at
the 39 and 59 ends, followed by sequencing with several internal
primers or were obtained from the GenBank database. Results
of parsimony analysis with PAUP suggested that the 16 studied
microsporidian species could be divided into four distinct
groups referred to as the Ichtyosporidium group, the Enceph-
alitozoon group, the Endoreticulatus group, and the Vairimor-
pha/Nosema group (Fig. 13). Delineation of Vairimorpha/
Nosema as a group was additionally supported by LSU rRNA
data obtained from several other species of Nosema and Vairi-
morpha (9). From the branching pattern in Fig. 13 and the
sequence difference between V. corneae (formerly N. corneum)
and N. bombycis (27.9%) it seems clear, that V. corneae is
unrelated to the other Nosema spp. Baker et al. (9, 10) placed
V. corneae in the Endoreticularis group because the parasite is
most closely related to Endoreticulatus schubergi (7.3% dis-
tance). This supports the reclassification of N. corneum to a
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA 273

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FIG. 13. Tree representing the phylogenetic relationship of several microsporidian species as determined by SSU rRNA sequence analysis. This was the most
pasimonious tree found by using the branch-and-bound option of PAUP. Reprinted from reference 101 with permission of the publisher.

new genus as V. corneae which was done based on the ultra-
structure of developmental stages in the livers of experimen-
tally infected atymic mice (the diplokaryotic arrangement of
the nuclei was the only character that conformed to the de-
scription of the genus Nosema) (324). Baker et al. (10) placed
in the Encephalitozoon group the genera which Cali et al.
placed in the family Encephalitozoonidae (47). By using parsi-
mony (branch-and-bound option of PAUP), E. intestinalis was
most closely related to E. cuniculi (Fig. 13), and by using
distance methods (unweighted pair group method with arith-
metic mean [UPGMA]), E. hellem was most closely related to
E. intestinalis (mean distance values, E. intestinalis/E. hellem 5
0.043, E. intestinalis/E. cuniculi 5 0.059, E. hellem/E. cuniculi 5
0.066), but in no case was E. hellem most closely related to E.
cuniculi (10). Because of the close relationship between the
three Encephalitozoon spp., Baker et al. proposed that E. in-
testinalis, originally described as Septata intestinalis by Cali et
al. (47), be designated Encephalitozoon intestinalis and that the
morphological definition of the genus be changed to reflect this
designation (10).
Similar conclusions were drawn by Hartskeerl et al. on the
basis of SSU rRNA sequence data (166). The SSU rRNA gene
of E. intestinalis, E. cuniculi, E. hellem, and E. bieneusi was
amplified with primers complementary to conserved regions at
the 39 and 59 end of the SSU rRNA gene (164, 166). Restric-
tion enzyme digestion of the amplified DNA with HphI and
BsiHKAI produced different restriction fragment length poly-
morphism patterns for all the species examined. The degree of
sequence homology was established by a pairwise comparison
with the Bestfit program. SSU rRNA gene sequences of E.
cuniculi and E. hellem showed a lower degree of homology
(89.7%) than did those of E. cuniculi and E. intestinalis (90.7%)
or E. hellem and E. intestinalis (91.4%). The E. intestinalis
sequence was only approximately 70% identical to those of
microsporidia of other genera such as E. bieneusi or V. necatrix.
Although sequence dissimilarity values are not suitable meth-
ods for phylogenetic analysis, the authors concluded that E.
cuniculi, E. hellem, and E. intestinalis belong to the same genus
and proposed the reclassification of Septata intestinalis to En-
cephalitozoon intestinalis (166).
The data reported by Hartskeerl et al. (166) and Baker et al.
(10) differed significantly from those of Zhu et al. (396). Harts-
keerl et al. and Baker et al. placed E. intestinalis within the
genus Encephalitozoon, while in the phylogeny of Zhu et al., E.
intestinalis was placed in a genus of its own (as originally
proposed by Cali et al. [47]). There are several possible expla-
274 FRANZEN AND MÜLLER
nations for this. First, Zhu et al. used sequence data of the 39
and 59 ends of the SSU and LSU rRNA genes and the inter-
genic spacer for their phylogenetic analysis, whereas Harts-
keerl et al. and Baker et al. used the sequence of the whole
SSU rRNA gene. However, Zhu et al. stated that their results
were unchanged when the whole SSU rRNA gene sequence
was used. Second, the alignment methods differed. Phyloge-
netic analysis depends on comparison of homologous charac-
ters, and only regions in which exact alignment is possible
should be used. Third, and this may be the most important
point, the E. cuniculi, E. hellem, and E. intestinalis sequences
obtained by Zhu et al. (393–396) differed significantly even in
conserved regions of the rDNA from those reported by Baker
et al. (10) and by Hartskeerl et al. (164, 166).
Cali et al. (49) did not agree with the reclassification of
Septata intestinalis into Encephalitozoon intestinalis. The sporo-
gony of this species is tetrasporous (47). On the other hand,
there are no supporting published data for tetrasporous devel-
opment of E. cuniculi, the type organism from which the genus
was established. Until E. cuniculi is demonstrated to be tetra-
sporous, the genus Encephalitozoon cannot be modified to in-
clude this feature, thus excluding Encephalitozoon (Septata)
intestinalis. They also noted that the morphological presence of
secretions from E. intestinalis and the lack of such secretions in
E. cuniculi was not addressed in the reclassification of E. in-
testinalis by Hartskeerl et al. (166) and Baker et al. (10). In
other microsporidia, this feature has been given taxonomic
value. For example, some genera in the family Pleistophoridae
were created on the basis of variations in secretion (49). Based
on these data, Cali et al. excluded E. intestinalis from the genus
Encephalitozoon until E. cuniculi is shown to be tetrasporous
(49).
Whereas molecular phylogenies suggest that the family En-
cephalitozoonidae should contain one genus, morphologic dif-
ferences between E. hellem, E. cuniculi, and E. intestinalis sug-
gest that three genera should be used. In cell culture, E. hellem
displays variable development (49, 102); some parasites occur
within a parasitophorous vacuole, while others develop in di-
rect contact with the host cell cytoplasm. This behavior has
never been observed for E. cuniculi. Such differences have
been used historically for genus-level classification in micro-
sporidia. Consequently, Cali et al. (49) proposed the removal
of E. hellem from the genus Encephalitozoon and its reclassi-
fication in a genus of its own, creating three genera in the
family Encephalitozoonidae. This would satisfy both morpho-
logical and molecular criteria (49).
Docker et al. (120) compared sequences of the SSU rRNA
gene, the intergenic spacer region, and the LSU rRNA gene of
N. salmonis with that of E. bieneusi amplified with primer pair
530f and 580r; the two sequences differed in composition by
19.8%. This genetic divergence between the two species was
sufficiently large to place the two species into two different
genera in the family Enterocytozoonidae (120, 197), as origi-
nally proposed by Hedrick et al. (169). This placement is sup-
ported by morphological data. Although N. salmonis exhibits
most of the distinguishing morphological characteristics of the
family Enterocytozoonidae, the distinctively different hosts and
sites of development support the placement of Nucleospora
and Enterocytozoon into separate genera within the family En-
terocytozoonidae (101, 120, 197).
Another SSU rRNA-based phylogeny of microsporidia has
been published by Malone and McIvor (233). Although they
included several other microsporidian species in their analysis
than were analyzed by Baker et al. (9, 10) the calculated phy-
logenetic tree, using the neighbor-joining method, showed a
CLIN. MICROBIOL. REV.
branching pattern comparable to the phylogeny of Baker et al.
(9, 10) for the species used in both analysis.
The phylogenetic relationships of microsporidia were also
analysed by riboprinting (280). The SSU rRNA and the inter-
genic spacer region of different microsporidia from fish, in-
sects, and shrimp were amplified and digested with restriction
enzymes. The generated riboprints were analyzed, and phylo-
genetic trees were constructed by using the MIX and DOL-
LOP programs of the PHYLIP package, in which each frag-
ment is considered a different character. While sequencing
gives more detailed information on the phylogenetic relation-
ship of different species and genera, riboprinting is a faster and
cheaper way of determining the identity and phylogeny of
microsporidia. This approach will work at the genus level, but
DNA sequencing should be used to define the relationships of
different species.
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a- and b-tubulin sequences obtained by amplification with
primer pairs directed against conserved sequences from differ-
ent microsporidian species (E. hellem, E. intestinalis, E. cunic-
uli, N. locustae, and Spraguea lophii) have been used by several
authors to construct phylogenetic trees (125, 126, 188, 221).
The b-tubulin sequences from E. hellem, E. intestinalis, and E.
cuniculi were closely related, but that of E. intestinalis was
more closely related to that of E. cuniculi than was that of E.
hellem (221), reinforcing the reclassification of E. intestinalis.
The generated a- and b-tubulin trees were nearly identical in
topology, but in several important aspects these trees are in-
consistent with phylogenetic trees constructed on the basis of
SSU rRNA sequence data. The few SSU rRNA-based molec-
ular trees for which microsporidian data are available suggest
that microsporidia are one of the most ancient eucaryotic lin-
eages (10, 11, 155, 217, 363), but the a- and b-tubulin trees
suggest that the microsporidia are close relatives of fungi,
which invites speculation that microsporidia evolved degenera-
tively from higher forms (126, 192, 221).
Phylogenetic analysis of the complete nucleotide sequence
of the genes putatively encoding translation elongation factors
1a and 2 from the fish microsporidium Glugea plecoglossi sug-
gests that G. plecoglossi represents the earliest branch of the
eukaryotes among the analyzed eukaryotic species (186, 187).
This reinforces the hypothesis that microsporidia are extremely
ancient eukaryotes that diverged before the occurrence of mi-
tochondrial symbiosis rather than evolving by degeneration
from higher forms.
One of the original reasons behind the proposal that micro-
sporidia are ancient eukaryotes is their lack of mitochondria
(64). The presence of mitochondrion-derived genes reveals
that the ancestor of a lineage contained a mitochondrion, even
though an organelle has not been identified in the lineage
(193). Recently, genes encoding a chaperone protein, Hsp70
(70-kDa heat shock protein), have been identified in the mi-
crosporidia N. locustae, V. necatrix, E. hellem, and E. cuniculi
(159, 173, 275a). Phylogenetic analyses have shown close rela-
tionships to Hsp70 proteins from mitochondria of other eu-
karyotes. In addition to the detection of genes encoding Hsp70
proteins in microsporidia, tRNA synthetase genes consistent
with the presence of mitochondria have been found in micro-
sporidia (381a). The simplest interpretation of these data is
that microsporidia have lost mitochondria while retaining ge-
netic evidence of their past presence. Interestingly, these mi-
crosporidian Hsp70 sequences appear as a sister group of fungi
in Hsp70 phylogenies, supporting the list of reasons to believe
that microsporidia are closely related to fungi, as a highly
specialized group that adapted extensively to an exclusively
intracellular parasitic mode of life by degenerative evolution
(193, 251a).
VOL. 12, 1999 MOLECULAR TECHNIQUES FOR ANALYSIS OF MICROSPORIDIA
The concept that microsporidia are ancient eukaryotes was
also supported by their apparent lack of spliceosomal introns.
The fact that the microsporidia N. locustae and V. necatrix
possess core spliceosomal components (U2 and U6 small nu-
clear RNAs) (115, 133a) is consistent with the recent diver-
gence of the microsporidia. It has been suggested that micro-
sporidia lost most of their spliceosomal introns during the
radical genome size reduction accompanying the adaptation to
an intracellular parasitic lifestyle (133a).
Phylogenetic analysis of 19 eukaryotic and archeal LSU
rRNA sequences, including the LSU rRNA sequence of E.
cuniculi, produced different trees depending on the maximum-
likelihood method used. FastDNAml produced a tree in which
three fast-evolving lineages including microsporidia are pre-
dicted to have diverged early from other eukaryotic lineages,
whereas the BASEML method placed the Encephalitozoon
sequence in a relatively late-emerging position with a very high
long terminal branch, indicating a high rate of evolution
(275b).
Why do different microsporidian genes suggest contradic-
tory phylogenies? The fact that a phylogeny can be obtained
for a group of organisms does not guarantee that it really
reflects the evolutionary history (381a). Much of our evolu-
tionary thinking is currently based on the phylogeny of SSU-
rRNA, which may be biased by the long-branch attraction
artefact. Like any other single-gene phylogeny, it may be mis-
leading in some aspects but correct in others. The phylogenies
presented above clearly point to the dangers of placing too
much confidence in the phylogny of a single gene, and more
DNA sequence data of protein-encoding sequences from mi-
crosporidia will be needed to answer this question.
Molecular Techniques for Susceptibility Testing
Several antibacterial protein synthesis inhibitors have anti-
parasitic activity, in particular the aminoglycoside paromomy-
cin. The paromomycin binding site includes several nucleotides
at the 39 end of the SSU rRNA, and mutational analysis has
specifically implicated the C1409 z G1491 base pair (E. coli num-
bering) at the base of the 47-479 hairpin (189). SSU rRNA
from all analyzed microsporidia lack this base pair, predicting
that they would be paromomycin resistant (189).
Several drugs, notably colchicine, vinca alkaloides, and ben-
zimidazoles, are effectors of microtubule polymerization.
Among these agents, the benzimidazoles are unique in being
selectively toxic to certain lower eukaryotes. The b-tubulin
subunit has been identified as the primary target of benzimid-
azole. Benzimidazoles such as albendazole act by disrupting
microtubules through b-tubulin binding, and six different
amino acid residues have been implicated in benzimidazole
susceptibility by mutational analysis of fungi (125, 188). Muta-
tions in His-6, Phe-167, Glu-198, Phe-200, and Arg-241 confer
resistance to the derivate benomyl, while a mutation in Ala-165
confers thiabendazole resistance (221). Both E. hellem and E.
cuniculi b-tubulins include His-6, Phe-167, Glu-198, Phe-200,
and Arg-241, predicting that these microsporidia would be
susceptible to benzimidazoles (125, 188). In addition, E. hellem
and E. intestinalis b-tubulins include Val-268, and mutational
and comparative sequence analyses with the yeast S. cervisiae
suggest that Val-268 also plays a role in benzimidazole suscep-
tibility (126).
FUTURE TRENDS
The genomes of microsporidia, especially E. cuniculi, are
very small. A genomic library of the smallest chromosome
275
(chromosome I) of E. cuniculi is already available, and system-
atic sequencing of this chromosome will be completed in the
near future (25, 124a). Complete mapping and gene sequenc-
ing of the whole genome of E. cuniculi are in progress (124a),
and this will be useful in advancing our knowledge of physio-
logically important genes and control regions of coding and
noncoding sequences in microsporidia.
rRNA data have suggested that microsporidia are one of the
most ancient eukaryotic lineages. On the other hand, the re-
latedness of microsporidia to fungi and animals, as suggested
by the a- and b-tubulin and mitochondrion-type Hsp70 se-
quences, invites speculation that microsporidia evolved degen-
eratively from higher forms. Examination of protein-coding
sequences from microsporidia will be of interest to answer this
question and is under way.
The taxonomy of microsporidia has undergone several
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changes during the last few years and will continue to change
significantly in the near future when new DNA-based data are
incorporated into new classification systems. As a first step,
molecular data suggest that the family Encephalitozoonidae
should contain only one instead of two genera, but morpho-
logical data and rules of taxonomy demand three genera. How-
ever, there is no longer justification for two genera within the
family Encephalitozoonidae.
Different strains of E. cuniculi and E. bieneusi have already
been identified, and more isolates from different hosts and
environmental samples are currently being examined to sub-
stantiate the belief that human microsporidiosis is a zoonotic
or waterborne disease. With respect to other species, no dif-
ferent strains have been identified so far, but examination of
different isolates from different geographic areas and hosts by
molecular techniques is under way.
Animal hosts have been identified for E. cuniculi and, most
recently, for E. hellem, E. intestinalis, and E. bieneusi. Further
examination of different hosts and environmental samples by
the highly sensitive molecular techniques is under way. These
studies may lead to the identification of further animal reser-
voirs for microsporidia which infect humans. The highly sen-
sitive molecular techniques may be also helpful for identifying
asymptomatic carriers of microsporidia.
At present only limited data have been published comparing
molecular techniques for diagnosis of microsporidia with tra-
ditional methods of determining the sensitivity and specificity
of these new techniques. Nevertheless, these studies indicate
that the sensitivity and specificity of molecular techniques can
be very high. One international, blinded multicenter study
comparing traditional light-microscopic techniques with PCR
has been published recently (290), but further studies are
needed.
CONCLUDING REMARKS
Microsporidia are an important cause of disease in HIV-
infected patients and are now increasingly also recognized as
pathogens in non-HIV-infected patients with or without im-
munosuppression. Therefore, methods for the diagnosis and
species differentiation of microsporidia should be available in
every laboratory performing parasitological examination of
clinical specimens.
Although molecular techniques, especially PCR, seem to be
very sensitive and specific and are useful for the diagnosis and
species differentiation of microsporidia, large comparative
blinded studies determining sensitivity and specificity are lack-
ing. Therefore, screening of clinical samples for microsporidia
by these techniques is not recommended at the moment. For
the detection of microsporidia in clinical samples, chemofluo-
276 FRANZEN AND MÜLLER
rescent stains or chromotrope-based stains should be used as
the methods of first choice. Nevertheless, accurate taxonomic
classification of microsporidia is essential because the different
genera and species exhibit different biological and epidemio-
logical characteristics, which influence approaches to preven-
tion and treatment. Application of PCR methods offers an
approach that can be adapted for processing large numbers of
specimens to define the epidemiological extent of microspo-
ridian infection in human populations (272). Therefore, iden-
tification of microsporidia by light microscopy should be fol-
lowed by confirmation and specifies differentiation with
molecular techniques.
As is true for many other new and emerging pathogens, we
have just scratched the surface of a complex and evolving
relation between the phylum Microspora and humans (382).
Application of molecular techniques to diagnosis, species dif-
ferentiation, and phylogentic analysis of microsporidia will
lead to an enormously increased knowledge of these organisms
in the near future.
ACKNOWLEDGMENTS
We acknowledge and appreciate the assistance of Britta Franzen
during preparation of the manuscript.
Portions of this work were supported by the Köln Fortune program/
Faculty of Medicine, University of Cologne, Cologne, Germany.
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