Microsporidiosis: human diseases and diagnosis

Microbes and Infection, 3, 2001, 389−400 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457901013958/REV

Microsporidiosis: human diseases and diagnosis Caspar Franzena*, Andreas Müllerb a

Department of Internal Medicine I, University of Cologne, Joseph Stelzmann Str. 9, 50924 Cologne, Germany b Department of Pediatrics, University of Cologne, Joseph Stelzmann Str. 9, 50924 Cologne, Germany

ABSTRACT – Microsporidia are considered opportunistic pathogens in humans because they are most likely to cause diseases if the immune status of a host is such that the infection cannot be controlled. A wide spectrum of diseases has been reported among persons infected with microsporidia and different diagnostic techniques have been developed during the last decade. © 2001 Éditions scientifiques et médicales Elsevier SAS microsporidiosis / microsporidia infections / complications / diagnosis

1. Introduction Microsporidia are obligate intracellular protozoan parasites infecting a broad range of vertebrates and invertebrates. In 1857 these parasites were first recognized as pathogens in silkworms, and long before they were described as human pathogens they were recognized as a cause of disease in many nonhuman hosts. The first human case of microsporidial infection was reported in 1959 [1] and only ten well-documented human infections with microsporidia were described until 1985, when a new species Enterocytozoon bieneusi was found in an HIVinfected patient [2]. Since then many infections with different species of microsporidia have been reported from all over the world and microsporidia are now frequently recognized as etiologic agents of opportunistic infections in persons with AIDS [3], and more recently, in non-HIVinfected patients [4, 5] and organ transplant recipients being treated with immunosuppressive drugs [6, 7]. The phylum Microsporidia consists of nearly 150 genera with more than 1 000 species [8], but only seven genera (Enterocytozoon, Encephalitozoon (including Septata), Pleistophora, Trachipleistophora, Vittaforma, Brachiola and Nosema) as well as unclassified microsporidia have been described as pathogens in humans.

2. Microsporidia detected in humans 2.1. Enterocytozoon bieneusi

E. bieneusi was first detected in 1985 following examination of an AIDS patient with chronic diarrhea [2]. During the last decade the number of reported cases has *Correspondence and reprints. E-mail address: [email protected] (C. Franzen). Microbes and Infection 2001, 389-400

steadily increased in nearly all parts of the world [3]. The organism develops in direct contact with the host cell cytoplasm. Meronts often have electron-lucent inclusions which are present throughout the life cycle. Sporonts form electron-dense precursors of the polar tube and the anchoring disk which develop before sporogonial plasmodia divide into sporoblasts (figure 1A, B). Multiple sporoblasts are formed by invagination of the plasma membrane of one large sporogonial plasmodium. Spores are oval and small, measuring only 1.1–1.6 × 0.7–1.0 µm, with five to seven coils of the polar tubule, arranged in two rows [9]. The parasite usually infects intestinal enterocytes but has also been detected in lamina propria cells of small bowel biopsies, biliary tree, gallbladder, liver cells, pancreatic duct, tracheal, bronchial and nasal epithelia. Sporadic case reports described E. bieneusi in non-HIV-infected immunodeficient and immunocompetent humans as well, and animal reservoirs (pigs and monkeys) have been identified recently [10, 11]. 2.2. Encephalitozoon spp.

E. cuniculi was the first microsporidium to be recognized as a parasite of mammals. First found in rabbits in 1922, this microsporidium was named by Levaditi et al. in 1923. Subsequently, this organism has been detected in many mammalian hosts, including man. All Encephalitozoon spp. develop within parasitophorous vacuoles. Meronts divide by binary fission and usually remain at the vacular membrane. Sporonts develop a thick surface coat which becomes the exospore of spores and sporonts divide into sporoblasts which will develop into spores (figure 1C–F). Spores measure 2.0–2.5 × 1.0–1.5 µm and the polar tubule has five to seven coils in a single row (figure 1C) [9]. Two pathogenic species of Encephalitozoon which infect humans, E. cuniculi and E. hellem, are morphologically similar by light and electron microscopy, and can 389

Current focus: Microsporidia, intracellular parasites causing emerging diseases

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Figure 1. Electron micrographs of different microsporidian species. (A) E. bieneusi. Two sporogonial plasmodia with several round nuclei and multiple electron dense precursors of the polar filament. (B) E. bieneusi. Cells containing spores that have sloughed from epithelial surface of duodenal mucosa. (C) E. cuniculi. A parasitophorous vacuole with two merogonial plasmodia, four sporogonial plasmodia, and four spores. (D) E. cuniculi. Spore within a parasitophorous vacuole. (E) E. hellem. Spore containing six cross sections of the polar filament in nasal discharge of an HIV-infected patient with disseminated infection. (F) E. intestinalis. Vacuole with one merogonial plasmodium, four sporogonial plasmodia, and four spores. The vacuole is separated by amorphous material which leads to septation of the parasitophorous vacuole.

only be distinguished by antigenic, biochemical, or nucleic acid analysis [12]. Several cases of Encephalitozoon infection were reported to occur in patients with and without AIDS prior to 1991. It was assumed that these infections were due to E. cuniculi following light and/or electron microscopic analysis. However, in 1991 a new species of Encephalitozoon, E. hellem was described by using biochemical and antigenic methods [12]. Since all subsequently published cases of Encephalitozoon infections in humans appeared to be caused by E. hellem, there was 390

some doubt as to whether E. cuniculi did in fact cause human infection. However, since 1995 several patients with disseminated E. cuniculi infection have been reported and in these cases species differentiation was confirmed by antigenic, biochemical, or nucleic acid based methods [13–15]. A third Encephalitozoon species, E. intestinalis, displays ultrastructural similarities with the genus Encephalitozoon. It was later designated as a new genus and species, Septata intestinalis on the basis of ultrastructural Microbes and Infection 2001, 389-400

Microsporidiosis: human diseases and diagnosis

Current focus: Microsporidia, intracellular parasites causing emerging diseases

differences [16]. Based on rRNA sequence data, it is now suggested to place it in the genus Encephalitozoon and rename it Encephalitozoon intestinalis [17]. E. intestinalis shows a unique parasite-secreted fibrillar network surrounding the developing parasites so that the parasitophorous vacuole appears septate (figure 1F) [9, 16]. 2.3. Pleistophora 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 muscles have been described in two HIVinfected patients and in a non-HIV-infected patient. The parasites develop within a vesicle, bounded by a thick parasite-formed coat named sporophorous vesicle. Spores measured 2.0–2.8 × 3.0–4.0 µm and had 10–12 coils of the polar tube [18]. 2.4. Trachipleistophora spp.

The genus Trachipleistophora was established for a microsporidium responsible for a case of myositis in a patient with AIDS. This parasite was cultivated in vitro and in athymic mice. Meronts had two to four nuclei and divided by binary fission. In sporogony the surface coat became separated from the plasma membrane and formed a sporophorous vesicle. The parasite differed from the genus Pleistophora because no multinucleate sporogonial plasmodium was formed at any stage. Thus, this organism was placed in a new genus as a new species Trachipleistophora hominis [19]. Two cases of infection with similar organisms have been reported, but the sporogony distinguishes this parasite from T. hominis as two different types of sporophorous vesicles and spores are formed and the parasite has been classified as a new species Trachipleistophora anthropophthera [20]. 2.5. Nosema spp.

Most Nosema species are parasitic in invertebrates. Their development takes place in direct contact with the host cell cytoplasm and nuclei are paired throughout the entire life cycle [9]. Although microsporidia of the genus Nosema are widespread parasites, only a few human infections with Nosema spp. have been reported. A case of systemic infection occurred in a 4-month-old thymic-deficient infant (Nosema connori) and another microsporidium was detected in the corneal stroma of an immunocompetent man (Nosema ocularum) [21]. 2.6. Vittaforma corneae

Microsporidian spores measuring 3.7 × 1.0 µm were identified in deep corneal struma of a otherwise healthy man with an 18-month history of unilateral progressive central keratitis and the parasite was isolated in cell cultures. Spores contained polar tubules with six coils and had nuclei in diplokaryotic arrangements. In cell culture all observed stages laid individually in the host cell cytoplasm. This organism was originally assigned to the genus Nosema and was named Nosema corneum. Based on the ultrastructure of developmental stages in liver cells of Microbes and Infection 2001, 389-400

experimentally-infected athymic mice (tetrasporoblastic sporogony, band-like sporonts, all stages are surrounded by a cisterna of host endoplasmatic reticulum), this organism was later transferred to a new genus as Vittaforma corneae [22]. The reclassification on ultrastructural grounds was later supported by small subunit rRNA gene sequence data which placed Vittaforma distant from Nosema. A case of disseminated V. corneae infection recently occurred in Swizerland [23]. 2.7. Brachiola spp.

A microsporidium infecting muscle cells of an HIVinfected patient has been described recently. Development took place in direct contact with the cell cytoplasm and contained one or two diplocaryotic pairs of nuclei. Spores were about 2.5–2.9 × 1.9–2.0 µm with 7–10 turns of the polar tubule. The features of this microsporidium are most closely aligned with the genus Nosema and the species N. algerae, a mosquito parasite, which, as molecular biology indicates, is phylogenetically distant from typical members of the genus Nosema [24], but Cali et al. proposed a new genus and species and named the organism Brachiola vesicularum [25]. One human case of keratitis caused by Nosema algerae has been reported [26], but N. algerae has been recently transferred to the genus Brachiola as well as B. algerae [27]. 2.8. Unclassified microsporidia

The collective group Microsporidium is an assemblage of identifiable species for which the generic positions are uncertain because details of their life cycles are missing. Microsporidium ceylonensis was identified in a corneal ulcer of an 11-year-old Tamil boy from Sri Lanka. Spores measured 1.5 × 3.5 µm, no meronts or sporonts were seen. Microsporidium africanum was detected in corneal stroma of a woman from Botswana suffering from a perforated corneal ulcer. Spores with 15–16 turns of the polar tubule measured 4.5 × 1.5 µm and no developmental stages of the parasite were seen.

3. Human diseases (table I) 3.1. Gastrointestinal and biliary tract infections

Intestinal infections with microsporidia have been found mainly in HIV-infected patients with most infections due to E. bieneusi or less frequently to E. intestinalis. Infections are most common in HIV-infected patients with severe immunodeficiency and a CD4+ T-cell count below 100/ µL. Parasites often cause a severe, nonbloody, nonmucoid diarrhoea with up to ten or even more bowel movements per day, slowly progressive weight loss, and malabsorption of fat, D-xylose, and vitamin B12. Intestinal infection is associated with lactase deficiency, a reduced activity of alkaline phosphatase and α-glucosidase at the basal part of the villus, and with reduced villus height and a villus surface reduction. Diarrhoea appears gradually and may continue for months. The patients are often reluctant to eat and may complain of nausea. Some patients have intermittent diarrhoea, but only a few excrete microsporidial 391

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Table I. Clinical manifestations of human microsporidial infections. Species

Clinical manifestation

E. bieneusi E. intestinalis

enteritis, diarrhea, cholangitis, cholecystitis, pneumonitis, bronchitis, sinusitis, rhinitis enteritis, diarrhea, small bowel perforation, cholangitis, cholecystitis, nephritis, urinary tract infection, sinusitis, rhinitis, bronchitis, keratoconjunctivitis, disseminated infection keratoconjunctivitis, sinusitis, rhinitis, pneumonitis/bronchiolitis, nephritis, ureteritis, prostatitis, urethritis, cystitis, disseminated infection hepatitisa, peritonitisa, encephalitis, intestinal infection, urinary tract infection, keratoconjunctivitis, sinusitis, rhinitis, disseminated infection cutaneous infection, hepatic failure, bone infection myositis, keratoconjunctivitis, sinusitis, rhinitis encephalitis, myositis, disseminated infection myositis keratitis, urinary tract infection keratoconjunctivitis disseminated infection myositis keratoconjunctivitis corneal ulcer corneal ulcer

E. hellem E. cuniculi Encephalitozoon spp. Trachipleistophora hominis T. anthropophthera Pleistophora spp. Vittaforma corneae Nosema ocularum N. connori Brachiola vesicularum B. (Nosema) algerae Microsporidium africanum M. ceylonensis a

Species not confirmed because classification was based only on ultrastructure morphology.

spores without having diarrhoea. In groups of patients with chronic diarrhoea who were negative for other enteric pathogens, prevalence rates of E. bieneusi have varied between 7 and 50%, depending on the study population and method of diagnosis [28]. 3.2. Hepatitis and peritonitis

Hepatitis and peritonitis caused by Encephalitozoon spp. that were classified as E. cuniculi on an ultrastructural basis were reported in two HIV-infected patients. The patients died and at autopsy microsporidia consistent in ultrastructure with E. cuniculi were discovered [29, 30]. Both cases were published before E. hellem was described as a new species. In both instances, diagnosis was made only on ultrastructural basis so that the exact species identification is uncertain. A second case of fulminant hepatic failure caused by an Encephalitozoon sp. was reported in a patient with AIDS. He suffered from microsporidial diarrhoea two months prior to development of fulminant hepatitis and died. The autopsy revealed disseminated microsporidial infection involving the liver, gall bladder wall, and a mediastinal lymph node [31]. Both E. bieneusi and E. intestinalis have been detected in nonparenchymal liver cells of several HIV-infected patients, but the patients did not show any signs of hepatitis. Disseminated infection involving several organ systems including the liver and the pancreas with T. anthropophthera was reported in an 8-year-old HIV-infected girl [32]. 3.3. Ocular infections

Besides gastrointestinal infection, ocular microsporidiosis is the most common manifestation of microsporidiosis in humans [33]. In HIV-infected patients, keratoconjunctivitis may be caused by all three Encephalitozoon spp. (E. hellem, E. cuniculi and E. intestinalis). Most patients present with bilateral conjunctival inflammation and also 392

exhibit bilateral punctate epithelial keratopathy leading to decreased visual acuity. Often the keratoconjunctivitis is asymptomatic or moderate, but it can be severe, and rarely leads to corneal ulcers. Other species (V. corneae, N. ocularum, T. hominis, M. ceylonensis, M. africanum) were reported only as single case reports [33]. 3.4. Sinusitis

Sinusitis is a common manifestation of human microsporidiosis [34]. All three Encephalitozoon spp. (E. hellem, E. cuniculi and E. intestinalis) have caused rhinosinusitis in several HIV-infected patients. Less frequently E. bieneusi and T. hominis have been detected in sinus biopsy specimens from HIV-infected patients. These patients suffered from severe rhinitis as well and nasal polyps are often present [28]. 3.5. Pulmonary infections

Pulmonary infections with microsporidia have been reported less frequently than other manifestations. Infection of the lower respiratory tract may be asymptomatic or is associated with bronchiolitis and rarely with pneumonia or progressive respiratory failure in HIV-infected patients. All three Encephalitozoon spp. have been detected in bronchial epithelial cells of HIV-infected patients with disseminated Encephalitozoon infection, whereas pulmonary involvement with E. bieneusi has been reported only sporadically [28]. 3.6. Urinary tract infections

Infections of the urinary tract are a common finding in HIV-infected patients with disseminated Encephalitozoon infections. Clinical presentation and consequences of the presence of microsporidia in the urinary system can vary remarkably and may be asymptomatic with or without microhematuria, can present as cystitis and intestinal nephritis with dysuria and gross hematuria, or may lead to progressive renal failure [35]. Microbes and Infection 2001, 389-400

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3.7. Myositis

Myositis caused by Pleistophora-like microsporidia and Trachipleistophora spp. has been described in a few immunocompromised patients [18, 36, 37]. One case of myositis led to the description of Brachiola vesicularum [25]. Patients suffered from generalized muscle weakness and fever and microsporidian spores were seen in muscle biopsies. 3.8. Cerebral infections

Two cases of disseminated Encephalitozoon infection with cerebral involvement were reported in a 9-year-old Japanese boy and in a 2-year-old Columbian boy [1, 38]. Both patients suffered from cerebral symptoms such as headache, vomiting, spastic convulsions, and convulsive seizures. Encephalitozoon-like organisms were found in urine from both patients and in cerebrospinal fluid of one patient. The exact species differentiation of these two parasites is uncertain. Cerebral microsporidiosis due to E. cuniculi was described in two HIV-infected patients. Microsporidia were classified as E. cuniculi by immunohistochemistry and molecular based analysis [15]. Cerebral involvement with T. anthropophthera was reported in two AIDS patients with seizures and cerebral lesions. At autopsy a pansporoblastic microsporidium was seen in several organ systems including the brain [32]. 3.9. Rare manifestations 3.9.1. Urethritis

Two cases of urethritis associated with microsporidia were reported in patients with AIDS who suffered from urethritis, sinusitis and diarrhea. 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 and in stool samples of the second patient [39, 40]. 3.9.2. Prostatic abscess

A prostatic abscess due to E. hellem was reported in an AIDS patient with disseminated E. hellem infection. The prostate was of normal size with a 1.5 × 1.8 cm central periurethral abscess containing necrotic prostatic tissue. Tissue Gram stain revealed Gram-positive microsporidian spores which were identified as E. hellem by an indirect fluorescence assay [41]. 3.9.3. Tongue ulcer

A shallow 1 cm ulceration on the dorsum of the tongue was observed in an HIV-infected patient with severe immunodeficiency (15 CD4+ T cells/µL) and disseminated infection due to E. cuniculi. Spores were identified in several samples and in soft tissue beneath the tongue ulcer. The microsporidian was identified as E. cuniculi by immunofluorescent staining, in vitro cultivation and molecular analysis of the small subunit-rRNA gene by PCR [13]. 3.9.4. Skeletal involvement

Two cases of skeletal involvement with microsporidia were reported in patients with AIDS. Both patients suffered from disseminated microsporidial infections. Species identification was not done [42]. Microbes and Infection 2001, 389-400

3.9.5. Cutaneous microsporidiosis

One case of nodular cutaneous microsporidiosis that resolved with oral clindamycin therapy was reported in an HIV-infected patient. Underlying osteomyelitis, that resolved under therapy as well, was not proven to be caused by the microsporidia. Species differentiation using polymerase chain reaction (PCR) techniques was not successful [43]. 3.10. Systemic infections

The first case of documented human microsporidial infection in 1959 was that of disseminated Encephalitozoon infection in a 9-year-old Japanese boy who suffered from recurrent fever, headache, vomiting and spastic convulsions [1]. Encephalitozoon-like organisms were found in cerebrospinal fluid and urine. 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 spp. were found in urine. Anti-E. cuniculi antibodies (IgG and IgM) were detected in serum samples [38]. Disseminated infections with all three Encephalitozoon spp. have been recognized in several severely immunosuppressed HIV-infected patients [44]. The spectrum of disease has expanded to include keratoconjunctivitis, bronchiolitis and pneumonia, sinusitis, nephritis, urethritis, cystitis, prostatitis, hepatitis, peritonitis, gastroenteritis and cholangitis, but there are clear differences in the typical distribution pattern for each microsporidian species. E. hellem mainly parasitizes the keratoconjunctivals, urinary tract, nasal sinuses and bronchial system. On the other hand, E. intestinalis appears to be mainly confined to the gastrointestinal and biliary tract with dissemination to the kidneys, eyes, nasal sinuses and sometimes the respiratory tract. E. cuniculi causes widely disseminated infections involving nearly all organ systems, but clinical manifestations vary substantially, ranging from no symptoms to severe disease [28, 35, 44]. Disseminated infections with other microsporidian species (N. connori, V. corneae, T. hominis, T. anthropophthera) have been reported only as single case reports.

4. Diagnosis During the last decade diagnostic procedures to detect microsporidia in humans have been improved remarkably and today several methods for detection and species differentiation of microsporidia are available [35, 45]. 4.1. Transmission electron microscopy

Originally, electron microscopy was necessary to diagnose microsporidial infections [45]. Today this technique remains the gold standard for confirmation and species identification of microsporidia especially in laboratories where molecular-based techniques are not established. Species differentiation using ultrastructural examinations is usually possible but two human microsporidia (E. cuniculi and E. hellem) are similar even at the ultrastructural level. The fine structure features of the spores with the unique coiled polar tube and of the proliferative 393

Current focus: Microsporidia, intracellular parasites causing emerging diseases

forms, the nature of host-parasite interface, and the method of division are criteria for diagnosis and species differentiation of microsporidia (table II). Electron microscopy has been successfully used for diagnosis of microsporidiosis in body fluids, but species differentiation is more difficult as in biopsy tissue because mostly proliferative stages are absent. In biopsy tissues the detection of microsporidia is very specific, but this technique has a low sensitivity because of the small amount of a sample that can be examined. Further disadvantages are the time consuming sample preparation and examination. 4.2. Light microscopy

In the last few years, an increasing number of publications have described new staining methods for histologic and cytologic examinations [45]. Light microscopy allows the diagnosis of microsporidiosis, but genus and species differentiation is uncertain. 4.2.1. Cytologic diagnosis and stool examinations

Microsporidian spores have been detected in several body fluids (e.g., urine, duodenal aspirates, respiratory specimens, cerebrospinal fluid) and stools. Many different staining techniques have been described but routine stains such as Gram and Giemsa are not suitable because they do not differentiate between microsporidia and other elements present in stool samples and other specimens [46]. A concentration procedure, e.g., ether sedimentation for stool specimens, seems to be helpful to increase the sensitivity [47]. Most suitable staining methods are the chromotrope-based stain including several modifications of this technique and different fluorescent staining methods (e.g., Uvitex 2B, Calcofluor) [35, 45]. Chromotrope-based stains involved a trichrome stain with a 10-times-higher concentration of chromotrope 2R [48]. The disadvantage of this technique is the timeconsuming procedure. Several modifications of the stain have been suggested for accelerating the process and to increase the contrast between spores and background. These modified chromotrope stains reached better staining characteristics due to increasing the staining temperature, decreasing the phosphotungstic acid level, and by substitution of the counterstain [45]. Furthermore, an improved hot Gram-chromotrope technique which shortens the total staining time to 5 min has been described [49]. Fluorescent staining methods were used several years ago to detect nonhuman microsporidia. These staining techniques use epifluorescence of the chitinous endospore after reaction with fluorochromes [50]. The advantage lies in the fact that it is an easy, rapid procedure the sensitivity of which is comparable to that of chromotrope-based stains, but the examinations require a fluorescence microscope. Specificity is dependent on the experience of the laboratory because other fecal elements (bacteria and fungi) were also stained with optical brighteners. In our opinion both the chromotrope-based and the fluorescent stains should be used simultaneously to increase the sensitivity and specificity of the procedure [35]. The staining characteristics of the different stains are described in table III. 394

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4.2.2. Histologic diagnosis

Various staining techniques have been used to detect microsporia in formalin-fixed and paraffin embedded tissues. In our laboratory we prefer the fluorescent stain with Uvitex 2B as an easy and quick technique with high sensitivity (figure 2) [51]. Other suitable techniques are tissue Gram stains (Brown-Hopps, Brown-Brenn), silver staining (Warthin-Starry) [52], and chromotrope-based staining methods [53] or staining of resin-embedded material (figure 3) [54]. Routine staining methods such as H & E have been used as well, but the sensitivity of this method depends on the experience of the pathologists [55]. If fresh material is available, chromotrope-based and fluorochrome staining techniques can be used on touch preparations of intestinal biopsies and endoscopic brush cytologic specimens [56]. 4.3. Cell culture and animal models

Several human infecting microsporidia (V. corneae, E. hellem, E. cuniculi, E. intestinalis, T. hominis) have been cultured in different mammalian cell lines (e.g., rabbit and monkey kidney cells, human fetal lung fibroblasts). This has been of enormous benefit in understanding the biologic aspects of microsporidia. However, E. bieneusi has not been established in long term culture, but short term culture has been described on human lung fibroblasts and Vero cells [57]. Animal models are necessary to study routes of transmission, immune responses, and therapeutic strategies, and are essential for producing mono- and polyclonal antibodies. Different immunodeficient mice (e.g., SCID, athymic mice), rabbits and simian immunodeficiencyinfected rhesus monkeys have been infected with several human infecting microsporidia including E. bieneusi [58]. However, for diagnostic approaches culture systems and animal models are not suitable methods, because these techniques are time-consuming and are available only in a few research laboratories. 4.4. Antigen-based methods

Immunofluorescent antibody tests with monoclonal antibodies seem to be useful tools that have been shown to recognize species-specific surfaces of the spores or polar tube proteins of microsporidia. Poly- and monoclonal antibodies against E. hellem, E. cuniculi and E. intestinalis have been used for diagnosis and species differentiation of microsporidia in clinical samples from humans. Recently, monoclonal antibodies (IgM, IgG) against spores of E. bieneusi were produced in BALB/c mice as well and were successfully tested with fecal specimens using an indirect immunofluorescence antibody test [59]. Polyclonal antibodies are used with limited specificity because cross-reactivity has been documented between Encephalitozon spp. and other microsporidian species. Comparative studies with routine staining methods have shown lower sensitivity of immunofluorescence detection procedures [60]. As a consequence, immunofluorescent antibody tests, which are not widely available, should be used only as confirmation and for species differentiation after initial diagnosis with routine staining methods such as fluorescent or chromotrope-based stain. Microbes and Infection 2001, 389-400

Encephalitozoon spp. Nosema spp.

Brachiola spp.

V. corneae

Pleistophora spp.

Trachipleistophora spp.

1.1–1.6 × 0.7–1.0

2.0–2.5 × 1.0–1.5

2.5–5.0 × 2.0–2.5

2.5–2.9 × 1.9–2.0

3.2–3.4 × 2.8

4.0 × 2.4

Coils of polar tubule 5–7 Arrangement of polar two rows tubule

5–7 one row

7–12 one row

7–10 one to three rows

3.05–4.55 × 0.77–1.27 5–7 one row

9–12 two rows

11 one or two rows

Nucleus

unikaryotic

unikaryotic

diplokaryotic

diplokaryotic

diplokaryotic

unikaryotic

unikaryotic

Vacuole

no vacuole, in direct septated contact with the host parasitophorous cell cytoplasm vacuole in Encephalitozoon intestinalis

no vacuole, in direct no vacuole, in direct no vacuole, in direct sporophorous vesicle sporophorous vesicle contact with the host contact with the host contact with the host cell cytoplasm cell cytoplasm cell cytoplasm

Special features

electron-lucent inclusion, development of the polar tubule beginning in the sporonts

diplosporoblastic sporogony

Size of spores (µm)

diplosporoblastic sporogony like Nosematidae

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

no multinucleate sporogonial plasmodium

395

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Table II. Morphological characteristics of microsporidia infecting humans.

Staining technique

Specimens

Features

Comments

References

Chemifluorescence

stool, body fluids, biopsy tissue

bright fluorescence of the chitinous spore wall

recommended and evaluated staining [50, 51] procedure, requiring a fluorescent microscope, fast and sensitive technique, other fecal elements (bacteria and fungi) are also stained, excellent in staining of biopsies, laboratory experience necessary

Chromotrope and modified chromotrope stool, body fluids, biopsy tissue

bright pinkish-red spore wall, pinkishred-stained belt-like stripe, background green or blue depending on the staining technique

recommended and evaluated staining procedure, original stain is timeconsuming, reduced staining time by using modified stains, different counterstain possible

[45, 48, 49, 53]

Giemsa

body fluids, biopsy tissue (touch preparations)

light blue staining, darkly stained nucleus

difficult interpretation because other fecal elements also stain blue

[56]

Gram

body fluids

Gram variable

difficult interpretation because other fecal elements were also stained

[46]

Modified Gram (Brown and Brenn, Brown and Hopps)

biopsy tissue

spores stain deep blue or red against a faint brown-yellow background

experienced pathologist necessary

[45]

Warthin-Starry

biopsy tissue

H&E

biopsy tissue

dark spores against red background

Toluidine blue, methylene blue-azure/ basic fuchsin

plastic-embedded biopsies

darkly blue-stained spores, background not routinely used blue

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396

Table III. Staining techniques for detection of microsporidia by light microscopy.

time-consuming, difficult interpretation [52] experienced pathologist necessary [54]

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Microsporidiosis: human diseases and diagnosis

Current focus: Microsporidia, intracellular parasites causing emerging diseases

to a tropical environment and infection with microsporidia (for review see [61]). A high seroprevalence of antibodies against E. intestinalis was also observed among Dutch blood donors (8%), pregnant French women (5%) and patients with various infectious and noninfectious diseases (4%) [62]. A study group from Slovakia examined sera of employees of a slaughterhouse for encephalitozoonosis with 5.1% positive results and suggested that these findings reflect the possible transmission of microsporidia from animals to man [63]. However, the sensitivity and specificity of serological tests for Encephalitozoon spp. are still unknown and crossreactivity between human microsporidia has been demonstrated in different studies. 4.6. Molecular-based methods

Figure 2. Resin-embedded semi-thin (1 µm) section of duodenal mucosa. Epithelial cells containing spores of E. cuniculi. Toluidine blue stain, original magnification × 1 000.

4.5. Serology

A variety of serological assays (carbon immunoassay (CIA), indirect immunofluorescence test (IFA), enzymelinked immunosorbent assay (ELISA), counterimmunoelectrophoresis (CIE), and Western blot) have been established to detect IgG and IgM antibodies to microsporidia, especially to E. cuniculi and E. intestinalis. These test systems are suitable for seroepidemiological studies, but antibody detection does not reflect an acute infection and it is impossible to differentiate between latent infections and former contact with microsporidian antigens. For E. bieneusi, the most prevalent microsporidian species, no serological test is available. Seroepidemiological surveys found antibodies to E. cuniculi and E. intestinalis in humans with and without HIV infection and suggested a possible link between exposure

Figure 3. Paraffin-embedded duodenal biopsy specimen from a patient with AIDS with intestinal E. bieneusi infection. The microsporidial spores are easily visualized within the enterocytes. Fluorescent microscopy after Uvitex 2B stain, original magnification × 1 000. Microbes and Infection 2001, 389-400

Many different genes of human- and animal-infecting microsporidia have been sequenced in the last few years (for review see [35, 64]). One milestone is the complete sequencing of the whole genome of E. cuniculi by the research group of C. Vivares in France. Molecular-based methods are important for taxonomic classification, phylogenetic studies and especially for detection and species differentiation of microsporidia in clinical samples of human patients. 4.6.1. PCR

PCR assays have been established for diagnosis and species differentiation of most human microsporidia. Several single and nested primer pairs have been described to amplify gene sequences of E. bieneusi, E. cuniculi, E. intestinalis and E. hellem (for review see [35]). Mostly, the small subunit rRNA was used as target gene, but some authors also used the large subunit rRNA and the intergenic spacer region as a target sequence. In our laboratory we used a primer system (V1/PMP2) which amplifies DNA sequences from all important human infecting microsporidia [65, 66]. Species differentiation is possible through the length of the amplicons between E. bieneusi and the Encephalitozoon spp. and can be further evaluated by restriction endonucleases or species-specific primer pairs. Both tissue biopsies and liquid material including stool samples can be used for PCR. DNA extraction from biopsies is easy to perform, but sufficient DNA extraction from spores is more difficult. Mechanical disruption and additional use of enzymes (lyticase, chitinase) is recommended by several authors to release DNA from spores [35, 67]. Comparison of molecular techniques with light microscopy is difficult. In a blinded multicenter study that compared PCR with light microscopy the greatest differences in sensitivity were found between individual laboratories rather than between different techniques [68]. Nevertheless, in combination with light microscopy, DNA amplification by PCR, especially from stool samples, offers an excellent approach to diagnosing microsporidiosis with the advantage of a simple species differentiation. 4.6.2. In situ hybridization

In situ hybridization has been established for the diagnosis of infections with E. bieneusi in humans and experimentally SIV-infected rhesus monkeys [69]. The described probes were directed against the SSU-rRNA of E. bieneusi 397

Current focus: Microsporidia, intracellular parasites causing emerging diseases

and allowed the identification of different microsporidian stages including plasmodia and spores in the cytoplasm of enterocytes [70]. However, this elegant but timeconsuming method has not been described for other human infecting microsporidia and sensitivity and specificity have not been evaluated.

5. Concluding remarks During the last decade our knowledge about the microsporidia has expanded remarkably. After the emergence of microsporidia in HIV-, and more recently in non-HIV-infected patients much research has been done to describe the clinical spectrum of human microsporidiosis and to develop reliable diagnostic tests. However, there are still major gaps in our knowledge of the disease. Natural reservoirs and mode of transmission remain to be investigated and information on the immunology and treatment of microsporidia is still very limited. With the new interest in microsporidia generated by the emergence of the latter as the cause of human disease, much research is ongoing and will provide continuing information concerning microsporidia and microsporidiosis in the future. We would like to thank Dr Britta Franzen for editorial assistance during preparation of the manuscript.

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