Pathogenesis And Clinical Significance

Almost all of the organs of the human body can be infected by one or more of the spectrum of 14 microsporidian species described in the previous section. Many tissues and cell types are involved (Table 8.1). According to site of infection, clinical manifestations may be diarrhoea, weight loss, cholecystitis, cholangitis, bronchitis, bronchiolitis, pneumonitis, sinusitis, rhinitis, hepatitis, peritonitis, nephritis, ureteritis, cystitis, urethritis, prostatitis, keratoconjunctivitis, corneal ulcer, myositis or encephalitis. The pathology has been reviewed by Weber et al. (1994) and Schwartz et al. (1996). Cardiac disease and probable pancreatic, parathyroid and thyroid dysfunction have been reported for T. anthropophthera (Yachnis et al., 1996). Without treatment, the outcome is likely to be fatal for severely immunocompromised hosts infected with the disseminating species.

Table 8.1 Sites of infection of microsporidia in immunocompromised and/or immunocompetent human patients

E. bieneusi E. intestinalis E. hellem E. cuniculi

V. corneae

M. ceylonensis M. africanum N. ocularum Pleistophora sp. T. hominis

T. anthropophthera

B. vesicularum B. connori

B. algerae

Epithelia of intestine, bile duct, gall bladder, pancreatic duct, trachea, bronchi, nasal sinuses and nose; non-parenchymal liver cells

Epithelia of intestine, bile duct, gall bladder and bronchi; macrophages, fibroblasts and endothelial cells of lamina propria; kidney tubule cells; non-parenchymal liver cells; nasal epithelium; corneal epithelium Epithelia of respiratory tract (trachea to bronchioles), nasal sinuses, nose, cornea and conjunctiva; kidney tubule cells and renal blood vessel endothelium; bladder; prostate; liver. Not intestine Epithelium (tubule cells) and endothelium of kidneys, adrenal glands, trachea; myocytes of heart; macrophages of brain, heart, urinary bladder, spleen and lymph nodes (suggestive of phagocytosis after release from other cells) (Mertens et al, 1997); epithelium of duodenum (transiently) and conjunctiva and detected in sputum, urine and stool (Franzen et al., 1995); liver; peritoneum Corneal stroma (Shadduck et al., 1990); urinary system (Deplazes et al., 1998). In experimentally infected athymic mice, liver, spleen, kidney, intestine, heart, lung, brain and retina (Silveira et al., 1993) Corneal stroma in macrophages and free between lamellae Corneal stroma in histiocytes and free between lamellae Corneal stroma (Bryan et al., 1990) Skeletal muscle myocytes (Ledford et al., 1985)

Skeletal muscle myocytes, nasal sinus (detected in nasal secretions) and conjunctival epithelium (Field et al.,

1996). Systemic infection (not including brain) in athymic mice (Hollister et al., 1996b) Brain astrocytes, endothelium, macrophages; heart myocytes, macrophages; kidney tubular and glomerular epithelium, endothelium, macrophages; pancreas endocrine and exocrine cells, Schwann cells; vascular smooth muscle; thyroid follicular epithelium; parathyroid epithelium, adipocytes; liver hepatocytes; unknown cells in bone marrow, lymph node and spleen (Yachnis et al., 1996) Skeletal muscle myocytes (Cali et al., 1998)

Disseminated, especially involving myocytes (myocardium, muscularis of gastrointestinal tract, walls of arteries in urinary bladder, kidney, liver, adrenals, heart and diaphragm) but also present in adrenal cortical epithelium, kidney tubules and foci of hepatocytes (Margileth et al., 1973) Cornea (Visvesvara et al., 1999)

Typical reactions to microsporidial infections in immunologically intact hosts are hypertrophy of infected cells and tissues, without inflammation as long as the infected cells remain intact, but with an inflammatory response once the spores are liberated. The response is in the form of a diffuse cellular infiltration, leading to granuloma formation, involving lymphocytes, plasma cells and macrophages. In immunocom-promised patients the cellular response may be similar but is sometimes minimal.

Intestinal Infection

Although alterations to the intestinal architecture are not universal in patients infected with E. bieneusi or E. intestinalis, there may be villus stunting, atrophy and other histological changes. E. intestinalis spreads throughout the epithelium and into the lamina propria (Figure 8.5B on Plate IV), while E. bieneusi is restricted to the entero-cytes between the brush border and the nucleus. Immunocompetent patients may suffer an acute, self-limited diarrhoea, while AIDS patients usually have chronic, intractable diarrhoea. Contact spread from the intestinal epithelium to the bile duct, gall bladder, pancreatic duct and respiratory surfaces, causing epithelial hyperpla-sia and associated clinical signs, has occurred with both species. E. hellem does not infect the gastrointestinal tract. E. cuniculi has only been found in the intestinal epithelium in a patient with disseminated infection but suffering no intestinal disorder, suggesting that infection of the intestine represents merely the route to the deeper viscera.

Renal Infection

The kidney is a site of predilection for all three Encephalitozoon spp. (Figure 8.5F on Plate IV) and was involved in disseminated cases of V. corneae, B. connori and T. anthropophthera. Infection is principally in the tubules but glomeruli may be involved. Breakdown of tubule epithelial cells stimulates an interstitial nephritis and debris accumulates in the tubule lumina. Kidney tissue destruction may be massive and there may be spread to the ureters and bladder. The spores can be detected in urine.

Muscular including Myocardial Infection

Infection in myocytes of the heart has been reported for B. connori, E. cuniculi and T. anthropophthera (Figure 8.5K on Plate IV). In all cases, each focus contained myriads of spores. Reactive cells were absent, except for macrophage activity after release of the spores, but areas with multiple lesions were associated with necrosis and fibrosis of adjacent tissue. Myocytes of skeletal muscle were the sites of infection with T. hominis (Figure 8.5J on Plate IV) and Pleistophora sp., while B. connori showed a predilection for the walls of blood vessels in most organs, occurring also in the kidney tubules and adrenal cortex, with no inflammatory response. However, a marked inflammatory infiltration was present in the severely infected muscularis of the diaphragm of the B. connori-infected immunocompromised infant.

Cerebral Infection

The brain has been reported only once in a human patient as a site of infection for E. cuniculi (Mertens et al., 1997). Spores were found free in parenchyma and perivascular spaces and others were in macrophages. Experience with animals has shown that the largest aggregates of spores occur in grey and white matter at all levels of the brain and that microgranulomata are formed after spore release. It is likely that brain infection also occurs regularly in human E. cuniculi infections but, in most of the few cases that have been diagnosed, parasites have been isolated from urine or bronchoalveolar lavage, the patients have responded to treatment and autopsies have not been performed. The most extensive catalogue of brain injuries due to microsporidia is that by Yachnis et al. (1996), who reported two cases of infection with the parasite now known as T. anthropophthera. Parasites were present in astro-cytes (Figure 8.5G on Plate IV) and endothelial cells in numerous lesions each measuring up to 2.5 cm (Figure 8.5L on Plate IV; Figure 8.3B). These lesions were characterised by a central necrosis with spores engulfed by macrophages, surrounded by infected astrocytes (Figure 8.5G on Plate IV).

Ocular Infection

There are two types of ocular microsporidiosis. Infections caused by E. hellem, E. cuniculi, E. intestinalis and T. hominis have been restricted to the corneal and conjunctival epithelia, and caused distressing bilateral punctate keratopathy with redness, irritation and decreased visual acuity. M. ceylonensis, M. africanum, V. corneae, B. algerae and N. ocularum have infected cells of the corneal stroma. The cases of M. ceylonensis and B. algerae led to severe ulceration and necessitated keratoplasty, while that of M. africanum required surgical removal of the eye.

Respiratory Infection

Respiratory disease is a common manifestation of E. hellem and less frequent involvement has been reported for E. intestinalis, E. cuniculi and E. bieneusi. E. bieneusi has been found in nasal and nasal sinus epithelia (Eeftinck Schattenkerk et al., 1993) and bronchial epithelium (Weber et al., 1992b), E. cuniculi in the tracheal epithelium (intense infection) (Mertens et al., 1997) and E. intestinalis in bronchial and nasal sinus epithelia (Molina et al., 1995). The extent of infection of E. hellem in the respiratory system and its absence from the intestine led Schwartz et al. (1992) to suggest that the respiratory epithelium was the port of entry. In one focus, parasites were found in subepithelial granulation tissue adjacent to capillaries, suggesting a route from the respiratory surface to the kidney. Manifestations of respiratory infection may be chronic cough and shortness of breath and finally even respiratory failure. Infection of the nasal and nasal sinus epithelia leads to formation of polypoid tissue and consequent nasal obstruction and discharge (Figure 8.3A).

Liver Infection

E. cuniculi has been reported once as a cause of hepatitis (Terada et al., 1987) and once as a cause of peritonitis (Zender et al., 1989) but both reports were made before E. hellem and E. intestinalis were described and the diagnosis remains unconfirmed. Surprisingly, liver parenchyma is rarely involved in human micro-sporidiosis.

mediator (Didier et al., 1994; Didier, 1995). Achbarou et al. (1996) confirmed the importance of IFNy in their mouse model for chronic infection with E. intestinalis, using IFNyR°/° mice, a strain with a deletion in the gene coding for the IFNy receptor. In their studies, spores of E. intestinalis were shed in increasing numbers by the IFNyR°/° mice during the experimental period, whereas there was a decrease in spore output in wild-type mice over the same period.

Pathogenic Mechanisms

The mechanisms of pathogenesis are little understood. In E. cuniculi, the formation of immune complexes undoubtedly contributes to disease in carnivores (Mohn and Nordstoga, 1975) and recently Sharpstone et al. (1997) proposed that elevated TNFa levels in the intestine of E. bieneusi-infected AIDS patients contributed to the diarrhoea, which could be alleviated by thalidomide, a TNFa inhibitor.

IMMUNOLOGY Cell-mediated Immunity

Current knowledge of immune responses to microsporidial infection have been summarised by Didier, Snowden and Shadduck (1998). It is not surprising that microsporidioses have emerged as important opportunistic infections in AIDS patients, as evidence derived from experimental infections of mice with E. cuniculi shows that cellmediated immune responses are paramount in controlling infection. Thus, transfer of sensitised syngeneic T cell-enriched spleen cells to athymic mice just prior to infection with E. cuniculi gave protection from lethal disease (Schmidt and Shadduck, 1984). SCID mice were similarly protected if the T cell transfer occurred before infection but they were only partially protected if the T cell transfer was effected after infection (Hermanek et al., 1993). Macrophage activation by cytokine release from lymphocytes, to stimulate phagocytosis and degradation of spores by nitrogen intermediates, has been demonstrated as one mechanism of protection, and interferon gamma (IFNy) has been implicated as the

Humoral Immunity

The role of antibodies in the control of micro-sporidial infection appears to be of secondary importance to the cell-mediated response. Nothing is known of the role, if any, of IgA in preventing infection via the intestine. Both IgM and IgG antibodies are produced in response to infection but are only likely to have an opsonis-ing effect on spores for uptake by macrophages (Niederkorn and Shadduck, 198°). In immuno-competent animals the persistence of high antibody levels after clinical recovery may indicate the presence of latent infections and this has been shown by reactivation of latent infections in mice by administration of hydrocortisone (Bismanis, 197°). However, it has yet to be determined whether the severe infections in AIDS patients are newly acquired or are reactivations of latent infections. Examination of serum taken from an AIDS patient before detection of E. hellem and at intervals after diagnosis showed a decline to almost non-detectable levels of specific antibody as the CD4+ T-cell count dropped, although the parasite burden remained high (Hollister et al., 1993b). Antibodies to Encephalitozoon spp. have been detected in several serological surveys using blood from healthy donors and patients suffering a variety of diseases (Hollister et al., 1991; Van Gool et al., 1997).

Two of the species first discovered in AIDS patients, E. bieneusi and E. intestinalis, have since been found in immunocompetent people as transient infections. This possibility was first signalled by Bretagne et al. (1993), who detected E. bieneusi spores in the stool of 8/99° children in Niger who were unlikely to have been HIV

positive. Spores of E. bieneusi have also been detected in a child in Tunisia who was experiencing severe diarrhoea (Aoun et al., 1997) and in a child in Zambia (Hautvast et al., 1997). E. bieneusi has also been found as a cause of traveller's diarrhoea in an otherwise healthy child (Sobottka et al., 1995) and an adult (Sandfort et al., 1994). Wanke et al. (1996) and Gainzairain et al. (1998) reported other adult cases and reviewed previous cases. E. intestinalis has been detected in several immunocompetent adults (Raynaud et al., 1998). Surprisingly, only two cases of microsporidiosis have been reported to date in people who were immunosuppressed after organ transplantation (Rabodonirina et al., 1996; Sax et al., 1995).

MOLECULAR BIOLOGY Gene Sequencing

The presence of 16S ribosomes in microsporidia has facilitated amplification of microsporidian ribosomal genes even without purification from host tissue. PCR amplification and sequencing of several microsporidian genes have now been achieved and the results have been used to examine the phylogenetic position of microsporidia (see above), contribute to an understanding of systematic relationships of genera, to epidemiology and above all to diagnosis (see below). Complete or partial sequences have been obtained for several genes, including the complete sequence of the rDNA unit of E. cuniculi comprised of the 16 S gene, ITS1 region, 5.8 S gene and 23 S gene plus flanking regions (Biderre et al, 1997c). Sequences are also available for the small subunit rDNA (many species), isoleucyl-tRNA synthetase in Nosema locustae (Brown and Doolittle, 1995), ß- and a-tubulin in E. hellem, E. cuniculi, E. intestinalis and Nosema locustae (Edlind et al., 1996; Li et al., 1996), a U2 RNA homologue in Vairimorpha necatrix (De Maria et al., 1996), elongation factor 1a in Glugea plecoglossi (Kamaishi et al., 1996), mitochon-drial-type heat-shock protein genes HSP70 in N. locustae (Germot et al., 1997) and V. necatrix (Hirt et al., 1997), and the largest subunit of RNA polymerase II (Hirt et al., 1999). Sequences of these genes have been used variously to deduce that microsporidia are primitive eukaryotes or highly derived fungi, with the balance of evidence in favour of fungal affinities (summarised in Canning, 1998; Weiss et al., 1999).

Molecular Karyotypes

Using another approach to species identification and, indeed, to investigation of microsporidial genomic organisation, molecular karyotypes have been obtained by pulsed field gel electrophoresis. Haploid genomes of only 2.9 Mb with 11 chromosomal bands in E. cuniculi (smaller than that of Escherichia coli at 4.7 Mb) (Biderre et al., 1995) up to 19.5 Mb with 16 bands for Glugea atherinae have been demonstrated (Biderre et al., 1997b). In hybridisation experiments, ribosomal DNA probes hybridised to all 11 chromosomes of E. cuniculi, while p-tubulin and aminopeptidase genes were each found on two chromosomes, and five other protein-encoding genes were found on only one chromosome (Biderre et al., 1997a).

The symbiont-like HSP70 genes identified in microsporidia would be expected to function in mitochondria, which are reportedly absent. These genes are unusual in having a peroxisomal targeting signal (Hirt et al., 1997), unlike all previously described HSP70 genes, and these organelles have also not been described. This is yet another highly unusual character of micro-sporidia and determination of the function of these genes is clearly a requirement for our understanding of microsporidian biology.

EPIDEMIOLOGY Transmission

Most microsporidia are transmitted directly between hosts by ingestion of spores, which are released into the environment via faeces or urine or by death and degeneration of the hosts e.g. of fish and invertebrates. There is evidence of transplacental transmission of E. cuniculi in rodents, rabbits and carnivores (see Canning and Lorn, 1986) but not in man. Strong circumstantial evidence was provided that E. hellem may enter via the respiratory system (Schwartz et al., 1992).

Spores released from patients or from animal sources could easily enter the water supplies, as the spores are small enough for all species reported in man to pass through the filters used in water purification. Indeed, evidence for the presence of microsporidian spores in river water and sewage effluent, including several species which infect man, has been obtained by combining water concentration, filtration through various pore sizes and PCR amplification of the residues (Sparfel et al, 1997; Dowd et al, 1998).

In considering the epidemiology of microspor-idiosis, there are many questions for which answers are still needed. How many of the 14 species already known to infect man are natural parasites which occur subclinically at low prevalence and are transmitted human-to-human through the general population? If directly human to human, what is the likely mode of transmission? How many species are examples of single, unfortunate encounters between parasite and host, when the host is unusually susceptible (immunocompromised)? In how many cases are alternative hosts involved? How many of the fulminant infections seen in AIDS patients are reactivations of latent infection and how many are newly acquired? To what extent can micro-sporidia of invertebrate hosts adapt to mammalian body temperature and pose a threat to immunocompromised people? If only a small fraction of the possible range of microsporidia capable of infecting man is known, what are the limiting factors and where should we look for potential sources of infection?

Enterocytozoon bieneusi

E. bieneusi is the most commonly occurring species in man with prevalences of 10-44% recorded in AIDS patients whose CD4+ cell counts have fallen below 100/ml3 and who suffer chronic diarrhoea. Similar spores detected in the stool of domestic pigs have been confirmed as E. bieneusi by 16S rRNA sequence data (Deplazes et al., 1996b) but contact between man and pigs is too limited to account for the recorded prevalences and none of the four genotypes found in pigs match those found in humans. However, rabbits and dogs have also been identified as hosts (del Aguila et al., 1999). Mansfield et al. (1997) reported the spontaneous occurrence of a microsporidium very close, if not identical, to E. bieneusi, in 35.2% (18/51) of several species of macaque monkeys with simian AIDS (SIV infection), which had shown signs of hepatobili-ary and intestinal disease at a primate centre. Again, monkeys cannot be considered as a source for human infection but an argument can be made that E. bieneusi is a species that naturally infects primates. Mansfield et al. (1997) stated that there was preliminary evidence that an E. bieneusi-like organism was present at subclinical levels in the colony of normal rhesus monkeys (Macaca mulatta) at the primate centre and proposed that the disease in macaques was due to reactivation after SIV infection. It is possible that, after a short, acute episode, E. bieneusi remains latent in man unless the immune constraints are removed. The demonstration of genetic diversity in E. bieneusi, with four types based on restriction (RFLP) analysis of PCR products (Liguory et al., 1998), further complicates the elucidation of E. bieneusi epidemiology.

Encephalitozoon spp.

Unlike E. bieneusi, which still cannot be maintained in vitro, culture of Encephalitozoon spp. has made it possible to conduct serological surveys to detect latent infections. Hollister et al. (1991), using whole spores of E. cuniculi as antigen in ELISA identified infections in numerous patients suffering tropical disease and confirmed their results by Western blotting of spore protein profiles with the patients' sera. They found that only 2/1002 healthy blood donors were positive. In contrast, Van Gool et al. (1997), using sonicated spore preparations of E. intestinalis as antigen in ELISAs and counter-immunoelectrophoresis, and germinated spores in immunofluorescence tests, found that 8% (24/ 300) of blood donors and 5% (13/276) of pregnant women had high antibody titres. The tests used by Van Gool et al. (1997) were genus-specific and indicated that one or more of the Encephalitozoon spp. occur as latent or past infections at significant levels in human population groups, thus providing a possible pool from which reactivations can occur in AIDS. Franzen et al. (1996c), using PCR amplification and Southern hybridisation with species-specific primers and probes, detected five cases of E. bieneusi, five cases of E. intestinalis and five double infections among 46 AIDS patients (33%). These results indicate a much higher prevalence of E. intestinalis (22%) than had previously been determined by parasitological examinations and echo those of Van Gool et al. (1994), who isolated E. intestinalis in vitro several times from the stool of AIDS patients, in whom the parasite had not been detected by direct faecal examination. E. cuniculi has been reported from two apparently immunocompetent children who suffered transitory neurological disorders (see Historical Introduction). Although these parasitological detections occurred before the other Encephalitozoon spp. had been recognised, it is likely that the diagnoses were correct because E. intestinalis infections are not associated with neurological damage, even in AIDS patients. E. intestinalis has recently been found in several immunocompetent people suffering diarrhoea (Raynaud et al., 1998) but E. hellem has only been found in people with AIDS.

Recently, a microsporidium contributing to morbidity and mortality in budgerigars (Melopsittacus undulatus) in a commercial aviary, has been identified by PCR and Southern blot analysis as E. hellem (Black et al., 1997). The infections were unusual in that the intestinal epithelium was heavily infected, a site not associated with E. hellem in man. Black et al. (1997) recorded that some of the AIDS patients with E. hellem infection had owned or been exposed to caged birds. Although yet to be confirmed as a source of infection, pet birds might be involved in the epidemiology of E. hellem infections. Recently, infections of E. intestinalis have been found in dog, donkey, pig, cow and goat faeces, suggesting that human infections may have a zoonotic origin (Bornay-Llinares et al., 1998).

The most complete evidence for zoonotic sources of human microsporidial diseases lies with E. cuniculi. This species has a wide host range among mammals. Canning and Lom (1986) recorded 25 hosts from several orders of mammals and these are probably just a few of the total of susceptible animals. Sequencing of the intergenic spacer region (ITS) of the ribosomal genes has revealed that isolates fall into three categories, based on the number of tetranucleo-tide repeats (5'-GTTT-3') in the ITS. The rabbit type has three repeats, mouse type has two and dog type has three (Didier et al., 1995b). Deplazes et al. (1996a) examined six isolates of E. cuniculi from AIDS patients in Switzerland and found that all were of the rabbit subtype, and concluded that E. cuniculi microsporidiosis in this situation was a zoonotic disease derived from rabbits. In contrast, other isolates of E. cuniculi from AIDS patients have been identified as dog subtypes (Hollister et al., 1993a, 1996a; Didier et al., 1996b). Dogs are, thus, a likely source of infection, as had been previously suggested by seroconversion of a child who had had close contact with dogs with overt encepha-litozoonosis (McInnes and Stewart, 1991).

Other Species

No firm data are available on the possible sources of infection of the remaining human microspor-idia. The Pleistophora sp. of Ledford et al. (1985), T. hominis and T. anthropophthera have some morphological features in common with the numerous Pleistophora spp. which are found in fish or crustaceans, mostly parasitising skeletal muscle (Canning and Lom, 1986). The finding of unidentified microsporidia, still enveloped by undigested muscle, in the stool of an AIDS patient with diarrhoea (McDougall et al., 1993) supports the concept of a dietary source for some of these species. Tocdlowski et al. (1997) found unidentified organisms, thought to be microspor-idia, in an extracellular position close to the caecal epithelium in puffin chicks (Fratercula corniculata), which had been captured and fed on silversides and krill. If their identification was correct it further shows that microsporidia can survive the digestive process in abnormal hosts.

Another possible route of infection to skeletal muscle is by direct inoculation by a bloodsucking invertebrate. Microsporidia are very common in mosquitoes, with many genera and species involved. When the sequences of the 16S rDNA of several genera of polysporous micro-sporidia derived from fish, crustacea and haematophagous insects were compared, the sequence closest to that of T. hominis was that of Vavraia culicis, a parasite of many species of culicine and anopheline mosquitoes (Cheney et al., 2000). Furthermore, T. hominis readily infects anopheline and culicine mosquitoes when spores are fed to larvae, and spores harvested from these larvae are infective to athymic mice (Weidner et al., 1999). Although T. hominis is morphologically distinct from V. culicis, it is possible that the human T. hominis was derived from a species closely related to V. culicis infecting another biting fly. Recently a well known parasite of mosquitoes, Brachiola algerae (=Nosema algerae), has been isolated from human cornea (Visvesvara et al., 1999) and ultrastructural data (Trammer et al., 1999) suggested that Brachiola vesicularum, which was described as a new species from a skeletal muscle biopsy taken from an AIDS patient, might actually be N. algerae. However, although N. algerae was transferred to the genus Brachiola, it was considered to differ from B. vesicularum (Lowman et al., 2000). The possibility that T. hominis has an insect origin raises issues of public health importance, and indicates that microsporidia of invertebrate origin should be investigated for their ability to adapt to human body temperature.

It was originally proposed that the corneal infection of V. corneae might have been acquired directly by swimming in a lake. However, subsequent investigation has shown that V. corneae gives rise to a systemic infection in athymic mice (Silveira and Canning, 1993) and in an AIDS patient (Deplazes et al., 1998), so that it is more likely that the corneal infection was secondary. However, an invertebrate origin for this species cannot be ruled out. Nothing is known about the generic status or likely sources of the other ocular infections due to M. ceylonensis, M. africanum or N. ocularum.

LABORATORY DIAGNOSIS Light Microscopy

Several staining techniques have proved particularly useful for detection of microsporidian spores in urine, faeces or tissue specimens. The original chromotrope-based stain (Weber et al.,

1992a) and modifications thereof (Ryan et al., 1993; Kokoskin et al., 1994) stain spores pinkish-red and can be used with light background counterstains to advantage on faecal and urine smears (Figures 8.5C,D on Plate IV) and tissue sections. An acid-fast chromotrope method has been developed that will stain both microspor-idian spores and Cryptosporidium oocysts, both of which may be present in stool (Ignatius et al., 1997). Warthin-Starry stains spores brownish-black and is best used on tissue sections, in which even single spores are easily detected (Figure 8.5B on Plate IV) (Field et al., 1993). Other useful stains are Gram's, which stain spores blackish-purple, and Ziehl-Neelsen, which stains them red (Figure 8.5F on Plate IV). Spores are more difficult to detect in Giemsa-stained smears but advantages of this method are that developmental stages are revealed and that the nuclei are visible (Figure 8.5I on Plate IV) to aid generic diagnosis. Haematoxylin and eosin used in routine histological processing is a poor method for microsporidia (Figure 8.5K on Plate IV) but the spores are clear when viewed with polarised light (Figure 8.5H on Plate IV). Toluidene blue gives excellent results on resin-embedded sections (Figure 8.5G,J on Plate IV).

The fluorescence brighteners (fluorochromes) Uvitex 2B (Van Gool et al., 1993) and Calcofluor M2R (Vavra et al., 1993) are without doubt the most sensitive for quick detection of spores in smears and sections. Both give brilliant blue-white fluorescence when examined with a fluorescence microscope at wavelengths of 390-415 nm (Figure 8.5E on Plate IV). However, as the fluorescence depends on the presence of chitin in the spore wall, fungal spores will also fluoresce and may give false positives in inexperienced hands. Chromotrope and fluorochromes have proved equally valuable in comparative tests (Didier et al., 1995a; Ignatius et al., 1997) and an excellent routine would be to scan specimens stained with Uvitex or Calcofluor and, if spores are suspected, to re-stain new preparations with Chromotrope.

Antibody Detection of Microsporidian Spores

Polyclonal and monoclonal antibodies raised against microsporidian species have been used as aids to detection and identification. It is likely that similar epitopes are present on the spore coat proteins of many microsporidia, so that polyclonal sera raised against spores will be cross-reactive. This was found when polyclonal sera raised against E. hellem or E. cuniculi bound strongly in immunofluorescence tests (IFAT) in homologous and heterologous reactions using fresh or formalin-fixed E. hellem, E. cuniculi, E. intestinalis and E. bieneusi (Aldras et al.,

1994). Surprisingly, a polyclonal serum raised against spores of E. cuniculi was highly specific and was used to identify this species in nasal discharge from an AIDS patient, there being no reaction of the spores with polyclonal sera raised against E. hellem or E. intestinalis (Franzen et al.,

1995). Species-specific polyclonal antisera have also been used to identify E. intestinalis in animals that may be a reservoir for human infection (Bornay-Llinares et al., 1998). Aldras et al. (1994) found that even monoclonal antibodies (Mabs) raised against E. hellem were cross-reactive with the other Encephalitozoon spp. and with E. bieneusi but not with N. corneum (=V. corneae). A similar level of specificity was found by Enriquez et al. (1997) for a Mab that reacted with all Encephalitozoon spp. in IFAT but, in this case, not with E. bieneusi or V. corneae. In contrast, one Mab raised against E. hellem by Croppo et al. (1998) was not cross-reactive, in IFAT or Western blots, with any other of the microsporidia tested and thus might be useful in identification of E. hellem in fixed tissues. Although IFAT is less convenient than chromotrope or fluorochromes for detection, it may become a valuable technique in species identification, when Mabs for all species are available.

Detection of Antibodies in Patient's Sera

Several serological tests have been designed to detect antibodies in human sera and thus determine the extent to which microsporidian infections occur in immunocompetent healthy people, as well as in those suffering AIDS or other diseases. Some results of serological surveys for E. cuniculi are presented in the section on Epidemiology (see above). In the absence of a satisfactory culture method for E. bieneusi, it has not been possible to develop serological tests for this species, based on E. bieneusi antigens. Ombrouck et al. (1995) reported binding of sera from E. bieneusi-infected, HIV-positive patients in Western blots of SDS-PAGE separated proteins of Glugea atherinae, a microsporidium derived from fish. Unfortunately, the binding patterns were highly variable and two of the most frequently recognised proteins were also recognised by sera from two of six patients uninfected with microsporidia and infected with Cryptosporidium. Clearly the development of serological tests for E. bieneusi awaits improvement in culture techniques.

Polymerase Chain Reaction (PCR)

PCR offers considerable promise both for detection of microsporidia in clinical samples and identification of species. Sequences are known for the 16 S rRNA genes of the Encephalitozoon spp., E. bieneusi, V. corneae and T. hominis and, from these, it is possible to design primers which will amplify all species (based on highly conserved regions), or are genus- or species-specific. When genus-specific primers are used, species identification can still be achieved by using species-specific oligonucleotide probes on Southern blots or by restriction digests. Vossbrinck et al. (1993) used primers for a region of the rDNA spanning part of the small subunit, the ITS region and part of the large subunit to amplify E. hellem, E. cuniculi and V. corneae from culture and differentiated these with restriction digests using Sau3a, EcoR1, Dra1 and Hinf1.

The first attempt to amplify microsporidian DNA from stool involved a lengthy (4 day) and complicated procedure involving mechanical and chemical disruption of spores (Fedorko et al., 1995). Later methods have shortened and simplified the procedure. Fresh, fixed or frozen tissue samples can be used for DNA extraction, with or without prior disruption by grinding. Stool samples can be processed after formalin fixation and dilution. Specimens are usually incubated in lysis buffer containing SDS and proteinase K. Ombrouck et al. (1997) recommended simple boiling of formalin-fixed faeces at 100°C and found that as few as 10 spores in a specimen could be detected.

PCR has been compared with standard staining techniques in several surveys. David et al. (1996) detected microsporidia (E. bieneusi or E. intestinalis) in 26/28 (93%) of intestinal biopsies from patients with proven micro-sporidiosis. Coyle et al. (1996) used PCR amplification with species-specific primers on intestinal biopsies for detection of E. bieneusi or E. intestinalis. They found that 25/68 patients with diarrhoea and 1/43 patients without diarrhoea were positive for E. bieneusi. Only 24 of these were positive by electron microscopy. Also E. intestinalis was detected in five out of the 68 patients with diarrhoea and none of the patients without diarrhoea, in accord with the TEM studies. Confirmation of the positive results was obtained by specific oligonucleotide probes on Southern blots. Franzen et al. (1996c), using PCR and Southern blots with E. bieneusi- and E. intestinalis-specific probes, detected five E. bieneusi, five E. intestinalis and five dual infections in 15 patients. The same technique was used to demonstrate the presence of E. intestinalis in stool samples, duodenal and bile juice, duodenal biopsies, urine, sputum, bronchiolar lavage and blood of one patient (Franzen et al., 1996b) and has also provided evidence for latent infection of E. intestinalis (Franzen et al., 1996a). The presence of E. intestinalis in blood is of special interest because it suggests that blood cells are used to transport the infection from the intestinal wall to the deeper viscera.

Amplification by PCR with non-specific primers, followed by restriction digests, have given good results with species identification. Raynaud et al. (1998) used Hinfl for identification of E. intestinalis and were the first to identify this species in immunocompetent patients with diarrhoea. This restriction enzyme was also useful in differentiating the three Encephalitozoon spp. and E. bieneusi (Delbac and Vivares, 1997). Didier et al. (1996a) used Fokl to identify E. hellem from a patient with conjunctival and renal infections. Other examples of progress in the use of PCR for microsporidian infections are: (a) specific amplification of part of the ITS region of E. bieneusi (Velasquez etal., 1996);(b)useofselec-ted primers which amplified all Encephalitozoon spp., E. bieneusi and V. corneae but which gave amplification products of different sizes according to species (Kock et al., 1997); (c) combination of PCR and RFLP to differentiate E. bieneusi, E. hellem, E. intestinalis and E. cuniculi from cultures (Katzwinkel-Wladarsch et al., 1997); (d) use of species-specific primers for the same range of species (del Aguila et al., 1997); and (e) use of E. intestinalis-specific primers to confirm identification of E. intestinalis infections in animals that may be a source of infection to man (Bornay-Llinares et al., 1998).

0 0

Post a comment