Journal of Infection (1993) 27, 229-236

REVIEW ARTICLE

H u m a n m i c r o s p o r i d i o s i s

Alan Curry and Elizabeth U. Canning

Public Health Laboratory, Withington Hospital, Manchester and Imperial College, London, U.K.

Accepted for publication I2 August I993

Since the advent of HIV infection, with its profound and progressive effect on the cellular immune system, a new group of human opportunistic pathogens has come into prominence, namely the microsporidia. These unicellular obligate intracellular parasites are transmitted by highly resistant spores. The spores contain the infective sporoplasm and a coiled tube (polar tube). On entering a suitable host, the polar tube is everted, the sporoplasm migrates through it and is injected into a host cell, primarily one in the gastrointestinal or respiratory tract. Once within the host cell, replication takes place (merogony) before commitment to spore production (sporogony). Dissemi- nation to other tissues may occur but the mechanism for this is not fully understood. Although eukaryotic, microsporidia are devoid of mitochondria, possess ribosomes similar to those of prokaryotes and are probably of ancient origin. Although they are ubiquitous as parasites of the lower orders of the animal kingdom (invertebrates) and f ish, 1'2 they were largely unknown as causes of human disease before HIV emerged.

A previous editorial on human microsporidioses, published in this journal, 3 outlined the situation as it was then known, but knowledge of this newly recognised group of human pathogens has developed rapidly in the last few years and a further review is now warranted.

Before the advent of HIV infection and AID S, few cases involving infection with microsporidia had been recorded. Together with recent cases in immunocompetent people, they included ocular, ~-v neurological, s, 9 muscular10 and disseminated n infections.

It is, however, in the late stages of HIV infection, when the CD4 T- lymphocyte count falls below ioo × i06/1 that microsporidia are a serious threat. 12 To date, five genera have been recognised in human beings. These are Encephalitozoon, Enterocytozoon, Nosema, Pleistophora and Septata. Nosema has not, as yet, been found in patients with AIDS but three species, Enterocytozoon bieneusi, Encephalitozoon hellem and Septata intestinalis were discovered because of the fulminant nature of the infections in such patients. Enterocytozoon bieneusi has since been reported in 1% children with diarrhoea in Niger who did not have AIDS but their HIV status was not established, t8 More recently, it has been detected in an HIV-negative man with severe diarrhoea. 14 Little or nothing is known about the epidemiology of these parasites but the discovery of an infection of E. bieneusi in an immuno-

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© I993 The British Society for the Study of Infection

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23 ° A. CURRY AND E. U. CANNING

competent man suggests that this species, at least, may be a more common human pathogen.

In an experimental investigation of the route and progression of infection with Encephalitozoon cuniculi in adult rabbits, 15 it was found that administrat ion of spores either orally or intratracheally induced serum antibodies within 3 weeks and excretion of spores in urine by 6 weeks. Similar information is not available for human microsporidial infections al though antibodies were detected in an HIV-posi t ive patient 2 years before micro- sporidia were detected in his ocular and nasal epithelia by retrospective examination of serum samples. 16

I f animals are hosts to any of these microsporidian species, the relationship has yet to be established. Pets, particularly dogs, may be implicated in some infections. 17 Encephalitozoon cuniculi is commonly found in animals 2 and antibodies to this organism have been found in surveys of the human population. 9,~s The faecal-oral route of infection (enteric infection), in- oculation of spores into abrasions (ocular infection) or inhalation of spores may all play a part in the infective process. However, the recent identification of E. hellem, ~9 which is morphologically similar to E. cuniculi, has made it difficult to establish whether some hum a n infections are due to E. cunicuH and has thus confused knowledge of a possible zoonotic relationship with dogs. Perhaps many of the microsporidial infections in A I D S require human to human spread.

T h e most common microsporidial infections in patients with A I D S are due to infections by E. bieneusi, E. hellem and S. intestinalis.

Prevalence figures for intestinal microsporidioses in patients with A I D S reveal the scale of human infection by this group of parasites. 2° Microsporidian infections of the intestine have been detected in Australia, the U.S.A., the Netherlands and England in I 5 - 3 0 % of patients with A I D S having diarrhoea. 2° In Africa, prevalence is lower (ref 2I and Hollister and Canning, unpubl ished observations).

Enterocytozoon bieneusi is largely restricted to the enterocytes of the small intestine 22'~3 where it causes villus atrophy and malabsorption. 24 Even so, there are a few reports of bile duct and gall bladder involvement associated with sclerosing cholangitis 25-~7 and of infection in the nasal sinuses 2s and bronchial epithelium. 29 These sites suggest that the routes of infection are by ingestion and /o r inhalation of spores.

Developmental stages of E. bieneusi in the small intestine lie in direct contact with host cell cytoplasm and seem always to occupy a position between the nucleus, which is sometimes indented by the parasite, and the microvillous border of the cell. Meronts become multinucleate and the elongated nuclei are often aligned with electron-lucent 'clefts ' in the cytoplasm of the parasite. These 'clefts ' probably represent expanded cisternae of endoplasmic ret iculum, and may be seen by light microscopy, so aiding diagnosis. Dur ing sporogony, which is polysporoblastic, electron-dense discs are secreted within the multinucleate sporogonial p lasmodium which later fuse to form the polar tubes. Around each nucleus is assembled a set of typical spore organelles, while division of the p lasmodium produces a variable number of sporoblasts. Secretion of both the exospore as the p lasmodium divides and the endospore

H u m a n microsporidiosis 231

layer (subsequently) completes the maturat ion of the spore. T h e spores of this organism are slightly larger than bacteria, i.e. r" 5 x o'9 #m.

A second enteric microsporidial parasite has been found more recently. Originally described as resembling E. cuniculi, 3°" 31 it has now been assigned to a new genus and named Septata intestinalis. 32 It also infects the intestinal enterocytes but disseminates more widely than E. bieneusi and has been found in the colon, kidney and liver. Unlike E. bieneusi, intracellular development takes place within a parasi tophorous vacuole bounded by a membrane. This has a distinctive scalloped margin and sporogonic stages are separated within the vacuole by septa composed of finely granular material. Enterocytozoon bieneusi and S. intestinalis have been found together in some patients with AIDS.30,33.34

Of the non-enter ic sites of microsporidial infection, ocular epithelia (corneal and conjunctival) appear to be the most common, 7'19'35'z6 with occasional involvement of the nasal sinus epithel ium or the epithelium of nasal polyps. 37 Presumably, infection can spread from the corneal epithelium through the lacrimal canaliculi and nasolacrimal ducts that drain secretions f rom the eyes into the nasal sinuses. Ocular infection, however, may possibly be acquired by reverse passage from a respiratory site since infection has been found th roughout the tracheo-bronchial epithelium. 38 Originally described in the U.S.A. as coming from the ocular epithelia of three patients with AIDS, the species involved was morphologically similar to E. cuniculi. Once established in cell culture and compared with a well characterised E. cuniculi isolate by S D S - P A G E analysis and Western blotting, however, the profiles obtained showed that the organism was distinct from E. cuniculi and was therefore established as a new species, E. hellem. Subsequent ultrastructural observ- ations on both E. cuniculi 39 and E. hellem 16 have shown morphological differences. In particular, the exospore coat of E. cuniculi is laid down on the sporont in narrow strips, whereas that of E. hellem is laid down in sheets. The spread of infection in both the corneal and sinus epithelia by E. hellem may be explained by the germination of mature spores while still within their parasi tophorous vacuoles, thus initiating and perpetuat ing infection in adjacent epithelial cells. 7'a°

Peritonitis ~1 and hepatitis 42 caused by uncharacterised E. cuniculi-like species, and myositis 43'4a caused by a species of Pleistophora have also been reported in patients with AIDS.

Unti l about r99o, diagnosis of enteric microsporidia required both light microscopic and electron microscopic examination of small intestinal biopsy sections 45 or touch preparations of biopsies. 4~ Electron microscopy of infected tissues may still be required for the definitive identification of the micro- sporidial species involved. T h e size of the spore, number of coils of the polar tube, number of nuclei (e.g. whether diplokaryotic or not) and whether or not development takes place in a vacuole are all important taxonomic features. A simpler, non-invasive, technique, however, is desirable for detecting infections. Several groups of workers have therefore developed microsporidial- spore-staining methods for examination of faecal samples. 3s,47,~8 Micro- sporidial spores are of the same order of size as bacteria so that staining has to differentiate between the spores and faecal bacteria. T h e methods must also

1 3 - 2

232 A. CURRY AND E. U. CANNING

allow the microscopist to differentiate between microsporidial spores and those of yeasts and fungi as well as the oocysts of coccidian protozoal parasites. T o achieve this end, use has been made of the staining properties of the microsporidial spore wall. T h e outer layer of the spore wall (exospore) is proteinaceous and the inner layer (endospore) is chitinous. Tr ichrome 3s and Giemsa ~7 staining as well as the use of fluorescent dyes 48 have been advocated for staining spores. Uvitex 2B (Ciba-Geigy) 48 and Calcofluor (Sigma) 49 are currently the fluorescent stains of choice. These are quick to use, but examination requires a fluorescence microscope with epi-i l lumination and fitted with a 35o-38o n m excitation filter and a high magnification objective lens (x Ioo oil fluorescence objective). Spores are identified by their size, shape (ovoid or ellipsoid) and fluorescent staining properties (brilliant blue- white). Since fungal spores also stain with these fluorescent brighteners, the microsporidian nature of putative positive samples identified initially should be confirmed by staining with Giemsa or Tr ichrome or by electron microscopy. Experience is required in order to ensure correct results. Th e tr ichrome stain developed by Weber et al28 stains the spores a characteristic pinkish red but the technique is lengthy. Al though satisfactory when spores are plentiful, spores are difficult to detect if sparsely distr ibuted in the sample.

Electron microscopic examination of thin-sectioned faecal samples also allows definitive identification of microsporidial spores but is expensive, t ime consuming and cannot deal with large numbers of specimens. I t may be less sensitive than light microscopy methods because of the small sample size that can be examined. Even so, electron microscopy remains useful for diagnosis whenever biopsy material is available. Indeed, as with other infections associated with A I D S such as Pneumocystis carinii and Gryptosporidium parvum, for which previously unknown sites of infection have been detected, 12 electron microscopy of all biopsies may reveal further information on the range of tissues which may be infected by microsporidia.

Disinfection of surfaces contaminated with microsporidia has received little attention. A rabbit isolate of E. cuniculi cultured in a rabbit choroid plexus (RCP) cell line has been used for in vitro experiments in testing the factors influencing the infectivity and replication of this organism in RCP cells2 ° Aliquots of organisms used for testing contained variable proport ions of spores, sporoblasts, sporonts and schizonts, al though a typical pool contained about 8o ~o spores, as judged by electron microscopy. Encephalitozoon cuniculi was not affected by penicillin, s t reptomycin or gentamicin nor was it affected by sonication, freezing and thawing or distilled water. Organisms survived 6o but not I2o min at 56 °C. They were killed after IO min of autoclaving at I2o °C or exposure to 2 % (v/v) lysol, IO % (v/v) formalin and 7o% (v/v) ethyl alcohol for IO min. It is not known whether all microsporidia are affected to the same degree as E. cuniculi by the physical conditions and chemicals used in the study.

Microsporidial infections are difficult to treat because of their intracellular habitat and the resistant nature of the spores. Enteric microsporidial infections have been treated with varying degrees of success with several drugs. The most promising anti-microsporidial drug to date is albendazole21 It does not eliminate infection with E. bieneusi but in many cases alleviates the diarrhoea.

Human microsporidiosis 233

The species which develop in parasitophorous vacuoles appear to be more susceptible to albendazole, as indicated by the disappearance of S. intestinalis from the intestine 52 and regression of hypertrophic nasal epithelium in a patient with sinusitis due to Encephalitozoon sp. 37 (later identified as E. hellem). 16 The direct effect of albendazole on microsporidia has been examined by addition of the drug to cell cultures infected with Encephalitozoon cuniculi.39 Many parasites became grossly enlarged, devoid of nuclei and traversed by abnormal bundles of tubules. Since albendazole prevents the polymerisation of microtubules, which are known to occur only within the intranuclear spindles of microsporidia, cell growth continues in the absence of nuclear division.

Treatment with itraconazole 53 was responsible, at least in part, for the resolution of a corneal infection with Encephalitozoon sp. (later identified as E. hellem) ~9 and a similar corneal infection, ultimately identified as being caused by E. hellem, 16 disappeared during treatment with broline (propamidine isethionate). The punctate keratopathy, however, returned when treatment was discontinued25

Successful culture of microsporidia in cell lines such as M D C K cells TM ~9 or infection of laboratory animals are important developments which enable new drugs or drug regimens to be tested before their clinical application.

Molecular techniques are beginning to be applied to microsporidial problems, particularly for use in the differentiation of species and their detection in clinical samples. Random amplified polymorphic D N A (RAPD) obtained by means of the polymerase chain reaction (PCR), as well as restriction enzyme digests (RFLP), have shown clear differences among isolates. 2°'5~'55 Ribosomal D N A gene sequences are available for E. cuniculi and E. hellem. 56

As a result of the fulminant nature of microsporidian infections in patients with AIDS, several previously unknown microsporidia have been found. It is not known whether these infections may be inapparent or clinically transient in immunocompetent persons since only a few infections have been detected in them. Increased awareness of these organisms, however, may reveal previously unrecognised disease associations in HIV-negative persons. New, less invasive and more convenient methods of detection and relatively effective drugs are important new developments in this field. Even so, ignorance of how these parasites are transmitted and whether animal or environmental sources play a part in their dissemination remain to be elucidated. Clearly, much basic work remains to be done in order to answer the questions of how this previously obscure group of organisms, in terms of human disease, has established itself as significantly pathogenic in the range of infections that are now associated with HIV infection and AIDS.

(Elizabeth Canning would like to acknowledge the support of the Wellcome Trust.)

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