Myositis due to the microsporidian Anncaliia (Brachiola) algerae in a lung transplant recipient.

Microsporidia are obligate intracellular parasites, more closely related to fungi than protozoa on molecular phylogenetic analysis, and are known to be a rare cause of opportunistic infection in immune compromised patients including human immunodeficiency virus-positive patients and solid organ transplant recipients. We report the first case to our knowledge of microsporidial myositis in a lung transplant recipient. He was 49 years old and had received a lung transplant in 2000 for cystic fibrosis. He presented in 2009 with fevers, chronic diarrhea, myalgia, and pancytopenia, and developed progressive weakness and neurological symptoms before his death 35 days after hospital admission. Multiple investigations, including stool culture, rectal biopsy, colonoscopy, cerebrospinal fluid examination, bone marrow biopsy, lung biopsy, and bronchoalveolar lavage, failed to reveal a definite cause for the patient's deterioration. The diagnosis of microsporidial infection was made on post-mortem light microscopic examination of tissue sections of the tongue and deltoid muscle. Light microscopy diagnosed a microsporidial myositis, confirmed by transmission electron microscopy, which suggested that the organism was Brachiola species. The identity of the organism was confirmed by polymerase chain reaction as Brachiola algerae (recently renamed Anncaliia algerae). The case highlights the need to consider protozoal organisms in the differential diagnosis of myalgia and multisystemic infections in immune compromised patients.

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria.

Microsporidia are small (1-20 micron) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2,3], and are reported to lack mitochondria [2,4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2,4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5-9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.

Reversible renal failure caused by a microsporidian infection.

OBJECTIVE: To report a case of renal failure associated with microsporidian infection in an HIV-seropositive patient. DESIGN: Case report. SETTING: Chelsea and Westminster Hospital, London, England, UK. PATIENT: An HIV-seropositive patient presented febrile with abdominal pain who developed renal failure. Renal biopsy and urinalysis showed infection with a microsporidian of the genus Encephalitozoon. INTERVENTION: Treatment with albendazole (400 mg) twice daily was associated with disappearance of infection from the urine, clinical improvement and return of renal function virtually to normal. CONCLUSION: HIV-seropositive individuals with renal failure should have urine screened for microsporidia. The administration of albendazole in such cases may reverse renal failure.

Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique.

AIMS: To assess the value of a new rapid fluorescence method for the diagnosis of microsporidiosis in HIV seropositive patients. METHODS: Microsporidian spores in stools were demonstrated by using the fluorochrome stain Uvitex 2B. The new technique was evaluated in three groups of HIV seropositive patients with diarrhoea. Group 1: 19 patients with biopsy confirmed E bieneusi infection (186 stool samples); group 2: 143 consecutive patients from whom faeces were submitted for routine investigation of diarrhoea (318 samples); group 3: 16 patients with small intestinal biopsy specimens negative for microsporidia (55 samples). The new method was used to monitor spore shedding during experimental treatment with paromomycin and albendazole in four patients. RESULTS: Brightly fluorescent spores were detected in all stool samples of patients in group 1. In group 2 16 (11%) patients had spores in their stool samples. E bieneusi was found in 11 patients; in the other five another genus of microsporidia, Encephalitozoon, was recognised. Encephalitozoon spores were also found in the urine of three of these patients and in the maxillary sinus aspirate of two of them, suggesting disseminated infection. The results were confirmed by electron microscopic examination. In group 3 negative biopsy specimens were confirmed by negative stool samples in all cases. Treatment with albendazole and paromomycin did not affect the spore shedding in three patients with E bieneusi infection. By contrast, in a patient with Encephalitozoon sp infection albendazole treatment resulted in clinical improvement together with complete cessation of spore excretion in the stool. CONCLUSION: The Uvitex 2B fluorescence method combines speed, sensitivity, and specificity for the diagnosis and treatment evaluation of intestinal and disseminated microsporidiosis.

Protozoan infections.

Protozoan infections, against which immunity is predominantly T cell mediated, are likely to be more severe in patients with the acquired immune deficiency syndrome (AIDS) than in immunocompetent hosts. Leishmaniasis, toxoplasmosis and cryptosporidiosis are examples, the last two being particularly common in AIDS patients. Cerebral toxoplasmosis almost always results from recrudescence of latent infections acquired earlier in life. Depletion of T-helper (CD4+) lymphocytes enables bradyzoites to survive if released from cysts in the brain of patients. In the absence of immune pressure bradyzoites revert to tachyzoites and multiply to cause a rapidly developing, necrotizing encephalitis which needs immediate treatment. AIDS patients, especially those who are negative for antibodies to Toxoplasma, should avoid cats, the source of oocysts, and undercooked meat which may contain tissue cysts, as primary infections may become systemic. Cryptosporidium infections are more likely to be primary infections. Sources of infection are other people, farm animals and pets and there is a significant risk from contaminated domestic water supplies. As infections cause a life-threatening secretory diarrhoea in AIDS patients, for which there is not satisfactory treatment at present, such patients should take steps to minimize the risk of infection.

Enterocytozoon bieneusi (Microspora): prevalence and pathogenicity in AIDS patients.

Microsporidia are unicellular organisms, which lack mitochondria. They have prokaryotic-like ribosomes and characteristic spores containing an extrusible polar tube which serves as a passage for inoculation of the infectious agent (sporoplasm) into host cells. Clinically apparent infections in man appear to be limited to immunoprivileged sites or immunocompromised patients. One species, Encephalitozoon cuniculi, has been reported several times in patients with neurological disorders and once causing a fatal hepatitis in an AIDS patient. The most recently discovered species, Enterocytozoon bieneusi, is known only from the small intestinal enterocytes of patients with the acquired immunodeficiency syndrome, and is easily differentiated from other microsporidia by the precocious development of spore organelles in the sporont and by the poor development of the endospore layer of the spore wall. Although only about 40 cases have been reported, circumstantial evidence suggests that E. bieneusi may be the cause of a severe watery diarrhoea, which responds only temporarily to treatment with metronidazole.

Observations on early and late post-sporozoite tissue stages in primate malaria. V. The effect of pyrimethamine and proguanil upon tissue hypnozoites and schizonts of Plasmodium cynomolgi bastianellii.

Two rhesus monkeys were each infected with 2.1 x 10(6) sporozoites of Plasmodium cynomolgi bastianellii; one was treated with 1.0 mg of pyrimethamine base per kg body weight for 5 d after sporozoite inoculation. A further 2 monkeys were each infected with 9.75 x 10(6) sporozoites of the same parasite; one was treated with 10 mg of proguanil per kg body weight for 4 out of 5 d after inoculation. The treated monkeys showed a delayed primary parasitaemia and relapses. In sections of liver biopsies taken 7.5 d after sporozoite inoculation, all monkeys showed numerous hypnozoites. However, there were no full grown schizonts and only rare retarded schizonts in the treated monkeys, in contrast to the untreated monkeys which had many mature or nearly mature schizonts. Later biopsies confirmed the continued presence of hypnozoites in all monkeys.

Mosquito (Diptera: Culicidae) host compatibility and vector competency for the human myositic parasite Trachipleistophora hominis (Phylum Microspora).

Microsporidian spores of Trachipleistophora hominis Hollister, isolated from a human, readily infected larval stages of both Anopheles quadrimaculatus Say sensu lato and Culex quinque-fasciatus Say. Mosquito infections with T. hominis were located, primarily, in abdominal muscles in segment numbers 4 through 6; other spores were found in the hemocoel and proboscis. Nearly 50% of the infected mosquito larvae survived to the adult stage. Spores recovered from adult mosquitoes were inoculated into mice and resulted in significant muscle infection at the site of injection. Preliminary observations also showed that T. hominis spores can be passively transferred from infected mosquitoes to a sugar water substrate.

A light- and electron-microscopic study of Amblyospora egypti n.sp. infecting Culex pipiens in Egypt.

A microsporidium was found infecting the fat body of larvae and adults of both sexes of Culex pipiens in Egypt. Developmental stages were found in larvae but only masses of spores were present in adults. The infection was easily visible in live mosquito larvae, as one or two blocks of opaque whitish fat body visible through the cuticle in each segment. Meronts were rounded cells, which were bounded by an unthickened unit membrane and divided by binary fission (rarely into four). At the onset of sporogony the surface membrane was thickened by electron dense deposits. This coat was sloughed off to form the sporophorous vesicle, the separation from the sporont surface being effected by the secretion of metabolic products into the sporophorous vesicle cavity. Division within the vesicle gave rise to eight uninucleate sporoblasts, then uninucleate spores. Spores exhibited an exospore of two membrane-like layers and a subtending layer of moderate electron density, appearing as eight to ten strata separated by fine lines and permeated by amorphous material, and an electron lucent endospore. The polar tube was anisofilar with 3-4 broad coils and 4-3 narrow coils. The development and spore structure were in accord with the genus Amblyospora Hazard and Oldacre, 1975 and, on the basis of spore size and number of coils of the polar tube, it is considered to be a new species, Amblyospora egypti n.sp.

Ultrastructure of Encephalitozoon sp. infecting the conjunctival, corneal and nasal epithelia of a patient with AIDS.

The ultrastructure of a species of Encephalitozoon infecting the ocular and nasal epithelia of a patient with AIDS is described. Development occurred in parasitophorous vacuoles, all stages had isolated nuclei and sporogony was disporoblastic. Spores had 5-8 coils of the polar tube, about 40 closely packed membranes forming the polaroplast, a single nucleus, abundant ribosomes and a posterior vacuole. Most of the mature spores in the conjunctival epithelium and many of those in the nasal epithelium had germinated within their parasitophorous vacuoles. The wall of the polar tube was made up of 2 membranes and eversion took place by an inner tube sliding through an outer tube. Polaroplast membranes became depleted in the spore at the onset of germination and may have contributed to polar tube formation. The sporoplasm, identified by its ribosomes, passed through the polar tube but was not enveloped by a membrane while doing so. Putative sporoplasms injected into host cell cytoplasm had acquired a surface membrane, possibly from the polaroplast, and an additional membrane, which became the boundary of the parasitophorous vacuole. As the emerging tube passed through cell boundaries it carried the plasma membranes forward with it and one of these could have been the origin of the parasitophorous vacuolar membrane. Early vacuoles, containing meronts and pre-spore stages, had a reticular matrix but vacuoles containing spores had a disrupted membrane and exhibited incursion of host cell organelles.

Nuclear division and chromosome cycle in microsporidia.

Nuclear division and chromosome cycle of microsporidia are reviewed in the light of recent speculation that the group diverged as an early branch in the eukaryotic line of descent. Microsporidia are primitive eukaryotes with simple cytoplasmic organisation, lacking mitochondria, peroxisomes and a classical Golgi apparatus. The ribosomes resemble prokaryotic ribosomes in size and in having sequences complementary to the eukaryotic 5.8S rRNA contained within their large (23S) ribosomal subunit, rather than a separate 5.8S molecule. Nuclei may be isolated or closely appressed as a diplokaryotic pair which divide synchronously. Mitosis is intranuclear: there are no centrioles but spindle termini are electron dense plaques in pores in the nuclear envelope. Some genera have isolated nuclei throughout the life cycle, while others have diplokaryotic nuclei throughout: meiosis is not known in either of these categories of genera. In contrast, certain polymorphic species, which are transmitted horizontally between copepods and mosquitoes and vertically between generations of mosquitoes, alternate between stages with isolated nuclei and stages with diplokaryotic nuclei. Haploid spores in copepods are infective to mosquito larvae, in which gametogenesis and plasmogamy occur to give diplokaryotic (diploid) stages. Stages remain diplokaryotic through transovarial transmission to the next generation, when an unusual form of meiosis is initiated in both nuclei of the diplokaryon (as indicated by synaptonemal complexes), and mingling of chromosomes occurs when the two nuclei fuse during pachytene.

Induction of protective immunity to Babesia divergens in Mongolian gerbils, Meriones unguiculatus, using culture-derived immunogens.

Immunogens derived from microaerophilous stationary phase (MASP) cultures of Babesia divergens grown in bovine erythrocytes were used to inoculate the laboratory host of B. divergens, the Mongolian gerbil, Meriones unguiculatus. Animals inoculated subcutaneously twice with preparations of freeze-thawed merozoites in complete Freund's adjuvant were fully protected against homologous challenge, as were gerbils immunised with a non-viable preparation of parasite-enriched lysed infected bovine erythrocytes. Animals which had been infected with small numbers of parasitised erythrocytes from cultures cooled to 4 degrees C, allowed to recover, then challenged, also survived. All three groups had high antibody titres which dropped immediately after challenge and then rose again. Gerbils given culture supernatants containing soluble merozoite protein coat antigens were partially protected only after receiving a third inoculation. Non-immunised animals all died 4 days after challenge.

Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis.

The production of monoclonal antibodies to the bloodstages of the haemoprotozoan parasites Babesia caballi and Babesia equi and the characterization of their corresponding antigens are described. Species specific and immunogenic proteins of both parasites were identified using SDS-PAGE, Western blotting and ELISA. These proteins were then electroeluted from SDS-PAGE gels and used to immunize BALB/c mice for hybridoma production. One monoclonal antibody (Mab), designated BC5.37.70.27 (BC5), recognized a 70 kDa protein of B. caballi as demonstrated by Western blotting under reducing conditions. Another Mab, BE1.24/2.95 (BEI), recognized a 34 kDa protein of B. equi. Both Mabs reacted specifically in indirect ELISA when isolated whole merozoites were used as antigen. Preliminary studies using the two Mabs in a competitive ELISA (cELISA) suggest that the cELISA for the detection of B. caballi infection is more sensitive than the commonly used complement fixation test but that refinement is necessary for the B. equi system.

Induction of proliferative kidney disease (PKD) in rainbow trout Oncorhynchus mykiss via the bryozoan Fredericella sultana infected with Tetracapsula bryosalmonae.

Proliferative kidney disease (PKD) is a serious infection of wild and farmed salmonids, affecting mainly the kidney and spleen but becoming systemic in most susceptible fish hosts. This report deals with the transmission of Tetracapsula bryosalmonae Canning, Curry, Feist, Longshaw & Okamura 1999 from naturally infected bryozoans Fredericella sultana Blumenbach 1779 to naive rainbow trout Oncorhynchus mykiss Walbaum 1792, thereby confirming the recent conclusion based on partial 18S rDNA sequence data that bryozoans are hosts of the myxozoan parasite T. bryosalmonae (formerly PKX organism) that causes the disease. Parasite transmission using T. bryosalmonae spores was successful by short-term exposure to disrupted bryozoans known to contain T. bryosalmonae spores and T bryosalmonae sacs liberated from the bryozoans, and by long-term cohabitation with infected bryozoan colonies. Infection was confirmed by examination of kidney imprints, detection of the parasite in stained tissue sections, PCR using T. bryosalmonae-specific primers, and comparison of amplified 18S rDNA sequences from the bryozoans and experimentally infected fish. Transmission was not apparent, nor was PKD induced, in fish challenged by intraperitoneal injection of spores isolated from F. sultana.

Ultrastructure of a microsporidian hyperparasite, Unikaryon legeri (Microsporida), of trematode larvae.

Unikaryon legeri (Dollfus, 1912) Canning and Nicholas, 1974, has been reexamined by electron microscopy from material collected in Portugal. It parasitises metacercariae of Meigymnophallus sp. in Cardium edule. A sporophorous vesicle forms around the sporonts, arising as a blister that separates from the electron-dense surface coat of the sporont. Sporogony is disporoblastic, giving rise to 2 spores that are retained in pairs within the sporophorous vesicle. Unikaryon piriformis, which is the type species of the genus and is also hyperparasitic in platyhelminth larvae, has not been examined by electron microscopy, and it is not known whether this species also produces sporophorous vesicles. If it does, then all that will be required is a simple addition of this character to the definition; if not, U. legeri will have to be transferred to a new genus and reclassified with other disporoblastic genera that sporulate in sporophorous vesicles.

Patterns of occurrence and 18S rDNA sequence variation of PKX (Tetracapsula bryosalmonae), the causative agent of salmonid proliferative kidney disease.

Recent progress in understanding the etiology of proliferative kidney disease (PKD) includes the identification of freshwater bryozoans as the natural hosts of the myxozoan parasite that causes the disease in salmonid fish and formal description of the parasite as Tetracapsula bryosalmonae. This paper presents data on patterns of occurrence of T. bryosalmonae and sequence variation among isolates. T. bryosalmonae infects bryozoans that range from primitive to more derived genera within the Phylactolaemata and that differ in growth form and habits. Infected bryozoans have been collected in diverse habitats including cold, clear streams and warm, eutrophic lakes. Temporal surveys reveal intra- and interannual variation in infection levels, and spatial variation in incidence of infection is implicit by the apparent absence of T. bryosalmonae from many bryozoan populations. The significance of minor variation in partial sequences of 18S rDNA requires further investigation. The information presented here provides the first significant insights into the ecology of T. bryosalmonae.

A strain of Babesia divergens, attenuated after long term culture.

The Weybridge strain of Babesia divergens became less virulent after 18 months in culture and was believed to be attenuated. Inoculations with the attenuated line using as many as 5 x 10(8) infected erythrocytes failed to raise a normal infection in Mongolian gerbils (Meriones unguiculatus) whereas, with the line that had been passaged from culture through gerbils at bimonthly intervals, inoculations of 10(7) infected erythrocytes gave rise to fatal infections. Gerbils were immunised with the attenuated line using doses ranging from 5 x 10(5) to 5 x 10(7) infected erythrocytes. Parasitaemias in the infected gerbils did not exceed 0.7 per cent after immunisation. All animals were protected when challenged with the virulent line four weeks later. Control animals died four days after challenge infection.

In vitro cultivation of the human microsporidium Vittaforma corneae: development and effect of albendazole.

When in vitro growth of Vittaforma corneae was tested using MDCK, MRC-5, XEN, L-929 and FHM cell lines, propagation occurred only in MDCK, MRC-5 and XEN cells. The intervals required for the various stages of the life cycle to develop were the same in all the cell lines tested. The MDCK cell line was selected to support the growth of V. corneae in vitro and provide the system for in vitro testing of drugs. The weekly output of V. corneae spores from the MDCK cell monolayer was monitored over a 61-week period during which there were fluctuations but no definite increase or decrease in output. Albendazole at 2.1 or 4.2 micrograms/ml in MEM was tested against V. corneae in MDCK cell monolayers and showed antimicrosporidial activity. The percentage of infected cells was reduced in the presence of the drug and there were ultrastructural abnormalities in all stages of the life cycle. The drug prevents parasite division.

Staining of microsporidian spores by optical brighteners with remarks on the use of brighteners for the diagnosis of AIDS associated human microsporidioses.

Conditions for the effective fluorescence labelling of microsporidian spores by optical brighteners, based on the presence of chitin in the spore wall, are described. Spores of Vairimorpha ephestiae, V. necatrix, V. plodiae, Nosema bombycis, N. apis, N. algerae, Encephalitozoon cuniculi and Enterocytozoon bieneusi were examined. The degree of binding of Calcofluor White M2R (CFW) to untreated spores depends on the conditions and time of storage and the degree of bacterial contamination of the spore sample. Unpurified spores, stored in water are unreliable as control material for the estimation of CFW labelling. However, spores subjected to alkaline treatment by NaOH before CFW application are visualized with ease under all experimental conditions by their bright, not quenching fluorescence in shortwave light (approximately 350 nm) in CFW dilutions of 10(-4) or even lower. Similar improvement in labelling is achieved by exposing spores to CFW dissolved in 0.5-1N NaOH. As well as Calcofluor White M2R other optical brighteners (e.g. Uvitex 2B, Ciba-Geigy or Rylux BA, Ostacolor) can be used for labelling of spores.

A species of Encephalitozoon isolated from an AIDS patient: criteria for species differentiation.

A microsporidium isolated in vitro from urine of an AIDS patient was identified as a species of Encephalitozoon. Investigation of the isolate by electron microscopy, amplification of DNA by RAPD PCR, protein analysis by SDS-PAGE and Western blotting and infectivity to athymic mice revealed that it differs from established species of the genus.