Elizabeth S. Didier

Microsporidiosis: current status

Purpose of reviewMicrosporidiosis is an emerging and opportunistic infection associated with a wide range of clinical syndromes in humans. This review highlights the research on microsporidiosis in humans during the previous 2 years. Recent findingsThe reduced and compact microsporidian genome has generated much interest for better understanding the evolution of these parasites, and comparative molecular phylogenetic studies continue to support a relationship between the microsporidia and fungi. Through increased awareness and improved diagnostics, microsporidiosis has been identified in a broader range of human populations that, in addition to persons with HIV infection, includes travelers, children, organ transplant recipients, and the elderly. SummaryEffective commercial therapies for Enterocytozoon bieneusi, the most common microsporidian species identified in humans, are still lacking, making the need to develop tissue culture and small animal models increasingly urgent. Environmental transport modeling and disinfection strategies are being addressed for improving water safety. Questions still exist about whether microsporidia infections remain persistent in asymptomatic immune-competent individuals, reactivate during conditions of immune compromise, or may be transmitted to others at risk, such as during pregnancy or through organ donation. Reliable serological diagnostic methods are needed to supplement polymerase chain reaction or histochemistry when spore shedding may be sporadic.

Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms.

ABSTRACT. Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans (Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi. Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis. Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many of the important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple origins.

Ribosomal Dna Sequences of Encephalitozoon Hellem and Encephalitozoon Cuniculi: Species Identification and Phylogenetic Construction

ABSTRACT. A segment of ribosomal DNA, about 1,350 base pairs long, was amplified from the microsporidian species Encephalitozoon hellem, isolated from AIDS patients, and Encephalitozoon cuniculi. the amplified DNA segment extends from position 530 in the small ribosomal RNA subunit to position 580 in the large ribosomal RNA subunit. A comparison of sequence data from this region for Encephalitozoon hellem and Encephalitozoon cuniculi shows relatively high sequence similarity, supporting the placement of these two organisms in the same genus. At the same time, sequence differences between the two organisms confirm that they are not the same species. Three separate isolates of E. hellem were sequenced in the highly variable intervening spacer region. the sequence was identical for all three isolates. Within the amplified DNA segment, regions were sequenced which yield highly variable, moderately variable and highly conserved sequence information, appropriate for comparison with other species in the phylum Microspora at all taxonomic levels. We suggest that sequence data from these regions be included in future species descriptions for the purposes of species identification and phylogenetic analysis. Restriction digests of the amplified region are presented and give a rapid method for distinguishing between the two Encephalitozoon species.

Microsporidiosis: Not just in AIDS patients

Purpose of review Microsporidia have emerged as causes of opportunistic infections associated with diarrhea and wasting in AIDS patients. This review describes recent reports of microsporidiosis in HIV-infected individuals and the growing awareness of microsporidiosis in non-HIV-infected populations. Recent findings Microsporidia were only rarely recognized as causes of disease in humans until the AIDS pandemic. Implementation of combination antiretroviral therapy (cART) to curtail HIV replication and restore immune status drastically reduced the occurrence of opportunistic infections, including those due to microsporidia, in HIV-infected individuals. In developing countries where cART is not always accessible, microsporidiosis continues to be problematic. Improvement of diagnostic methods over the previous 25 years led to identification of several new species of microsporidia, many of which disseminate from enteric to systemic sites of infection and contribute to some unexpected lesions. Among non-HIV-infected but immune-suppressed individuals, microsporidia have infected organ transplant recipients, children, the elderly, and patients with malignant disease and diabetes. In otherwise healthy immune-competent HIV seronegative populations, self-limiting diarrhea occurred in travelers and as a result of a foodborne outbreak associated with contaminated cucumbers. Keratitis due to microsporidiosis has become problematic and a recent longitudinal evaluation demonstrated that non-HIV-infected individuals seropositive for microsporidia who had no clinical signs continued to intermittently shed organisms in feces and urine. Summary Greater awareness and implementation of better diagnostic methods are demonstrating that microsporidia contribute to a wide range of clinical syndromes in HIV-infected and non-HIV-infected people. As such, microsporidia should be considered in differential diagnoses if no other cause can be defined.

Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites.

Microsporidiosis in mammals

Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.

Genetic and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis.

The relationships between the Encephalitozoon-like Septata intestinalis and other microsporidia that occur in humans; notably Encephalitozoon cuniculi and Encephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects of S. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of the S. intestinalis rrs gene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplified rrs genes and establishment of the degree of sequence identity between rrs genes of various microsporidian species. The unique sequence of rrs of S. intestinalis as well as the distinct RFLP and SDS-PAGE profiles indicate that S. intestinalis is clearly different from other human microsporidian species. However, its rrs gene shared about 90% sequence identity with rrs of both Encephalitozoon spp., E. cuniculi and E. hellem. This is remarkably higher than the about 70% identity observed between rrs of microsporidian species which belong to different genera and thus suggests that S. intestinalis should be regarded as a species of the genus Encephalitozoon.(ABSTRACT TRUNCATED AT 250 WORDS)

Biology of microsporidian species infecting mammals.

Microsporidia (phylum Microspora) are obligate intracellular protozoan parasites that infect a wide range of vertebrate and invertebrate hosts. Over 1000 species have been classified into approximately 100 genera, and at least 13 species have been reported to infect mammals. Phylogenetically, the microsporidia are early eukaryotes because they have a true nucleus, possess prokaryote-like ribosomes, and lack mitochondria. The species that infect mammals are relatively small, measuring 2.0-7.0 microns long and 1.5-5.0 microns wide. The mature organism is the spore, which is enclosed by a chitinous coat, making it relatively resistant to the environment. Infections often occur by fecal-oral or urinary-oral transmission, although vertical transmission is quite common in the carnivores. Host cells become infected through a process of germination in which the spore propels its contents through the everting and unwinding polar filament into the host cell. The polar filament is unique to the microsporidia. With a few exceptions, microsporidiosis is typically chronic and subclinical in immunologically competent hosts. Young carnivores infected with microsporidia, however, develop severe and sometimes lethal renal disease, and immunodeficient laboratory animals (e.g. athymic and SCID mice) develop ascites and die from microsporidiosis. This review describes the morphology, life cycle, taxonomy, and host-parasite relationships of the species of microsporidia that infect mammals.

Characterization of Encephalitozoon (Septata) intestinalis isolates cultured from nasal mucosa and bronchoalveolar lavage fluids of two AIDS patients

ABSTRACT. Microsporidia are obligate intracellular protozoan parasites that can cause opportunistic infections in AIDS patients. Species from five genera of microsporidia are presently known to infect man. One species, Septata intestinalis originally was detected in stool specimens of individuals with chronic diarrhea and subsequently was found to disseminate to the kidneys, lungs, and nasal sinuses. This organism has since been reclassified as Encephalitozoon and in this study, we report the culture of Encephalitozoon intestinalis from a bronchoalveolar lavage specimen and a nasal mucus aspirate of two AIDS patients living in the USA. The bronchoalveolar and nasal microsporidian isolates grew in several continuous cell lines including RK‐13, MDCK, HT‐29, Caco‐2, Vero, and 1047. Transmission electron microscopy of the clinical and cell culture specimens revealed that the new isolates appeared to be E. intestinalis based on morphology and growth of organisms in septated membrane‐bound parasitophorous vacuoles. The new E. intestinalis isolates were characterized and compared with the first isolated E. intestinalis that was cultured from stool to confirm their identity and to determine if there existed any minor differences, as seen in the closely related Encephalitozoon cuniculi strains. By the methods of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis staining for proteins and carbohydrates, Western blot immunodetection, and polymerase chain reaction‐based methods with restriction endonuclease digestion, double‐stranded DNA heteroduplex mobility shift analysis, and DNA sequencing of the ribosomal DNA intergenic spacer region, the new isolates were identical to each other and to the reference isolate of E. intestinalis. In addition, with any of these methods, the E. intestinalis organisms could be distinguished from the three E. cuniculi strains, Encephalitozoon hellem, and Vittaforma corneae, which is important for diagnostics, therapeutic strategies, and epidemiology.

Encephalitozoon hellem in Budgerigars (Melopsittacus undulatus)

Microsporidiosis with concurrent megabacteriosis in budgerigar (Melopsittacus undulatus) chicks contributed to significant economic loss in a commercial pet bird aviary in Mississippi. Three budgerigar chicks, 1-2 weeks old, from the aviary were necropsied. Microscopic lesions in the chicks consisted of heavy infection of enterocytes with microsporidia (2/3; autolysis precluded critical evaluation of the intestine of chick No. 2), multifocal hepatic necrosis and inflammation with intralesional microsporidia (1/3), spherical clusters of microsporidia in the hepatic sinusoids in the absence of inflammation (1/3), and gastric megabacteriosis (3/3). The ultrastructure of the microsporidian spores was consistent with an Encephalitozoon species. The polymerase chain reaction and Southern blot analysis were used to identify the microsporidian as Encephalitozoon hellem, an organism that has only been identified in humans. Encephalitozoon hellem causes keratoconjunctivitis and respiratory infections in humans with acquired immunodeficiency syndrome. This report presents the first confirmed case of microsporidiosis in budgerigars. The finding of E. hellem in pet birds may be important in elucidating the epidemiology of human infections with this organism.