Human Microsporidiosis Research Articles

Host-specific segregation of ribosomal nucleotide sequence diversity in the microsporidian Enterocytozoon bieneusi

Enterocytozoon bieneusi is a unicellular enteric fungal pathogen and the most common cause of human microsporidiosis. The frequent detection of this organism in animals, including companion animals, livestock and wildlife, has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen. A partial sequence of the ribosomal internal transcribed spacer (ITS) has been widely used as a genetic marker for studying the molecular epidemiology of E. bieneusi. With the aim of comparing E. bieneusi ITS genotypes originating from different host species, and assess the potential for zoonotic transmission, E. bieneusi ITS sequences retrieved from GenBank were analyzed using two metrics of diversity, rarefaction and phylogenetic distance. In spite of the human ITS sample being geographically more diverse, ITS sequence diversity in animals exceeded that of humans. In both host groups much of the ITS diversity remains to be sampled. Using quantitative phylogenetic tests we found evidence for a partial but significant segregation of E. bieneusi ITS sequences according to host species. Host-specific segregation was confirmed by hierarchical analysis of molecular variation. To improve our understanding of the epidemiology of human microsporidiosis and strengthen the study of E. bieneusi populations, efforts to genotype additional E. bieneusi isolates from wildlife and companion animals should be prioritized and the geographic and species diversify of animal samples should be increased. Due to the possibility of genetic recombination in this species, additional unlinked genetic markers need to be developed and included in future studies.

Examination of genotoxic effects of fumagillin in vivo

Fumagillin is an antibiotic derived from the fungus Aspergillus fumigatus. It has been used successfully for the treatment of intestinal microsporidiosis in HIV-positive humans, as well as in those suffering from intestinal amebiasis and microsporidial keratoconjunctivitis. In veterinary medicine it is approved for the treatment of microsporidiosis in bees and fish. In this research fumagillin was tested for the ability to provoke chromosomal aberrations in mouse bone marrow cells. BALB/c mice were administered fumagillin by gastric probe in doses of 5, 10 and 20 mg/kg b.w. Water-sugary syrup was the negative and cyclophosphamide (15 mg/kg b.w.) the positive control. Significantly increased frequencies (p<0.01 or p<0.001) of numerical chromosomal aberrations (aneuploidies and poliploidies) was observed both in the medium (10 mg/kg b.w.) and the highest (20 mg/kg b.w.) dose of fumagillin. Structural chromosomal aberrations (gaps, breaks and insertions) were noticeably more frequent in comparison to negative control only in the highest experimental dose of dycikloheksilamine. These results clearly showed that fumagillin in concetrations 10 and 20 mg/kg b.w. had a genotoxic potential in vivo.

Ophthalmic microsporidiosis: Emerging pathogens or emerging awareness?

Abstract Purpose We report and discuss an apparent increase in the number of ocular and adnexal microsporidial infections. Methods We present cases of ocular microsporidial infection which were diagnosed by light (LM) and electron microscopy (EM) at our departments and review the literature. Results Cases involved Encephalitozoon, Vittaforma, Trachipleistophora and Nosema sp. with infection of ocular surface, corneal stroma, canaliculi and nasolacrimal apparatus. Conclusion Microsporidia are minute obligate intracellular parasites which infect via a polar tube. Human microsporidiosis is normally HIV/AIDS related, most infections being enteric with few ocular cases. With the success of antiretrovirals, the incidence of enteric microsporidiosis is now much lower. Ocular infection is now more prevalent and not usually HIV‐associated. Our cases had infection of ocular surface, cornea and lacrimal canal/sac. The diagnosis was not initially considered clinically. LM may be diagnostic if characteristic refractile ZN‐positive microspores are seen. If they are few or are not identified, LM is not diagnostic. LM does not permit speciation which usually requires EM. Diligent search for organisms may be necessary as distribution may be focal. Insufficient data exist to allow PCR‐based diagnosis of most microsporidial species. Sources and mechanisms of infection remain speculative. Insect infection is abundant. Spores are present in water. Microsporidia are poikilothermic. Microsporidiosis occurs in animals. Does the eye's immune status and lower surface temperature favour opportunistic infection by organisms adapted to lower temperatures in insects? Are animals vectors? Is travel to "exotic" places a factor? Are microsporidia emerging pathogens or are they simply not recognised?

Effects of host temperature and gastric and duodenal environments on microsporidia spore germination and infectivity of intestinal epithelial cells

Approximately 14 of the more than 1,000 species of microsporidia infect humans, only two of which, Enterocytozoon bieneusi and Encephalitozoon intestinalis, cause intestinal microsporidiosis. Clinical isolates of three microsporidia species, E. intestinalis, Encephalitozoon hellem, and the insect parasite, Anncaliia (Brachiola, Nosema) algerae were used in a spore germination assay, and enterocyte attachment and infection assays were performed to model the potential roles of gastric and duodenal environments and host temperature in determining why only one of these microsporidia species causes intestinal microsporidiosis. Enterocyte infection with A. algerae spores was 10% that of the Encephalitozoon species, a difference not attributable to differences in spore attachment to host cells. Prior spore treatment with pepsin in HCl, pancreatic enzymes, or ox bile did not inhibit germination or enterocyte infection by the three microsporidia species. While the Encephalitozoon species differentiated to mature spores within 3 days, the time taken for many enterocytes to turn over, A. algerae took 3-5 days to produce mature spores, near the upper limit for enterocyte turnover in vivo. Thus, host temperature may contribute to A. algerae not causing human intestinal microsporidiosis, but none of the factors tested account for the inability of E. hellem to cause such an infection.

Microsporidian Infection Is Prevalent in Healthy People in Cameroon

Most studies of opportunistic infections focus on those with weak immune systems, such as human immunodeficiency virus (HIV)/AIDS patients and children. However, there is a lack of information on these infectious agents in healthy people worldwide. In the present study, stool samples from both HIV patients and healthy people were examined to begin filling in this serious gap in the understanding of human microsporidiosis, particularly the enteric parasite Enterocytozoon bieneusi. Specimens were obtained from 191 individuals living in Yaoundé, the capital city of Cameroon, in sub-Sahara Africa, including 28 HIV-positive patients who also had tuberculosis (TB). E. bieneusi prevalence was 35.7% among the HIV(+) TB patients, whereas it was only 24.0% among 25 HIV(-) TB patients in the same hospital. Unexpectedly, the prevalence (67.5%) of microsporidiosis was found to be even higher for 126 immunocompetent individuals than for those with TB (healthy people compared to HIV(+) TB and HIV(-) TB patients; P < 0.001). The immunocompetent group included people ranging from 2 to 70 years of age living in four different neighborhoods in Yaoundé. The highest prevalence (81.5%) was among teenagers, and the highest mean infection score (+2.5) was among children. Additional studies of immunocompetent people in other parts of Cameroon, as well as in other countries, are needed to better understand microsporidiosis epidemiology. There is still much more to be learned about the natural history of microsporidia, the pathogenicity of different strains, and the role of enteric microsporidia as opportunistic infections in immunodeficient people.

Asymptomatic Enterocytozoon bieneusi microsporidiosis in captive mammals

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic transmission of microsporidian spores. A survey utilizing chromotrope 2R stain and fluorescent in situ hybridization techniques for testing feces from 193 captive mammals demonstrated that 3 animals (1.6%) shed Encephalitozoon bieneusi spores. These include two critically endangered species (i.e., black lemurs, Eulemur macaco flavifrons; and Visayan warty pig, Sus cebifrons negrinus) and a threatened species (mongoose lemur, Eulemur mongoz). The concentration of spores varied from 2.7 x 10(5) to 5.7 x 10(5)/g of feces, and all infections were asymptomatic. The study demonstrates that E. bieneusi spores can originate from captive animals, which is of particular epidemiologic importance because the close containment of zoological gardens can facilitate pathogen spread to other animals and also to people such as zoo personnel and visitors.

Microsporidiosis: current status

Microsporidiosis 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. The 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. Effective 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.

System for Expression of Microsporidian Methionine Amino Peptidase Type 2 (MetAP2) in the Yeast Saccharomyces cerevisiae

Microsporidia are parasitic protists of all classes of vertebrates and most invertebrates. They recently emerged as important infections in various immunosuppressed and immunocompetent patient populations. They are also important veterinary and agricultural pathogens. Current therapies for microsporidiosis include benzimidazoles, which bind tubulin-inhibiting microtubule assembly, and fumagillin and its derivatives, which bind and inhibit methionine amino peptidase type 2 (MetAP2). Benzimidazoles are not active against Enterocytozoon bieneusi, the most common cause of human microsporidiosis. Fumagillin is active against most microsporidia, including E. bieneusi, but thrombocytopenia has been a problem in clinical trials. There is a pressing need for more-specific microsporidian MetAP2 inhibitors. To expedite and facilitate the discovery of safe and effective MetAP2 inhibitors, we have engineered Saccharomyces cerevisiae to be dependent on Encephalitozoon cuniculi MetAP2 (EcMetAP2) for its growth, where EcMetAP2 is harbored on an episomal uracil-selectable tetracycline-regulated plasmid. We have also constructed a leucine-selectable tetracycline-regulated expression plasmid into which any MetAP2 gene can be cloned. By utilizing a 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain, a yeast strain can be generated whose growth is dependent on MetAP2 from any organism. The level of heterologous MetAP2 gene expression can be controlled by the addition of tetracycline to the growth medium. These yeast strains should permit high-throughput screening for the identification of new inhibitors with high specificity and activity toward microsporidian MetAP2.

Enterocytozoon bieneusi (microsporidia) in clinical samples from immunocompetent individuals in Tenerife, Canary Islands, Spain

Microsporidia are newly emerging pathogens of humans and animals, with Enterocytozoon bieneusi being the most common causal agent in human microsporidiosis. To determine the presence of E. bieneusi, 273 clinical samples (40 urine, 156 stools, 37 sputum, 9 bronchial aspirates, 5 bronchial washes and 26 pleural fluids) from immunocompetent patients, mainly suffering diarrhoea or pneumonia, in Tenerife, Canary Islands, Spain were analysed using light microscopy after staining with Weber's chromotrope and by PCR/hybridisation with a specific probe designed to increase the sensitivity of the identification. In this study, detection of E. bieneusi after PCR/hybridisation is reported in 18 (11.54%) of 156 stool samples, 1 (2.5%) of 40 urine samples and 6 (16.22%) of 37 sputum samples. To our knowledge, these are the first reports of E. bieneusi in this subtropical region, showing the increased importance of these parasites as emerging pathogens worldwide.

The First United Workshop on Microsporidia from Invertebrate and Vertebrate Hosts

The phylum Microsporidia is a large group of parasitic unicellular eukaryotes that infect a wide range of invertebrate and vertebrate taxa. These organisms are significant human and veterinary pathogens with impacts on medicine, agriculture and aquaculture. Scientists working on these pathogens represent diverse disciplines that have had limited opportunities for detailed interactions. A NATO Advanced Research Workshop 'Emergent Pathogens in the 21st Century: First United Workshop on Microsporidia from Invertebrate and Vertebrate Hosts' was held July 12-15, 2004 at the Institute of Parasitology of the Academy of Sciences of the Czech Republic to bring together experts in insect, fish, veterinary and human microsporidiosis for the exchange of information on these pathogens. At this meeting, discussions were held on issues related to taxonomy and phylogeny. It was recognized that microsporidia are related to fungi, but the strong opinion of the participants was that the International Code of Zoological Nomenclature should continue to be applied for taxonomic descriptions of the Microsporidia and that they be treated as an independent group emerging from a paraphyletic fungi. There continues to be exponential growth in the pace and volume of research on these ubiquitous intracellular protists. The small genomes of these organisms and the reduction in the size of many of their genes are of interest to many disciplines. Many microsporidia are dimorphic and the mechanisms underlying these morphologic changes remain to be elucidated. Epidemiologic studies to clarify the source of human microsporidiosis and ecologic studies to understand the multifaceted relationship of the Microsporidia and their hosts are important avenues of investigation. Studies on the Microsporidia should prove useful to many fields of biologic investigation.

Microsporidiosis in humans: parasitology, clinical features and treatment

Microsporidiosis in humans: parasitology, clinical features and treatment

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae.

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.

Diagnosis and clinical aspects of human microsporidiosis.

Diagnosis and clinical aspects of human microsporidiosis.

Determination of types of Enterocytozoon bieneusi strains isolated from patients with intestinal microsporidiosis.

To determine the types of Enterocytozoon bieneusi strains associated with intestinal microsporidiosis, we developed a rapid and efficient approach for typing parasites obtained from stool specimens by PCR-restriction fragment length polymorphism (PCR-RFLP). Typing was based on DNA polymorphism of the ribosomal DNA internal transcribed spacer (ITS) region of E. bieneusi. RFLPs generated with two restriction enzymes (NlaIII and Fnu4HI) in PCR-amplified ITS products were used to classify strains into different lineages. This approach was successfully used to differentiate 78 strains that had been obtained from the stools of 65 patients with intestinal microsporidiosis. Among the 78 strains, we found four genetically unrelated lineages, showing the genetic diversity of E. bieneusi. Type I strains of E. bieneusi were found in a majority of the samples, accounting for 51 (78%) of the 65 microsporidiosis cases. In contrast, type II, III, and IV strains were found in only 8 (12%), 3 (5%), and 3 (5%) cases, respectively. All strains of E. bieneusi we have tested so far fall into one of four different lineages, and this study shows that human intestinal microsporidiosis is most often associated with type I strains. PCR-RFLP analysis of the ITS region of E. bieneusi should be useful for epidemiological studies.

Examination of the prevalence and seasonal variation of intestinal microsporidiosis in the stools of persons with chronic diarrhea and human immunodeficiency virus infection.

The epidemiology of human microsporidiosis is poorly understood and environmental factors affecting transmission of the organism have not been fully elucidated. Temporal variation in the prevalence of microsporidia in the stool of patients with human immunodeficiency virus (HIV) infection and diarrhea was studied to evaluate the role of water-borne transmission. From January 1993 to December 1996, 8,439 stools from HIV-infected individuals were examined for microsporidia spores in southern California. Yearly positivity rates were 8.8% in 1993, 9.7% in 1994, 6.6% in 1995, and 2.9% in 1996. An analysis for linear trend showed a statistically significant decrease in stool positivity rates over time (chi2 = 81.9, P = 0.001). No significant seasonal variation in the prevalence of microsporidiosis was seen over that time period. These results suggest the constant presence of microsporidia in the environment, rather than a seasonal association with recreational water use or seasonal contamination of the water supply, and a real decrease in yearly prevalence of microsporidia related diarrhea. Factors related to a progressive decrease in prevalence are subjects of future investigation.

Encephalitozoon intestinalis:Humoral Responses in Interferon-γ Receptor Knockout Mice Infected with a Microsporidium Pathogenic in AIDS Patients

El Fakhry, Y., Achbarou, A., Desportes-Livage, I., and Mazier, D. 1998.Encephalitozoon intestinalis: Humoral responses in interferon-γ receptor knockout mice infected with a microsporidium pathogenic in AIDS patients.Experimental Parasitology,89,113–121. IFN-γ receptor knockout mice and wild-type mice were infectedper oswithEncephalitozoon intestinalis. Both groups developed an infection that was chronic in the mutant mice whereas it was only transient in wild-type mice. The infection of mutant mice was characterized by the continual shedding of spores in feces, splenomegaly, the enlargement of the biliary tract, and the occurrence of numerous nodules in the liver and in the small intestine wall. The humoral response was studied by ELISA, IFA, and Western blotting. ELISA titers of anti-E. intestinalisantibodies of IgG, IgM, and IgA isotypes were higher in IFN-γ R0/0mice than in wild-type mice and they increased in time after infection. Levels of IgG2a were inferior to those of IgG1 in mutant mice in contrast to wild-type mice. High levels of parasite specific antibodies were accompanied by an increase in type 2 cytokines (IL-4 and IL-10) secretion in the duodenum of IFN-γ R0/0mice. TheE. intestinalisspore wall was recognized by IgM, IgG, and IgA from all infected mice whereas the extruded polar tube only reacted with IgG and IgA from IFN-γ R0/0mice after 45 days of the infection. IFN-γ R0/0mice IgG and IgA reacting with polar tube identified also a series of proteins which could be components of this structure. On the proteins recognized by all infected mice sera, two were first recognized by IgM at day 15 and then by IgG at day 30 in wild-type (WT) mice. The persistent reactivity of all proteins in mutant mice is consistent with the chronicity of the infection in these animals; in contrast, their resorption at day 30 in WT animals corroborates the transient character of the infection in these mice. The correlation between the evolution of the proteic pattern and the development of the infection provides evidence of the validity of this murine model to study human microsporidiosis. Indeed the reported results confirm the potential value of serological methods for diagnosingE. intestinalisinfection in immunocompetent and in immunocompromised human subjects, for elucidating the age pattern of the microsporidiosis and also for identifying risk groups.

Human microsporidiosis in the acquired immunodeficiency syndrome

OURADisciplinas de Doencas Infecciosas e Parasitarias e de Parasitologia da Faculdade de Ciencias Medicas do Hospital Universitario PedroErnesto, Universidade Estadual do Rio de Janeiro, Rio de Janeiro, RJ.Tabela 1 — Classificacao dos microsporidiaPhylum MicrosporaClasse MicrosporeaOrdem MicrosporidiaSubordem Pansporoblastina ApansporoblastinaGeneros Pleistophora NosemaSeptata EnterocytozoonEncephalitozoonEspecies S. intestinalis * N. connoriN. corneumE. bieneusiE. cuniculiE. hellem

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for species-specific detection of Encephalitozoon cuniculi. Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Enterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected with Enterocytozoon bieneusi (n = 9), Encephalitozoon spp. (n = 2), and Encephalitozoon intestinalis (n = 1) as well as stool spiked with spores of Encephalitozoon cuniculi and Encephalitozoon hellem and tissue cultures of Encephalitozoon cuniculi and Encephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected with Encephalitozoon cuniculi and Encephalitozoon hellem. Moreover, identification of Encephalitozoon spp. could be specified as Encephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates species-specific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.

Longevity and effects of temperature on the viability and polar-tube extrusion of spores of Glugea stephani, a microsporidian parasite of commercial flatfish.

Food contamination with respect to microsporidiosis in humans (associated with acquired immunodeficiency syndrome) and in marine fish farming deserves particular attention. For the first time, a study on the longevity and resistance to both heat and freezing of the spores of a vertebrate microsporidian, Glugea stephani, parasitizing a commercial flatfish was carried out. As judged on the basis of determinations of the extrusion rate, the resistance of the spores to temperature stress was remarkable. The extrusion rate, which can be directly related to infectivity, was always lower than the viability (membrane integrity). It should be pointed out that neither heat (60 degrees C for 30 min) nor freezing (-19 degrees C for 24 h) caused a complete reduction in the extrusion rate or viability. Consequently, the ingestion of poorly cooked or raw fish (even if previously frozen) represents a danger for aquaculture and, probably, for immunodepressed patients.

Activity of Benzimidazoles against Cryptosporidiosis in Neonatal BALB/c Mice

The need for an effective compound for the prevention and treatment of cryptosporidiosis in humans and animals has led to the testing of benzimidazoles based on reports that albendazole was clinically effective against related protozoan parasites causing microsporidiosis in humans. Albendazole and other benzimidazole derivatives were tested for prophylactic efficacy against cryptosporidiosis at dosage levels 1-3x the levels found effective for treatment of cattle or sheep for helminth infections. Daily dosage levels of thiabendazole, parbendazole, oxibendazole, mebendazole, and albendazole, as high as 200, 30, 10, 15, and 15 mg/kg of body weight, respectively, were not efficacious in neonatal mice. Although the number of parasites in histologic sections of intestine from mice mediated with 15 mg albendazole/kg of body weight was significantly lower than in unmedicated control mice, suggesting activity against the parasite, a high percentage of epithelial cells in the medicated mice were infected.