Evaluation of an immunofluorescent-antibody test using monoclonal antibodies directed against Enterocytozoon bieneusi and Encephalitozoon intestinalis for diagnosis of intestinal microsporidiosis in Bamako (Mali).

A 2-month study was carried out in Mali to evaluate an immunofluorescent-antibody test (IFAT) using monoclonal probes specific for Enterocytozoon bieneusi or Encephalitozoon intestinalis. Sixty-one human immunodeficiency virus (HIV)-seropositive adult patients and 71 immunocompetent children were enrolled. Microsporidia were detected in stools from 8 of 61 patients (13.1%) seropositive for HIV. A single species, E. bieneusi, was identified. All the children were negative for microsporidia. The sensitivity and specificity of IFAT were 100% compared with those of PCR, which was used as the "gold standard." Moreover, species identification by IFAT was more rapid and less expensive than that by PCR. These results show the suitability of IFAT for detection of microsporidia in developing countries.

In vitro activities of two antimitotic compounds, pancratistatin and 7-deoxynarciclasine, against Encephalitozoon intestinalis, a microsporidium causing infections in humans.

The antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with Encephalitozoon intestinalis, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC(50)s) of pancratistatin and 7-deoxynarciclasine for E. intestinalis were 0.18 microM and 0.2 microM, respectively, approximately eightfold lower than the IC(50)s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.

One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption.

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.

Dissemination of Encephalitozoon intestinalis, a causative agent of human microsporidiosis, in IFN-gamma receptor knockout mice.

The dissemination of Encephalitozoon intestinalis, a microsporidium causing intestinal diseases and systemic infection in humans, was investigated in IFN-gamma Ro/o mice. Although lesions were seen in organs of autopsied animals, the parasites were rarely detected using histological examination. Nevertheless, infection of the duodenum, liver, kidneys and lungs was demonstrated by polymerase chain reaction. This method also enabled the detection of the parasite in the brain and the heart. The development of E. intestinalis in RK13 cell cultures to which cell suspensions from liver, kidney, lung or brain of infected IFN-gamma Ro/o mice were added, confirmed the spread of intestinal microsporidiosis to these organs. No dissemination was observed in wild-type mice. These results confirm those of previous studies and emphasize the low morbidity of the infection in IFN-gamma Ro/o mice and confirm the role of IFN-gamma in the control of E. intestinalis infection. These mice infected with E. intestinalis offer important information about this interesting and important parasitic disease of man and animals.

Trial of oral fumagillin for the treatment of intestinal microsporidiosis in patients with HIV infection. ANRS 054 Study Group. Agence Nationale de Recherche sur le SIDA.

OBJECTIVE: Intestinal microsporidiosis caused by Enterocytozoon bieneusi is a cause of chronic diarrhoea in patients with HIV infection for which there is no current therapy. This study was designed to assess the safety and efficacy of oral fumagillin in this infection. DESIGN: A dose-escalation trial. METHODS: Twenty-nine HIV-infected patients with E. bieneusi infection were consecutively enrolled in the trial. Oral doses of fumagillin were given to four groups of patients for 14 days: 10 mg/day (group 1), 20 mg/day (group 2), 40 mg/day (group 3), and 60 mg/day (group 4). Patients were seen at weeks 1, 2, 4 and 6 to assess safety and efficacy. Efficacy was assessed primarily by the clearance of microsporidia from stools and follow-up duodenal biopsies. RESULTS: Thirteen patients complained of abdominal cramps, vomiting or diarrhoea during the study, and three patients had fumagillin withdrawn because of adverse events. Thrombocytopenia, neutropenia and hyperlipasaemia were the most frequent biological adverse events. Twenty-one out of 29 patients transiently cleared microsporidia from their stools during the study. By week 6, however, all patients in groups 1, 2 and 3 had parasitic relapse. Interestingly, eight out of 11 (72%) patients treated with 60 mg/day (group 4) apparently cleared microsporidia from their gastrointestinal tract and gained weight. No parasitic relapse was documented in these eight patients during a mean follow-up of 11.5 months. CONCLUSION: Treatment with fumagillin at 60 mg/day for 14 days has promise as an effective oral treatment for E. bieneusi infections.

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study.

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.

Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi.

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.

Encephalitozoon intestinalis: humoral responses in interferon-gamma receptor knockout mice infected with a microsporidium pathogenic in AIDS patients.

IFN-gamma receptor knockout mice and wild-type mice were infected per os with Encephalitozoon 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. intestinalis antibodies of IgG, IgM, and IgA isotypes were higher in IFN-gamma R0/0 mice 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-gamma R0/0 mice. The E. intestinalis spore 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-gamma R0/0 mice after 45 days of the infection. IFN-gamma R0/0 mice 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 diagnosing E. intestinalis infection in immunocompetent and in immunocompromised human subjects, for elucidating the age pattern of the microsporidiosis and also for identifying risk groups.

Albendazole for treatment and prophylaxis of microsporidiosis due to Encephalitozoon intestinalis in patients with AIDS: a randomized double-blind controlled trial.

A double-blind placebo-controlled trial was conducted to assess the efficacy and safety of albendazole (400 mg twice daily for 3 weeks) for the treatment of Encephalitozoon intestinalis infection in patients with AIDS. Clearance of microsporidia from the intestinal tract was obtained in 4 of 4 patients in the albendazole group versus 0 of 4 in the control group (P = .01, one-sided Fisher's exact test) and was associated with significant clinical benefit. All 4 controls subsequently cleared microsporidia following open-labeled albendazole treatment. To investigate the effect of albendazole in preventing relapse, these 8 patients were then randomly assigned to receive either albendazole (400 mg twice daily) or no treatment for the next 12 months. Albendazole significantly delayed the occurrence of relapse (P = .04, one-sided log-rank test). In human immunodeficiency virus-infected patients with E. intestinalis infection, albendazole has parasitologic and clinical efficacy and reduces the risk of relapse.

Human microsporidioses.

Recent developments in microsporidiosis research include the increased utilization of molecular techniques for the investigation of clinical specimens as well as for epidemiological and phylogenetic studies. Special attention is given to studies reporting severe disseminated microsporidioses involving most organs in AIDS patients, and the increased number of HIV-seronegative and immunocompetent individuals with microsporidiosis. The potential efficacy of fumagillin (Sanofi Recherche, Gentilly, France) in infections caused by Enterocytozoon bieneusi, and the remission of intestinal microsporidiosis in HIV infected patients with antiretroviral therapy are also highlighted.

Potential efficacy of fumagillin in intestinal microsporidiosis due to Enterocytozoon bieneusi in patients with HIV infection: results of a drug screening study. The French Microsporidiosis Study Group.

OBJECTIVE: Intestinal microsporidiosis due to Enterocytozoon bieneusi is a frequent cause of chronic diarrhoea in patients with HIV infection for which there is no available therapy. This study was designed to search for a drug with activity against this organism. DESIGN: Prospective open-labelled Phase II multicentre study. SETTING: University hospitals. PATIENTS: Sixty HIV-infected men with intestinal E. bieneusi infection. INTERVENTIONS: Ten drug regimens were consecutively tested orally for 3 weeks: albendazole plus metronidazole, sulphadiazine plus pyrimethamine, atovaquone, doxycycline plus nifuroxazide, itraconazole, flubendazole, chloroquine, paromomycin, sparfloxacin and fumagillin. Nine evaluable patients per regimen were required, but each patient could be enrolled up to three times in the study. OUTCOME MEASURE: Efficacy was assessed primarily by the clearance of E. bieneusi from stools and intestinal biopsies. The safety of each regimen was also assessed. RESULTS: Only purified fumagillin was able to clear E. bieneusi from stools as well as intestinal biopsies, whereas all other regimens failed to show antiparasitic efficacy. However, only four patients received fumagillin because of drug-induced thrombocytopenia. The four patients who received fumagillin remained free of E. bieneusi infection after a mean follow-up of 10 months. CONCLUSION: Eradication of E. bieneusi from the intestinal tract of patients with HIV infection and persistent immunosuppression is an achievable goal. Our study allowed the identification of oral fumagillin as a potential treatment for intestinal microsporidiosis due to E. bieneusi.

[Human intestinal microsporidiosis in Bamako (Mali): the presence of Enterocytozoon bieneusi in HIV seropositive patients]. / Microsporidiose intestinale humaine à Bamako (Mali): présence d'Enterocytozoon bieneusi chez les patients séropositifs pour le VIH.

A study was conducted between 1993 and 1996 in Bamako to determine the rate of occurrence of microsporidia in 88 patients. Most (80%) had chronic diarrhea associated with weight loss and 87.5% were HIV-positive. Intestinal microsporidia were detected in 32% of the patients infected with HIV-1, HIV-2, or coinfected with both strains. Microsporidiosis was also diagnosed in three of the eleven HIV-negative individuals (27%). Microsporidiosis was confirmed by electron microscopy in 6 HIV-positive patients and 1 HIV-negative individual. Enterocytozoon bieneusi was detected in each case. These results suggest that microsporidia are common pathogens in HIV-positive patients in Bamako. Cases of microsporidiosis have been reported for the first time in HIV-2-infected patients. The proportion of women microsporidiosis patients is higher in Mali than in industrialized countries. The presence of microsporidia in HIV-negative patients suggests that these parasites may be an underestimated cause of enteritis in developing countries.

Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients.

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species.

Cell invasion by the microsporidium Encephalitozoon intestinalis.

The ultrastructural study of the invasion of cells (THP1 and RK13) by E. intestinalis shows that infective stages enter host cells via a phagocytic process initiated at the contact of the apical part of spores with host cell membrane. The polar tube is extruded within an invagination of the host cell membrane that extends inside a pseudopod containing microfilaments. These observations suggest that microsporidia as well as other intracellular pathogens can induce host cell alterations facilitating the invasion.