Development of a novel in vitro method for drug development for fish; application to test efficacy of antimicrosporidian compounds.

Few drugs are approved for treating diseases caused by parasites in minor species such as fish. This is due, in part, to the expense of drug development and to the comparatively small market. In vivo effectiveness trials for antiparasitic drugs are costly, time consuming and require ethics approval, therefore an in vitro screening approach is a cost-effective alternative to finding promising drug candidates. We developed an in vitro testing system to test antimicrosporidial compounds against a microsporidian pathogen Heterosporis saurida. Five antiparasitic compounds, albendazole, fumagillin, TNP-70, nitazoxanide and lufenuron, were assayed for antimicrosporidial activity. All compounds reduced the number of H saurida spores in infected cells when applied at a concentration that did not appear to be toxic to the host cells. Albendazole inhibited replication of H saurida by >60 per cent, fumagillin and its analogue TNP-470 inhibited H saurida >80 per cent, nitazoxanide and lufenuron inhibited growth >70 per cent. The data suggest that both fumagillin and its analogous TNP-70 hold the best promise as therapeutic agents against H saurida. The ability to use fish cell cultures to assess drugs against H saurida demonstrates an approach that may be helpful to evaluate other drugs on different microsporidia and host cells.

Novel alkylpolyamine analogues that possess both antitrypanosomal and antimicrosporidial activity.

A novel series of alkyl- or aralkyl-substituted polyamine analogues was synthesized containing a 3-7-3 polyamine backbone. These analogues were evaluated in vitro, and in one case in vivo, for activity as antitrypanosomal agents, and for activity against opportunistic infection caused by Microsporidia. Compound 21 inhibits trypanosomal growth with an IC(50) as low as 31nM, while compound 24 shows promising activity in vitro against trypanosomes, and against Microsporidia in vitro and in vivo.

Lectin-reactive components of the microsporidian Glugea plecoglossi and their relation to spore phagocytosis by head kidney macrophages of ayu Plecoglossus altivelis.

We investigated the reactivity of lectins to spores of Glugea plecoglossi from ayu Plecoglossus altivelis. Smear preparations of purified spores were treated with 8 kinds of lectins. Lectin blots were used to detect glycoproteins of spore lysates. In addition, lectin-treated spores were applied to head kidney macrophages of ayu, and the percentage of phagocytosis (PP) was calculated and compared with the control. Two lectins (ConA, WGA) reacted with the surface of the spores, and a major band (55 kDa) and some minor bands were visualized on blots after treatment with these. PP was decreased after ConA treatment. From these results, we suggest that G. plecoglossi spores can be phagocytized by ayu head kidney macrophages via ConA-reactive glycoprotein-mediated recognition.

Quinine hydrochloride treatment delays xenoma formation and dissolution in rainbow trout challenged with Loma salmonae.

An experiment was designed to study the effect of dietary quinine hydrochloride (61 mg/kg of fish/day), on the rate of xenoma formation in the gills of Loma salmonae-infected juvenile rainbow trout maintained at a water temperature of 15 degrees C. Almost all (90.9%) control fish had developed xenomas by week 6 post-exposure (PE), but significantly fewer (18.2%) of the medicated fish were similarly affected (P < 0.0001). By week 8 PE, 100% of control fish had xenomas, but only 57.5% of quinine-treated had xenomas (P < 0.0001). However, by week 9 there was no difference between treated and control fish. Xenoma dissolution and branchitis, two crucial events in the pathogenesis of L. salmonae infection in farm-reared Pacific salmon, were present at week 10 in control fish. In contrast, comparable lesions did not develop in treated fish until week 14. These findings are of potential significance for the control of L. salmonae infection in farmed salmon.

Enterocytozoon bieneusi multiorgan microsporidiosis in a HIV-infected patient.

Multiorgan microsporidiosis due to Enterocytozoon bieneusi was diagnosed in an HIV-infected patient. The parasite was found and identified as E. bieneusi by transmission electron microscopy in stools, duodenal biopsy, nasal discharge and sputum. No clinical improvement or parasite eradication was obtained after albendazole therapy, but the patient remained alive 9 months after diagnosis.

[Bilateral microsporidial keratitis in an HIV-positive patient with AIDS stage infection]. / Beidseitige Mikrosporidien-Keratitis bei einer HIV-positiven Patientin im Stadium AIDS.

INTRODUCTION: Microsporidia are spore-forming, obligate intracellular protozoa. Humans seem to be infected only by 4 genera of microsporidia. Microsporidial keratoconjunctivitis in immunodeficient patients has a characteristic appearance. CASE REPORT: 34 year old woman with AIDS complained of bilateral blurred vision. The visual acuity was 0.6 on both eyes. She had a mild conjunctivitis and disseminate, not very prominent intraepithelial corneal opacities. She was treated with propamidine isethionate 0.1% 5 times daily and artificial tears under presumptive diagnosis of microsporidial keratoconjunctivitis. We discontinued this treatment because of no improvement. Within 6 months the visual acuity decreased to 0.05. A conjunctival smear was positive for microsporidia. Local Fumagillin-eye-drops 0.07 mg/ml 7 times daily were given. Within 2 weeks an impressively improvement was seen. Because of an persisting diarrhea 400 mg Albendazol twice daily was added orally without success. DISCUSSION: The biomicroscopically changes of microsporidial keratoconjunctivitis are characteristical and lead to the clinical diagnosis. Fumagillin is in vitro and clinically a potent antimicrosporidian agent with an extremely broad therapeutic range.

Screening of compounds for antimicrosporidial activity in vitro.

Relatively few effective compounds are available for treating microsporidiosis in humans. In this study, several compounds were assayed for activity against Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) and Vittaforma corneae Shadduck, Meccoli, Davis et Font, 1990 in vitro. Of the benzimidazoles tested, albendazole was most effective and the MIC50 values were 8.0 ng/ml and 55.0 ng/ml for E. intestinalis and V. corneae, respectively. Fumagillin and its analogue, TNP-470 were nearly equally effective against both E. intestinalis and V. corneae. The MIC50 values of fumagillin were 0.52 ng/ml and 0.81 ng/ml, and the MIC50 values of TNP-470 were 0.35 ng/ml and 0.38 ng/ml for E. intestinalis and V. corneae, respectively. In addition, 12 of 44 purines and pteridines with putative tubulin binding activity that were synthesized at Southern Research Institute (SRI), inhibited microsporidial replication by more than 50% at concentrations that were not toxic to the host cells. Several chitin synthesis/assembly inhibitors inhibited growth of the microsporidia in vitro but were toxic for the host cells making it difficult to interpret the results. One exception was lufenuron, which caused no significant toxicity to the host cells and expressed approximate MIC50 values of 2.95 micrograms/ml and 6.3 micrograms/ml against E. intestinalis and V. corneae, respectively. These results warrant further studies on albendazole, fumagillin, TNP-470, lufenuron, and the selected SRI purines and pteridines for developing therapeutic strategies for microsporidiosis.

Treatment of fish parasites. 11. Effects of different benzimidazole derivatives (albendazole, mebendazole, fenbendazole) on Glugea anomala, Moniez, 1887 (Microsporidia): ultrastructural aspects and efficacy studies.

Three different benzimidazole derivatives, albendazole [methyl-5-(propylthio)-2-benzimidazolcarbamate], mebendazole (methyl-5-benzoyl-2-benzimidazolcarbamatic acid methyl ester), and fenbendazole [methyl-5-(phenylthio)-2-benzimidazolcarbamate] were tested in vivo against Glugea anomala parasitizing the connective tissue of sticklebacks (Gasterosteus aculeatus). Naturally infected sticklebacks were incubated in aerated plastic aquaria (10 1) at 22 degrees C in water containing 0, 1, 5, 10, or 50 micrograms of either albendazole, mebendazole or febendazole for 2 or 6 h. For intermittent treatment, 2 micrograms substance was administered three times for 6 h at intervals of 36 h. At the ultrastructural level, at all developmental stages of G. anomala there were no significant differences in the kind of damage caused by either albendazole, mebendazole, or febendazole. Starting with a dose of 1 microgram/ml for 2 h, each of the drugs irreversibly damaged uni- and multinucleate meronts, sporogonial plasmodia, and sporoblasts. Disorganized spores were also observed. Treatment with higher doses (10 micrograms/ml, 2 or 6 h) caused malformations of the merogonic and the sporogonic stages, a significant reduction in the number of ribosomes, and disruptions of the nuclear membranes. The first recognizable treatment effect was an enlargement of the smooth endoplasmic reticulum. In the sporogonial plasmodia, the membranes of the sporophorous vesicle envelopes were lumpy or even completely destroyed. After incubation with the highest dose (50 micrograms/ml, 6 h), microtubules were apparent within the karyoplasm of the uninucleate meronts. After interval treatment, all forms of damage were intensified, especially in the mature spores. When treatment was done three times at low doses (3 x 2 micrograms/ml, 6 h, 36-h intervals), spore infectivity was drastically lowered. Therefore, it seems likely that an intermittent regimen of medicinal baths can be successfully applied against susceptible Microsporidia in fish.

Persistent damage to Enterocytozoon bieneusi, with persistent symptomatic relief, after combined furazolidone and albendazole in AIDS patients.

AIM: To investigate morphological changes in Enterocytozoon bieneusi and the duration of symptomatic relief after combination treatment with furazolidone and albendazole in AIDS patients. METHODS: Four severely immunocompromised AIDS patients with symptomatic E bieneusi infection of the gut received an 18 day course of combined furazolidone and albendazole (500 + 800 mg daily). All patients were monitored for parasite shedding in stool by light microscopy at the end of treatment and monthly during follow up. At the end of treatment, duodenal biopsy specimens obtained from three patients were studied by transmission electron microscopy by two pathologists blind to the patients' treatment or clinical outcome. Duodenal biopsy specimens obtained from one of the patients two months after completion of treatment were also studied electronmicroscopically. RESULTS: All patients had long lasting symptomatic relief, with a major decrease--or transient absence--of spore shedding in stools from completion of treatment. After treatment, changes in faecal spores were persistently found by light microscopy in all cases, and there was evidence of both a substantial decrease in the parasite load and ultrastructural damage in the parasite in all biopsy specimens. The treatment was well tolerated, and no patient had clinical or parasitological relapse during follow up (up to 15 months). CONCLUSIONS: The long lasting symptomatic relief observed in all four treated patients correlated with the persistent decrease in parasite load both in tissue and in stool, and with the morphological changes observed in the life cycle of the protozoan. These data suggest that combined treatment with furazolidone and albendazole is active against E bieneusi and may result in lasting remission even in severely immunocompromised patients.

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.

Effects of chitinolytic and proteolytic enzymes on in vitro phagocytosis of microsporidians by spleen macrophages of turbot, Scophthalmus maximus L.

The serum of many teleosts, including turbot, contains chitinolytic and proteolytic enzymes. In the present study, the possible role of these enzymes in nonspecific immune responses to microsporidian infection was investigated. The rate of phagocytosis of Glugea caulleryi spores by turbot splenic macrophages was significantly reduced after pretreatment of spores with proteolytic or chitinolytic enzymes, suggesting that alteration of surface glycoproteins affects spore recognition. However, intracellular superoxide production by macrophages was significantly higher after stimulation with protease-treated spores, or with untreated spores plus normal turbot serum (NTS), than after stimulation with untreated spores in the absence of NTS. These results support the view that the chitinolytic and proteolytic activities in teleost serum may play a role in defence against microsporidian infection.

Effects of an asymmetric triazine derivative, HOE 092 V, on Glugea anomala, Moniez, 1887 (Microsporidia) parasitic in the three-spined stickleback Gasterosteus aculeatus.

An asymmetric triazine derivative, HOE 092 V,2-[3,5-alpha-dichloro-4-(4-methyl-sulfonylphenoxy)-phenyl]- 1-methyl-hexahydro-1,2,4-triazine-3,5-dion, was tested in vivo against Glugea anomala parasitizing the connective tissue of sticklebacks (Gasterosteus aculeatus). Naturally infected sticklebacks were incubated in 10-1 plastic aquaria in water containing 0, 2.5, 5, and 10 micrograms HOE 092 V/ml for 2, 3, and 4 h at 22 degrees C. As seen at the ultrastructural level, the drug caused severe damage to all developmental stages of G. anomala except the mature spores. Starting with a dose of 2.5 micrograms/ml, the drug caused significant damage on uni- and multinucleate meronts, sporogonial plasmodia, and sporoblasts. The damage mainly consisted in a decrease in the number of ribosomes, an enlargement of the smooth endoplasmic reticulum and a vacuolization of the cytoplasma. When treatment was done with 5 micrograms for 2 h, multinucleate meronts and sporogonial plasmodia were no longer detectable, and the sporoblasts and the prespore stages except the mature spores had shrunk. After incubation of the infected fish with 10 micrograms HOE 092 V/ml and 4 h exposure, uninucleate meronts were no longer detectable by means of transmission electron microscopy. In the sporoblast mother cells, vacuolization of the cytoplasma and lysis of the nuclei occurred. However, mature spores were not affected. It seems likely that HOE 092 V can be successfully applied in medicinal baths against Microsporidia in fish. The infected fish should be incubated in separate, aerated containers.

Clinical and microscopical features of small-intestinal microsporidiosis in patients with AIDS.

Intestinal microsporidiosis by Enterocytozoon bieneusi is an increasingly recognized infection in AIDS patients. We report eight cases of microsporidiosis. All patients were severely immunodepressed. Clinical features were highly variable. Patients were followed up for a mean period of 7.8 months. All patients had persistent infection during the follow-up and spore excretion remained constant. Two patients became asymptomatic during the follow-up. None of the patients presented clinical and echographic signs of biliary involvement. Treatment with albendazole, metronidazole or paromomycin failed to produce a durable clinical response or to eradicate the organism. Cases were identified by stool examination and additionally investigated with light and electron microscopy. It was found that light microscopy was a sensitive method, while electron microscopy was less sensitive but allowed the definition of the infecting species. The modified trichrome stain was a satisfactory method for diagnosis on fecal smears. The calcofluor stain and the combination of DAPI with calcofluor was a rapid and simple staining method for screening.

Multiorgan microsporidiosis: report of five cases and review.

We describe five cases and review 34 reported cases of multiorgan microsporidiosis. Most of the patients with multiorgan involvement have been adults with AIDS. Organs most commonly infected include the small intestine, urinary tract, biliary tree, and eye; involvement of the respiratory tract, nasal sinuses, and central nervous system is also described but appears to be less frequent. Although patients with multiorgan disease may be asymptomatic, clinical presentation usually relates to the involved organs. Enterocytozoon bieneusi and Septata intestinalis are the most frequently identified species of pathogens. An affinity for certain tissues is observed among different microsporidial species. In all but one case of E. bieneusi infection, infection was limited to intestinal and hepatobiliary tracts, a finding suggestive of local extension. In contrast, the patients infected with S. intestinalis had widespread involvement, suggesting true hematogenous or lymphatic dissemination. Treatment may have to be based on findings regarding which organs and specific microsporidial species are involved. Further investigation of the pathogenic tendencies and route of acquisition of these organisms and the therapeutic agents active against them is needed.

Disseminated microsporidiosis due to Septata intestinalis in nine patients infected with the human immunodeficiency virus: response to therapy with albendazole.

Disseminated microsporidiosis due to the newly described species Septata intestinalis in nine patients infected with human immunodeficiency virus is described. All patients were male homosexuals; the mean age was 41 years (range, 35-58 years). They were all severely immunocompromised, with a mean CD4 lymphocyte count of 15/mm3 (range, 0-32/mm3). Infection by S. intestinalis was seen in duodenal biopsy specimens from all patients, and dissemination was demonstrated by the presence of microsporidial spores in urine (9 of 9 patients), sinonasal secretions and/or nasal mucosal biopsy specimens (6 of 6), and sputum (6 of 6). Seven patients were treated with albendazole (400 mg twice daily), resulting in significant dissipation or complete resolution of diarrhea for six patients and abatement of symptoms for the six patients with chronic rhinosinusitis. There was a parallel parasitological response, with clearance of S. intestinalis infection from almost all sites.

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.

Electron microscopic changes in Enterocytozoon bieneusi following treatment with albendazole.

AIM: To identify and describe electron microscopic changes occurring in Enterocytozoon bieneusi in patients treated with albendazole. METHODS: Eighteen HIV seropositive patients with E bieneusi infection of the small intestine were treated with albendazole 400 mg twice a day for one month. Duodenal biopsy specimens obtained before and at the end of treatment were examined electron microscopically by a pathologist who was unaware of the clinical response. A semiquantitative assessment of the parasite load and description of the parasite morphology was made. RESULTS: A complete resolution of diarrhoea occurred in nine patients and a further three had a greater than 50% reduction in baseline stool frequency or volume. Three of the non-responders were also infected with cryptosporidium. There was a reduction in parasite load in those responding to treatment and an increase in abnormal forms in both responders and non-responders. CONCLUSION: The clinical response to albendazole treatment seen in some patients with small intestine microsporidiosis may be due to damage to the developmental stages, causing a partial inhibition of parasite reproduction.