Safety and efficacy of fumagillin for the treatment of intestinal microsporidiosis. A French prospective cohort study.

BACKGROUND: Intestinal microsporidiosis due to Enterocytozoon bieneusi is a cause of chronic diarrhoea in immunocompromised patients. Fumagillin has been approved in France for its treatment. OBJECTIVES: To investigate the efficacy and safety of fumagillin in a real-life setting. METHODS: As required by the French Medicine Agency, all patients receiving fumagillin were enrolled in a prospective study to evaluate its efficacy and safety. Stool examination with identification of E. bieneusi by PCR was performed at baseline, end of treatment and monthly thereafter for 6 months. Safety was monitored up to 6 months and full blood counts were monitored up to 42 days after treatment initiation. The primary endpoint was safety. Parasite clearance and relapses were secondary endpoints. RESULTS: From 2007 to 2018, 166 patients received fumagillin, including 6 children. Patients were transplant recipients (84%), HIV-infected patients (13%) or had another cause of immunosuppression (5%). Serious adverse events were reported in 41 patients (25%), mainly thrombocytopenia (15%) and neutropenia (5%), with two haemorrhagic events leading to one death. Severe thrombocytopenia (<50 G/L) developed in 50 patients (29.6%), neutropenia (<1 G/L) in 20 patients (11.8%) and severe anaemia (<8 g/dL) in 21 patients (12.4%). At the end of treatment, 94% of patients with available stool examination (n = 132) had no spores detected. Among 99 patients with available follow-up after the end of treatment, three parasite relapses were documented. CONCLUSIONS: E. bieneusi microsporidiosis was mainly diagnosed in transplant recipients. Fumagillin was associated with haematological toxicity but showed high efficacy with a low relapse rate.

Therapeutic targets for the treatment of microsporidiosis in humans.

INTRODUCTION: Microsporidia have been increasingly reported to infect humans. The most common presentation of microsporidiosis is chronic diarrhea, a significant mortality risk in immune-compromised patients. Albendazole, which inhibits tubulin, and fumagillin, which inhibits methionine aminopeptidase type 2 (MetAP2), are the two main therapeutic agents used for treatment of microsporidiosis. In addition, to their role as emerging pathogens in humans, microsporidia are important pathogens in insects, aquaculture, and veterinary medicine. New therapeutic targets and therapies have become a recent focus of attention for medicine, veterinary, and agricultural use. Areas covered: Herein, we discuss the detection and symptoms of microsporidiosis in humans and the therapeutic targets that have been utilized for the design of new drugs for the treatment of this infection, including triosephosphate isomerase, tubulin, MetAP2, topoisomerase IV, chitin synthases, and polyamines. Expert opinion: Enterocytozoon bieneusi is the most common microsporidia in human infection. Fumagillin has a broader anti-microsporidian activity than albendazole and is active against both Ent. bieneusi and Encephaliozoonidae. Microsporidia lack methionine aminopeptidase type 1 and are, therefore, dependent on MetAP2, while mammalian cells have both enzymes. Thus, MetAP2 is an essential enzyme in microsporidia and new inhibitors of this pathway have significant promise as therapeutic agents.

First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target.

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.

Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae.

The honeybee disease nosemosis type C is a serious problem since its causative agent, microsporidium Nosema ceranae, is widespread among adult honey bees. Some of the feasible alternative treatments that are used to control this disease are plant extracts. The aim of the present work was to evaluate the effects of essential oils of Chilean plant species, such as Cryptocarya alba, which is used against N. ceranae, and to identify and quantify the majority active compounds in the EO as well as their potential use for the control of nosemosis. Essential oils were obtained using the stripping steam technique with Clevenger equipment and were subsequently analyzed by Gas chromatography-mass spectrometry. Mortality was recorded daily over at least 8days as worker honeybees were exposed to a range of doses of EO dispersed in a sucrose solution. C. alba oil appears to be nontoxic to A. mellifera adults at the tested concentration (the same concentration inhibits the growth of N. ceranae), showing that this oil can be used for the treatment of nosemosis. EO effectiveness was demonstrated against N. ceranae by calculating the percentage of decrease in infected bees from untreated infected groups vs infected groups treated with EO or the reference drug fumagillin. It was determined that a dose of 4µg EO/bee was most effective in controlling N. ceranae development. We determined innocuous doses of C. alba essential oil for honeybees. We demonstrated the antifungal activity of C. alba EO at 4µg/bee against N. ceranae and compared it to its major monoterpenes, such as ß-phellandrene (20µg/bee), eucalyptol (20µg/bee) and α-terpineol (20µg/bee). The major compounds of C. alba EO, α-terpineol, eucalyptol and ß-phellandrene, had significant effects against Apis mellifera infection by N. ceranae, but the antifungal effect of the complete essential oil on N. ceranae was larger than the effect of α-terpineol, eucalyptol or ß- phellandrene separately, showing that C. alba oil may be a candidate for the treatment or prevention of nosemosis.

Fumagillin control of Nosema ceranae (Microsporidia:Nosematidae) infection in honey bee (Hymenoptera:Apidae) colonies in Argentina.

Information on the long­term consequences of Nosema ceranae to honey bee lifespan and effectiveness of Nosema control with fumagillin is scarce and not always consistent. Our objective in this study was to evaluate the effectiveness of the antibiotic fumagillin to control N. ceranae in hives in East­Central Argentina. Honey bee hives were assigned to 3 experimental treatments, a control group with un­treated hives, a preventive strategy group with hives treated monthly, and a monitoring strategy group with hives treated according to a N. ceranae threshold level. Apiaries were monitored monthly during Fall­Winter 2009 and 2010 and N. ceranae spore intensity and honey bee colony strength measures were estimated. Fumagillin­treated colonies had reduced N. ceranae spores load in 2010 compared to control colonies. However, there was no significant difference between treated and control groups for colony strength measures including adult bee population, bee brood availability, honey, or pollen. Fumagillin treatment reduced N. ceranae intensities but had little effect on colonies. The bee population during Winter was reduced in treated as well as in control colonies. Our results clarify that fumagillin treatment should be at least reviewed and that further research should be conducted to acquire a more complete perspective of Nosemosis disease.

Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

Successful treatment with fumagillin of the first pediatric case of digestive microsporidiosis in a liver-kidney transplant.

We report the first successful use, to our knowledge, of fumagillin alone in a pediatric patient to cure intestinal microsporidiosis in a liver-kidney transplanted child. Detection of Enterocytozoon bieneusi in stool became negative from the first post-therapeutic control, while digestive symptoms disappeared in 4 days. During a 9-month follow-up, polymerase chain reaction and direct examinations remained negative for microsporidia in her feces. No major undesirable effects were noted during the anti-microsporidial therapy.

[Microsporidium spp. infection in an immunocompromised child diagnosed by polymerase chain reaction]. / Immün sistemi baskilanmis bir çocukta Microsporidium spp. Enfeksiyonunun polimeraz zincir reaksiyonu ile tanimlanmasi

Microsporidium spp. may lead to a variety of clinical pictures like sinusitis, keratoconjunctivitis, hepatitis, myositis, peritonitis, nephritis, encephalitis and pneumonia in case of immune deficiencies. In this report, a case of diarrhea due to Microsporidium spp. has been presented. A four years old male patient who was followed with the diagnosis of myotonic dystrophia, was admitted to the hospital with the complaints of respiratory distress and fever. Due to the history of recurrent infections, further investigations was carried out to clarify the immunological status of the patient, and the total IgA and IgM levels were found as 14 mg/dl and 30 mg/dl, respectively (normal values were; 18-160 and 45-200 mg/dl, respectively). Following bronchoscopy done to enlighten respiratory distress, the patient developed high fever and watery diarrhea. Since bacteriological cultures of the stool yielded Shigella spp., antimicrobial therapy with ciprofloxacin was initiated. Parasitological examination of the stool done by Weber's modified trichrome dye, yielded Microsporidium spp. microscopically and albendazole was added to the treatment. Presence of Microsporidium spp. was confirmed by polymerase chain reaction with the use of C1 and C2 primers (Metabion, Germany) targeted to Microsporidium spp. and besides a 270 bp band specific for Encephalitozoon intestinalis was also obtained. This case emphasized that in case of diarrhea the stool samples of the immunocompromised patients should be evaluated in terms of Microsporidium spp. in addition to the routine parasitologic examinations.

Fumagillin for treatment of intestinal microsporidiosis in renal transplant recipients.

We report 10 cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in renal transplant (RT) recipients who were treated with fumagillin. All patients presented with afebrile subacute diarrhea (median of 2 weeks), associated with abdominal cramps (n = 5), and weight loss (n = 6), a mean of 68 months after RT. The diagnosis was made by the identification of microsporidial spores in stools with the use of appropriate staining and confirmed by a specific polymerase chain reaction assay for E. bieneusi in 7 patients. Median CD4 cell count was 292 cells/mm(3). All patients received a median of 14 days of oral fumagillin (20 mg tid), and four patients also discontinued or tapered their immunosuppressive regimen (mycophenolate mofetil in 3, and azathioprine in 2). Clinical symptoms resolved rapidly with the clearance of microsporidial spores from stools in all patients. A severe but reversible thrombocytopenia was observed in one patient during fumagillin therapy, and another patient presented with abdominal cramps. Trough levels of tacrolimus measured in seven patients dropped below 5 ng/mL in six of them after 7-14 days of fumagillin. Intestinal microsporidiosis can cause subacute diarrhea in RT recipients. Fumagillin is an effective treatment with an acceptable safety profile, but monitoring of tacrolimus levels is warranted.

Therapeutic evaluation of polyamine analogue drug candidates against Enterocytozoon bieneusi in a SCID mouse model.

Enterocytozoon bieneusi is the most common cause of chronic diarrhea in individuals with human immunodeficiency virus infection or AIDS, and there is no effective therapy. The inhibitory activities of polyamine analogues (PG-11157, PG-11158, and PG-11302) against E. bieneusi infection were evaluated in SCID mice preconditioned with anti-gamma interferon monoclonal antibody intraperitoneally (i.p.). Mice were challenged orally with 10(4) E. bieneusi spores, and groups of mice were treated orally or i.p. 14 days later for 7 days. The inhibitory activities of the drugs against infection were determined by enumerating the E. bieneusi spores in feces three times a week by an immunofluorescence assay. Immunohistochemistry staining confirmed the infection within enterocytes. Oral administration of the analogues PG-11157 (at 150 or 75 mg/kg of body weight/day) and PG-11302 (at 250 mg/kg/day) had significant inhibitory activity (96.2 to 99.6%) that was slightly better than that of fumagillin (1 mg/kg/day; 93.7%). The inhibitory activity with i.p. injection was significant only with PG-11302 at 20 mg/kg/day. While the treatments considerably reduced the levels of spore excretion, neither polyamine analogues nor fumagillin was able to completely eliminate E. bieneusi, as excretion reappeared within 7 days after the end of treatment. Drug toxicity was apparent during treatment, but it disappeared at the end of treatment. These results warrant further examination of the analogues PG-11157 and PG-11302.

Microsporidiosis in solid organ transplant recipients: two Enterocytozoon bieneusi cases and review.

Microsporidiosis first came to prominence as an opportunistic infection in patients with acquired immunodeficiency syndrome. Microsporidia are now emerging pathogens responsible for severe diarrhea during solid organ transplantation. Two main clinical entities can be identified: infection by Enterocytozoon bieneusi, causing diarrhea with limited treatment options; and infection by Encephalitozoon intestinalis, which may disseminate and usually responds to albendazole treatment. We describe here 2 cases of microsporidiosis caused by E. bieneusi in a renal and a liver transplant recipient, respectively, in whom complete clinical efficacy of a short course of fumagillin therapy was obtained. Long-term microbiological eradication was assessed using classical methods and monitored using a real-time quantitative polymerase chain reaction-based method. Both patients experienced drug-induced thrombocytopenia, which resolved after withdrawal of the treatment. We also review the 18 other previously reported cases of microsporidiosis in transplant recipients. In case of persistent diarrhea in solid organ transplant patients, microsporidiosis should be considered. Based on the present experience, treating E. bieneusi infection with 7 days of fumagillin therapy is adequate to eradicate E. bieneusi in this context.

Antimicrosporidial activities of fumagillin, TNP-470, ovalicin, and ovalicin derivatives in vitro and in vivo.

Therapies for microsporidiosis in humans are limited, and fumagillin, which appears to be the most broadly effective antimicrosporidial drug, is considered to be moderately toxic. The purpose of this study was to apply an in vitro drug screening assay for Encephalitozoon intestinalis and Vittaforma corneae and an in vivo athymic mouse model of V. corneae infection to assess the efficacy of TNP-470 (a semisynthetic analogue of fumagillin), ovalicin, and eight ovalicin derivatives. TNP-470, ovalicin, and three of the ovalicin derivatives inhibited both E. intestinalis and V. corneae replication by more than 70% in vitro. Another three of the ovalicin derivatives inhibited one of the two microsporidian species by more than 70%. None of the treated athymic mice survived the V. corneae infection, but they did survive statistically significantly longer than the untreated controls after daily treatment with fumagillin administered at 5, 10, and 20 mg/kg of body weight subcutaneously (s.c.), TNP-470 administered at 20 mg/kg intraperitoneally (i.p.), or ovalicin administered at 5 mg/kg s.c. Of two ovalicin derivatives that were assessed in vivo, NSC 9665 given at 10 mg/kg i.p. daily also statistically significantly prolonged survival of the mice. No lesions associated with drug toxicity were observed in the kidneys or livers of uninfected mice treated with these drugs at the highest dose of 20 mg/kg daily. These results thus support continued studies to identify more effective fumagillin-related drugs for treating microsporidiosis.

Antimicrosporidial activity of (fluoro)quinolones in vitro and in vivo.

Microsporidia are a cause of emerging and opportunistic infections in humans and animals. Although two drugs are currently being used to treat microsporidiosis, concerns exist that albendazole is only selective for inhibiting some species of microsporidia that infect mammals, and fumagillin appears to have been found to be toxic. During a limited sequence survey of the Vittaforma corneae genome, a partial gene encoding for the ParC topoisomerase IV subunit was identified. The purpose of this set of studies was to determine if fluoroquinolones, which target topoisomerase IV, exert activity against Encephalitozoon intestinalis and V. corneae in vitro, and whether these compounds could prolong survival of V. corneae-infected athymic mice. Fifteen fluoroquinolones were tested. Of these, norfloxacin and ofloxacin inhibited E. intestinalis replication by more than 70% compared with non-treated control cultures, while gatifloxacin, lomefloxacin, moxifloxacin, and nalidixic acid (sodium salt) inhibited both E. intestinalis and V. corneae by at least 60% at concentrations not toxic to the host cells. These drugs were tested in vivo also, where gatifloxacin, lomefloxacin, norfloxacin, and ofloxacin prolonged survival of V. corneae-infected athymic mice (P < 0.05), whereas moxifloxacin and nalidixic acid failed to prolong survival. Therefore, these results support continued studies for evaluating the efficacy of the fluoroquinolones for treating microsporidiosis and for characterizing the target(s) of these fluoroquinolones in the microsporidia.

Effects of Nosema bombi and its treatment fumagillin on bumble bee (Bombus occidentalis) colonies.

We examined the effects of Nosema bombi (Microsporidia: Nosematidae) on colonies of bumble bees, Bombus occidentalis Greene (Hymenoptera: Apidae), used to pollinate tomatoes in commercial greenhouses. We assessed methods of detecting N. bombi and tested the effectiveness of fumagillin to control this parasite. N. bombi did not affect adult population size or amount of brood in B. occidentalis colonies. Fumagillin was not effective against N. bombi at the doses we tested, and frass samples did not provide accurate estimates of the intensity of N. bombi infections. The number of N. bombi spores per bee was highly variable among bumble bees within colonies, and accurate estimates could only be obtained by sampling a large proportion of bees in each colony. Therefore, whole bee and frass sampling is useful for determining if N. bombi is present or absent, but not for obtaining accurate estimates of the intensity of N. bombi infections.

Fumagillin treatment of intestinal microsporidiosis.

BACKGROUND: Intestinal microsporidiosis due to Enterocytozoon bieneusi is a cause of chronic diarrhea, malabsorption, and wasting in immunocompromised patients. Currently, there is no effective treatment. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of fumagillin (60 mg per day orally for two weeks) in patients with chronic E. bieneusi infection. Efficacy was assessed primarily by the clearance of microsporidia, as evidenced by analysis of stool specimens. Patients in whom microsporidia were not cleared received treatment for two weeks with open-label fumagillin. After clearance of the parasite, follow-up stool examinations were performed monthly to detect relapses. RESULTS: Twelve patients were enrolled in this study, 10 with the acquired immunodeficiency syndrome and 2 who had received organ transplants. Clearance of microsporidia occurred in all six of the patients in the fumagillin group, as compared with none of the six in the placebo group (P=0.002). Treatment with fumagillin was also associated with increases in absorption of D-xylose (P=0.003) and in Karnofsky performance scores (P<0.001) and with decreases in loperamide use (P=0.01) and total stool weight (P=0.04). There were serious adverse events (neutropenia and thrombocytopenia) in three patients in the fumagillin group; one patient in the placebo group had severe diarrhea. All six controls subsequently had clearance of microsporidia after open-label treatment with fumagillin. Relapses of the infection were identified in two patients during follow-up (median follow-up, 10 months). CONCLUSIONS: Fumagillin is an effective treatment for chronic E. bieneusi infection in immunocompromised patients.

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.

A preliminary investigation of alternatives to fumagillin for the treatment of Loma salmonae infection in rainbow trout.

The effects of the following six treatments against Loma salmonae, a microsporidian gill pathogen, were examined in rainbow trout: fumagillin (high dose), pyrimethamine + sulphaquinoxaline, albendazole, amprolium, fumagillin (low dose), and metronidazole. The fish were infected by mouth and the treatments were administered at intervals for a period of several weeks. The results were assessed on the basis of (1) delay in the formation of xenomas, and (2) the number of xenomas per gill arch. The first five treatments, in descending order of efficacy, delayed the formation of xenomas (P<0.01), but metronidazole had no such effect. Fumagillin (high or low dose) and albendazole both reduced the number of xenomas present 10 weeks after infection (P<0.01), but the other three treatments did not do so. From these results, both fumagillin and albendazole appeared to be of potential value in controlling L. salmonae infection in trout.

Efficacy of the fumagillin analog TNP-470 for Nucleospora salmonis and Loma salmonae infections in chinook salmon Oncorhynchus tshawytscha.

Oral treatment with fumagillin is effective for controlling various microsporean and myxosporean infections in fish. We tested a synthetic analog of fumagillin, TNP-470 (Takeda Chemical Industries), for its efficacy against 2 microsporean pathogens of salmon: Loma salmonae and Nucleospora salmonis. Chinook salmon Oncorhynchus tshawytscha were experimentally infected with either L. salmonae (per os) or N. salmonis (intraperitoneal, i.p., injection) and held in fresh water at 15 degrees C. Fish were then divided into 3 replicate groups: untreated or treated orally at 1.0 mg or at 0.1 mg drug kg-1 fish d-1. With L. salmonae, the high dose fish had 0.32 xenomas mm-2 of gill tissue compared to controls at 24.5 xenomas per mm2. With N. salmonis infections, untreated fish exhibited 100% infection, showed prominent clinical signs (e.g. renal swelling, anaemia), and high mortality. In contrast, fish treated at 1.0 mg kg-1 showed no clinical signs, and 16% of those treated at 0.1 mg kg-1 showed only mild gross pathological changes. With the treated groups, over 50% of the fish exhibited extremely light infections, even with high dose treatments, but no mortalities were attributed to N. salmonis infections. Uninfected fish treated at 1.0 mg drug kg-1 fish d-1 for 5 wk appeared clinically normal and showed no reduction in growth. However, about half of these fish exhibited atrophy of the renal interstitial hematopoietic tissue.