Encephalitozoon cuniculi Genotype III Evinces a Resistance to Albendazole Treatment in both Immunodeficient and Immunocompetent Mice.

Of four genotypes of Encephalitozoon cuniculi, E. cuniculi genotype II is considered to represent a parasite that occurs in many host species in a latent asymptomatic form, whereas E. cuniculi genotype III seems to be more aggressive, and infections caused by this strain can lead to the death of even immunocompetent hosts. Although albendazole has been considered suitable for treatment of Encephalitozoon species, its failure in control of E. cuniculi genotype III infection has been reported. This study determined the effect of a 100× recommended daily dose of albendazole on an Encephalitozoon cuniculi genotype III course of infection in immunocompetent and immunodeficient mice and compared the results with those from experiments performed with a lower dose of albendazole and E. cuniculi genotype II. The administration of the regular dose of abendazole during the acute phase of infection reduced the number of affected organs in all strains of mice and absolute counts of spores in screened organs. However, the effect on genotype III was minor. Surprisingly, no substantial effect was recorded after the use of a 100× dose of albendazole, with larger reductions seen only in the number of affected organs and absolute counts of spores in all strains of mice, implying variations in albendazole resistance between these Encephalitozoon cuniculi genotypes. These results imply that differences in the course of infection and the response to treatment depend not only on the immunological status of the host but also on the genotype causing the infection. Understanding how microsporidia survive in hosts despite targeted antimicrosporidial treatment could significantly contribute to research related to human health.

Effects of Homeopathic Phosphorus on Encephalitozoon cuniculi-Infected Macrophages In-Vitro.

INTRODUCTION: Encephalitozoon cuniculi (E. cuniculi), a fungus that acts as an intracellular pathogen, causes a marked neurological syndrome in many host species and is a zoonotic concern. Although no well-established treatment for this syndrome is known, previous successful clinical experience using homeopathic phosphorus has been described in which symptom remission with no mortality occurred in 40/42 animals by means of unknown immunological mechanisms. The latter observation was the main motivation for this study. OBJECTIVE: To verify, in an in-vitro model, if macrophages infected with E. cuniculi can change in function after treatment with different potencies of phosphorus. MATERIALS AND METHODS: RAW 264.7 macrophages were infected with E. cuniculi in-vitro and treated with various homeopathic potencies of phosphorus. The vehicle was used as a control solution (0.06% succussed ethanol). After 1 and 24 hours, the following parameters were analyzed: parasite internalization (by the Calcofluor staining method), lysosome activity (by the acridine orange method), cytokine/chemokine production (by the MAGPIX system), and cell ultrastructure. Automatic image analysis was used when applicable, and the experiments were performed in triplicate. RESULTS: Treatment with vehicle alone increased interleukin (IL)-6, tumor necrosis factor alpha and monocyte chemotactic protein -1 production (p ≤ 0.05) and reduced the number of internalized parasites (p ≤ 0.001). A progressive and time-dependent increase in RANTES (regulated on activation, normal T-cell expressed and secreted) and lysosome activity (p ≤ 0.002) was observed only after treatment with the highest potency of phosphorus (Phos 200cH), together with decreased apoptosis rate, intense parasite digestion, and the presence of non-internalized spores. CONCLUSIONS: Phos 200 cH has a modulatory action on the activity of infected macrophages, especially a specific increase in RANTES, a key element in the prognosis of E. cuniculi-infected and of immunosuppressed patients bearing infections.

The course of infection caused by Encephalitozoon cuniculi genotype III in immunocompetent and immunodeficient mice.

Encephalitozoon cuniculi is probably the most common microsporidia which infects a wide range of vertebrates, including human. So far, four genotypes of this parasite have been identified based on the rRNA internal transcribed spacer variations. The course of infection caused by E. cuniculi III had very massive onset in immunocompetent host characterized by the presence of this parasite in all organs and tissues within one week after peroral infection. Encephalitozoonosis caused by E. cuniculi III had very progressive spreading into all organs within first week post inoculation in immunocompromised SCID mice and led to the death of the host. The experimental treatment with albendazole of immunocompetent BALB/c mice infected with E. cuniculi III have shown very weak effect. Our findings clearly showed that the different course of infection and response to treatment depends not only on the immunological status of the host, but also on the genotype of microsporidia. It could be very important especially for individuals under chemotherapy and transplant recipients of organs originating from infected donors.

Effects of selected Indonesian plant extracts on E. cuniculi infection in vivo.

The present study was conducted to evaluate the methanolic extracts from several plant leaves widely used in traditional medicine to cure digestive tract disorders and in the self-medication of wild animals such as non-human primates, namely Archidendron fagifolium, Diospyros sumatrana, Shorea sumatrana, and Piper betle leaves, with regard to their antimicrosporidial activity against Encephalitozoon cuniculi in immunocompetent BALB/c mice determined using molecular detection of microsporidial DNA (qPCR) in various tissues and body fluids of infected, treated mice. Of the plant extracts tested, Diospyros sumatrana provided the most promising results, reducing spore shedding by 88% compared to untreated controls. Moreover, total burden per 1 g of tissue in the D. sumatrana extract-treated group reached 87% reduction compared to untreated controls, which was comparable to the effect of the standard drug, Albendazole. This data represents the baseline necessary for further research focused on determining the structure, activity and modes of action of the active compounds, mainly of D. sumatrana, enabling subsequent development of antimicrosporidial remedies.

TNP-470 is an effective antimicrosporidial agent.

Therapy for microsporidia, which cause diarrhea and a wasting syndrome in persons with AIDS, has had limited success. Fumagillin, a naturally secreted water-insoluble antibiotic, has in vitro activity against microsporidia and has been used successfully in the treatment of superficial keratitis in patients with AIDS, but systemic therapy has been limited by toxicity of the currently available fumagillin salt. TNP-470, a semisynthetic analogue of fumagillin, was studied in vitro and in the athymic nude mouse model of microsporidiosis. RK13 cells were infected with microsporidia of the family Encephalitozoonidae and treated at day 3 with TNP-470. This agent was highly effective, with an ID50 (50% inhibitory dose compared with control) of 0.001 microg/mL. TNP-470 also demonstrated in vivo activity against Encephalitozoon cuniculi, with prolonged survival and the prevention of the development of ascites in infected athymic mice. These data suggest that the fumagillin derivative TNP-470 is a promising agent for the treatment of microsporidiosis.

Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin.

A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.

Topical fumagillin in the treatment of microsporidial keratoconjunctivitis in AIDS.

OBJECTIVE: To report microsporidial keratoconjunctivitis in a patient infected with HIV who was treated with topical fumagillin. CASE SUMMARY: A 37-year-old white man who was experiencing redness, pain, irritation, decreased vision, and a foreign body sensation occurring in both eyes was referred to the ophthalmology clinic. The patient had a medical history significant for AIDS, Pneumocystis carinii pneumonia, and Cytomegalovirus retinitis. Conjunctival smears were taken and stained positive for the presence of Microsporidia. The patient was diagnosed with bilateral microsporidial keratoconjunctivitis and fumagillin therapy was initiated. After 5 days of therapy, the patient reported significant improvements characterized by a decrease in blurred vision with only slight blurring in the left eye, decrease in headache, and decreased foreign body sensation. The patient continued topical fumagillin therapy for more than 14 months, with only slight blurring in the left eye and no apparent ocular toxicity as a result of fumagillin therapy. DISCUSSION: Although rare in occurrence, increasing numbers of documented microsporidial infections are being reported in the medical literature, particularly in individuals who are seropositive for HIV. Clinicians need to be cognizant of microsporidial keratoconjunctivitis as another opportunistic infection in this patient population. CONCLUSIONS: Although a curative agent has yet to be discovered, fumagillin represents a safe, effective, low-cost, topical agent for the treatment of microsporidial keratoconjunctivitis.

Successful treatment of microsporidial keratoconjunctivitis with topical fumagillin in a patient with AIDS.

There recently have been several reports of microsporidial keratoconjunctivitis caused by the organism Encephalitozoon hellem. However, treatment of this infection has been largely ineffective. We report a case of a 35-year-old Hispanic woman with AIDS and E. hellem keratoconjunctivitis confirmed with light, electron, and immunofluorescence microscopy that resolved promptly with topical fumagillin, a crystalline antibiotic with proven efficacy against Encephalitozoon species. No corneal or systemic toxicities were noted using the dosage and preparation employed (10 mg/ml suspension in balanced salt solution). An easily prepared, topical fumagillin suspension appears to be a safe, effective treatment for E. hellem keratoconjunctivitis.

Treatment of microsporidial keratoconjunctivitis with topical fumagillin.

Encephalitozoon hellem is a newly described cause of microsporidial keratoconjunctivitis, occurring chiefly in patients with significantly diminished CD4+ T-lymphocyte levels. This disorder is symptomatically disabling and generally recalcitrant to topical antimicrobial therapy. Two homosexual men with E. hellem keratoconjunctivitis diagnosed by Gram stain, transmission electron microscopy, and specific indirect immunofluorescent assay were treated with topical fumagillin (Fumidil B). Both patients had marked symptomatic improvement with reduction of clinical findings. Symptoms and signs recurred with temporary discontinuation of the drug. Both patients, however, remained symptom-free on maintenance levels of topical fumagillin with no evidence of toxic side effects.