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Ocular, peroral, intraperitoneal, intramuscular, and subcutaneous inoculation of severe combined immunodeficient (SCID) mice with spores of the human isolate (CDC: V404) of Brachiola algerae (syn. Nosema algerae) (Phylum Microspora) revealed that the microsporidium develops in viscera of the immunodeficient mouse host, but only after the ocular administration of spores. It is hypothesized that the physico-chemical milieu of the conjunctiva and cornea helped to adapt the originally 'poikilothermic microsporidian' to the conditions within the homoiothermic organism. Ocular application of spores caused no clinical signs of disease at the application site. However, severe infection in the liver was found 60 days after infection, manifested as hepatosplenomegaly and multifocal miliary necroses and granulomas containing parasites. No microsporidia were found in any other tissues. Transmission electron microscopy revealed characteristic tubulovesicular 'secretory materials' on the plasma membrane of all developmental stages of B. algerae except sporoblasts and spores. These formations increase the parasite surface and allow more efficient metabolic communication of the parasite with the host cell. It is hypothesized that the presence of these structures is a factor helping the parasite to grow in a variety of hosts and tissues. Ultrastructural characters support the likelihood that B. algerae and B. vesicularum are conspecific, and that there exists a relationship between species of the genera Brachiola and Anncaliia.

Objective: Enterocytozoon bieneusi is the most prevalent microsporidian causing chronic diarrhea in patients with acquired immunodeficiency syndrome. The current methods used for routine diagnosis of infections caused by microsporidia are based on microscopic detection of the microorganism spores in stained smears. We evaluated the usefulness of the polymerase chain reaction (PCR) technique as a tool to diagnose Enterocytozoon bieneusi infections, using the species-specific diagnostic primer pair EBIEF1/EBIER1 on stool samples that were also analyzed by optical microscopy., Design: To perform PCR in such samples, we developed a novel protocol to obtain DNA free of PCR inhibitors. This protocol was based on disruption of spores using glass beads and overnight digestion with proteinase K; final purification was accomplished with the RapidPrep Micro Genomic DNA isolation Kit for Cells and Tissues (Pharmacia Biotech Inc, Piscataway, NJ). We also evaluated this approach on aliquots of a sample fixed in formalin from 1 to 10 days. PATIENTS AND SAMPLES: We evaluated the PCR technique on 64 stool samples obtained from patients with acquired immunodeficiency syndrome who had persistent chronic diarrhea. Patients were from Spain, Brazil, Germany, and the United States., Results: Using this approach, we could confirm the presence of E bieneusi in all 17 positive samples; no false-positive results were observed. We could also amplify E bieneusi DNA in 10 aliquots of one sample fixed up to 10 days in 10% formalin., Conclusion: We conclude that PCR technology is very suitable for species identification of microsporidia in stool samples and may have a potential application in prospective studies in formalin-fixed samples.

Objective: This report describes a new and improved "quick-hot Gram-chromotrope" staining technique that detects microsporidian spores in clinical specimens, such as stool, urine, saliva, nasopharyngeal fluid, and bronchoalveolar lavage samples, as well as in formalin-fixed and paraffin-embedded tissue sections., Design: In this procedure, the samples are stained in heated (50 degrees C to 55 degrees C) solutions of crystal violet and iodine used in Gram's stain, followed by a modified chromotrope solution (heated to 50 degrees C to 55 degrees C). The modified stain is composed of chromotrope 2R (1%), fast green (0.15%), and phosphotungstic acid (0.25%)., Results: With this stain and the new protocol, microsporidian spores are stained dark violet against a pale green background, and the total staining time is shortened to 5 minutes., Conclusions: This new technique is fast, reliable, and simple. It can be easily adapted for use in clinical laboratories.