Microsporidia are obligate intracellular eukaryotic parasites that utilize a unique mechanism to infect host cells. One of the main characteristics of all microsporidia is that they produce spores containing an extrusion apparatus that consists of a coiled polar tube ending in an anchoring disc at the apical part of the spore. With appropriate conditions inside a suitable host, the polar tube is discharged through the thin anterior end of the spore, thereby penetrating a new host cell for inoculating the infective sporoplasm into the new host cell. This method of invading new host cells is one of the most sophisticated infection mechanisms in biology and ensures that the microsporidia enter the host cell unrecognized and protected from the host defence reactions. Recent studies have shown that microsporidia gain access to host cells by phagocytosis as well. However, after phagocytosis, the special infection mechanism of the microsporidia is used to escape from the maturing phagosomes and to infect the cytoplasm of the cells. Gaining access to cells by endocytosis, and escaping destruction in the phago-/endo-/lysosome by egressing quickly from the phagocytic vacuole to multiply outside the lysosome, is a common phenomenon in biology that has been evolved several times during evolution. How this is put into execution by the microsporidia is an inimitable principle by which an obligate intracellular organism has managed this problem. The extrusion apparatus of the microsporidia has obviously ensured the success of this phylum during evolution, resulting in a group of obligate intracellular organisms, capable of infecting almost any type of host and cell.
Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We used sequence data of the rRNA gene for the identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi, and E. hellem in different biological samples of HIV-infected patients by PCR, Southern blot hybridization, restriction endonuclease digestion analysis, cloning, and comparative genetic sequencing. One primer pair was used for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E, intestinalis, and E. hellem from samples with electron microscopy confirmed infection. The amplified 1.2 kb SSU-rRNA gene fragments were ligated into a pMOSBlue T-vector, transfected into pMOSS/ме competent cells, and were used as positive controls. Several primer pairs and hybridization probes were used to amplify and identify microsporidian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron microscopy was done on a subset of patient samples. DNA products were obtained from all samples with confirmed microsporidial infections. The identity of the DNA fragments was determined by Southern blot hybridization or by restriction endonuclease digestion analysis or by DNA sequencing. The results show that PCR is a reliable and sensitive indicator for the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differentiation of microsporidia, even between species which cannot be differentiated by light and/or electron microscopy.