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Polymerase Chain Reaction (PCR)
PCR offers considerable promise both for detection of microsporidia in clinical samples and identification of species. Sequences are known for the 16 S rRNA genes of the Encephalitozoon spp., E. bieneusi, V. corneae and T. hominis and, from these, it is possible to design primers which will amplify all species (based on highly conserved regions), or are genus- or species-specific. When genus-specific primers are used, species identification can still be achieved by using species-specific oligonucleotide probes on Southern blots or by restriction digests. Vossbrinck et al. (1993) used primers for a region of the rDNA spanning part of the small subunit, the ITS region and part of the large subunit to amplify E. hellem, E. cuniculi and V. corneae from culture and differentiated these with restriction digests using Sau3a, EcoR1, Dra1 and Hinf1.
The first attempt to amplify microsporidian DNA from stool involved a lengthy (4 day) and complicated procedure involving mechanical and chemical disruption of spores (Fedorko et al.,
1995). Later methods have shortened and simplified the procedure. Fresh, fixed or frozen tissue samples can be used for DNA extraction, with or without prior disruption by grinding. Stool samples can be processed after formalin fixation and dilution. Specimens are usually incubated in lysis buffer containing SDS and proteinase K. Ombrouck et al. (1997) recommended simple boiling of formalin-fixed faeces at 100°C and
found that as few as 10 spores in a specimen could be detected.
PCR has been compared with standard staining techniques in several surveys. David et al. (1996) detected microsporidia (E. bieneusi or E. intestinalis) in 26/28 (93%) of intestinal biopsies from patients with proven micro-sporidiosis. Coyle et al. (1996) used PCR amplification with species-specific primers on intestinal biopsies for detection of E. bieneusi or E. intestinalis. They found that 25/68 patients with diarrhoea and 1/43 patients without diarrhoea were positive for E. bieneusi. Only 24 of these were positive by electron microscopy. Also E. intestinalis was detected in five out of the 68 patients with diarrhoea and none of the patients without diarrhoea, in accord with the TEM studies. Confirmation of the positive results was obtained by specific oligonucleotide probes on Southern blots. Franzen et al. (1996c), using PCR and Southern blots with E. bieneusi- and E. intestinalis-specific probes, detected five E. bieneusi, five E. intestinalis and five dual infections in 15 patients. The same technique was used to demonstrate the presence of E. intestinalis in stool samples, duodenal and bile juice, duodenal biopsies, urine, sputum, bronchiolar lavage and blood of one patient (Franzen et al., 1996b) and has also provided evidence for latent infection of E. intestinalis (Franzen et al., 1996a). The presence of E. intestinalis in blood is of special interest because it suggests that blood cells are used to transport the infection from the intestinal wall to the deeper viscera.
Amplification by PCR with non-specific primers, followed by restriction digests, have given good results with species identification. Raynaud et al. (1998) used HinfI for identification of E. intestinalis and were the first to identify this species in immunocompetent patients with diarrhoea. This restriction enzyme was also useful in differentiating the three Encephalitozoon spp. and E. bieneusi (Delbac and Vivares, 1997). Didier et al. (1996a) used FokI to identify E. hellem from a patient with conjunctival and renal infections. Other examples of progress in the use of PCR for microsporidian infections are: (a) specific amplification of part of the ITS region of E. bieneusi (Velasquez etal., 1996); (b) use ofselec-ted primers which amplified all Encephalitozoon spp., E. bieneusi and V. corneae but which gave
amplification products of different sizes according to species (Kock et al., 1997); (c) combination of PCR and RFLP to differentiate E. bieneusi, E. hellem, E. intestinalis and E. cuniculi from cultures (Katzwinkel-Wladarsch et al., 1997); (d) use of species-specific primers for the same range of species (del Aguila et al., 1997); and (e) use of E. intestinalis-specific primers to confirm identification of E. intestinalis infections in animals that may be a source of infection to man (Bornay-Llinares et al., 1998).
CLINICAL MANAGEMENT AND PREVENTION
In immunocompetent people microsporidial infections are likely to be self-limiting and require no treatment but chemotherapy can be given if necessary. In immunocompromised people, E. bieneusi causes chronic disorders (diarrhoea, cholecystitis, etc.) and, in the absence of any really effective drug, only palliative measures, such as non-specific antidiarrhoeal medication are useful. Microsporidioses due to the Encepha-litozoon spp. are progressive, with potentially fatal outcome if untreated.
The first anti-microsporidial drug, fumagillin, was used for the control of Nosema disease (Nosema apis) of honey bees (Bailey, 1953) and was later found to inhibit proliferation of E. cuniculi in vitro without killing the spores (Shadduck, 1980). It is an antibiotic produced by Aspergillus fumigatus but, both as the water-insoluble native product and as its water-soluble bicyclohexyl amino salt, Fumidil B, it is highly toxic if given systemically. Nevertheless, it has shown potent activity when applied topically in cases of keratoconjunctivitis caused by E. hellem and E. intestinalis (reviewed by Didier, 1997). A solution containing 3 mg/ml Fumidil B (= 70 pg/ ml fumagillin), applied hourly as drops, brought about relief of symptoms and reduction of epithelial damage in two patients with E. hellem ocular lesions but did not eliminate the spores, which were able to initiate proliferation on