Polymerase Chain Reaction for Diagnosis and Species Differentiation of Microsporidia
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FOLIA PARASITOLOGICA 45: 140-148, 1998. Polymerase chain reaction for diagnosis and species differentiation of microsporidia Caspar Franzen1, Andreas Müller1, Pia Hartmann1, Petra Hegener1, Matthias Schrappe2, Volker Diehl1, Gerd Fätkenheuer1 and Bernd Salzberger1 1Department of Internal Medicine I, University of Cologne, D-50924 Cologne, Germany; 2Quality Management, University of Cologne, D-50924 Cologne, Germany Key words: microsporidia, Enterocytozoon bieneusi, Encephalitozoon, polymerase chain reaction Abstract. 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 pMOSBlue 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. Microsporidia are obligate intracellular protozoan smears, bronchoalveolar lavage fluid, sputum, nasal parasites that infect a broad range of vertebrates and discharge, and biopsy tissues. invertebrates. These parasites were first recognized as Although the diagnosis and identification of pathogens of silkworms (Naegeli 1857), and had been microsporidia by light microscopy has greatly improved recognized as a cause of disease in many non-human during the last years, species differentiation is often hosts including insects, mammals, and fish (Canning impossible using these techniques. This is critical and Lom 1986) long before they were described as because albendazole is effective against human pathogens. Only ten well documented human Encephalitozoon spp. but not against E. bieneusi (Owen microsporidial infections had been reported up to 1985, 1997). Immunfluorescent staining techniques have been when a new species Enterocytozoon bieneusi has been developed for species differentiation of microsporidia, described in an HIV-infected patient from France but antibodies used in these procedures are not yet (Desportes et al. 1985, Modigliani et al. 1985). Since available outside research laboratories (Schwartz et al. then many cases of human microsporidiosis have been 1994). A variety of serological tests have been reported from all over the world, and microsporidia are developed to detect antibodies to microsporidia but the now recognized as one of the most common pathogens sensitivity and specifity of these tests are unknown and in HIV-infected patients (Weber et al. 1994). they are not suitable methods to diagnose infections in For most infectious diseases, techniques involving immunosuppressed persons (Hollister et al. 1991, Van microbiological isolation and growth characterization Gool et al. 1997). Cell culture systems are used for in offer the most rapid and specific determination of the vitro cultivation of microsporidia but this is not a ethiologic agent. This is not a suitable procedure for suitable technique for routine use (Schwartz et al. 1994, microsporidia, being obligate intracellular parasites that Van Gool et al. 1994a). require cell culture systems for growth. Therefore, During the last 10 years, the diagnosis of agents of diagnosis of human microsporidiosis is dependent on infectious diseases has begun to include the use of the identification of spores in clinical samples, and nucleic acid-based technologies. Diagnosis of parasitic microsporidia have now been described from virtually organisms is the last field of clinical microbiology to every tissue and body fluid in humans including stool incorporate these techniques and ribosomal RNA specimes, duodenal/bile juice, urine, conjunctival (rRNA) data are now increasingly used for diagnosis, Address for correspondence: C. Franzen, Department of Internal Medicine I, University of Cologne, Joseph-Stelzmann Str. 9, D-50924 Cologne, Germany. Phone: ++ 49 221 4784433; Fax: ++ 49 221 4786456; E-mail: [email protected] 140 Franzen et al.: PCR for diagnosis of microsporidia species differentiation, and phylogenetic analysis of Species-specific primers. The primer pair V1 and EB450 microsporidia (Schuitema et al. 1993, Vossbrink et al. (5’-ACTCAGGTGTTATACTCACGTC-3’) was used for 1993, Zhu et al. 1993, 1994, Weiss et al 1994, Baker et species-specific amplification of a 348-bp DNA fragment of al. 1995, Fedorko et al. 1995, Franzen et al. 1995, the SSU-rRNA gene of E. bieneusi at an annealing temperature of 48°C (Zhu et al. 1993) and the primer pair V1 1996a, 1996b, Da Silva et al. 1996, David et al. 1996, and SI500 (5’-CTCGCTCCTTTACACTCG-3’) was used for Didier et al. 1996a, Katzwinkel-Wladarsch et al. 1996). species-specific amplification of a 370-bp DNA fragment of This paper reviews data generated in our laboratory on the SSU-rRNA gene of E. intestinalis at an annealing the use of molecular-based techniques for diagnosis and temperature of 58°C (Weiss et al. 1994). Two primer pairs species differentiation of microsporidia. EHEL-F/EHEL-R (5’-TGAGAAGTAAGATGTTTAGCA-3’/ 5’-GTAAAAACACTCTCACACTCA-3’) and ECUN-F/ ECUN-R (5’-ATGAGAAGTGATGTGTGTGTGCG-3’/5’- MATERIALS AND METHODS TGCCATGCACTCACAGGCATC-3’) were used for species- Microsporidia and DNA isolation specific amplification of DNA from the SSU-rRNA gene of E. hellem and E. cuniculi, respectively (Visvesvara et al. 1994). Encephalitozoon intestinalis spores were obtained from PCR. Amplifications were done in 50 µl reactions under urine of a patient with transmission electron microscopy the following conditiones: 25 pmol of each primer, 200 µM of confirmed infection and grown as described (Visvesvara et al. each dNTP, 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM 1994, 1995, Franzen et al. 1996c) in MRC-5 human lung MgCl2, and 2.5 U Taq DNA polymerase (Perkin Elmer). One fibroblasts in MEM containing 10% FBS (Sigma). Cells were tenth to 1.0 µg genomic DNA (10 µl) and 40 µl mineral oil fed every three days and the supernatant which contains the were used. Reactions were run in a Perkin-Elmer thermocycler spores was harvested and stored at –20°C until processing. using a step cycle programm. After initial denaturation of the Intestinal biopsies from HIV-infected patient were obtained DNA at 94°C for 3 min, 35 cycles were run: 94°C for 1 min, from the distal duodenum by flexible fibreglass endoscopy. 40-60°C for 2 min, and 72°C for 3 min with a 10 min 72°C Samples were snap frozen in liquid nitrogen and stored at extention after the 35 cycles. A 10 µl aliquot from each -80°C until processing. Stool samples, urine, nasal discharge, reaction was run on a 3% NuSieve 3 : 1 electrophoresis-grade sputum, bronchoalveolar lavage fluid, duodenal/bile juice, and agarose gel (FMC) in 1 × TAE buffer (0.04 M Tris, 0.001 M blood from HIV-infected patients were stored at –20°C until EDTA) with ethidium bromide (0.5 mg/ml) to visualize the amplified PCR-products under UV-illumination (Franzen et al. processing. 1995, 1996a) Biopsies were incubated in digestion buffer with 400 µg Because of the fact that PCR testing is susceptible to cross proteinase K (Qiagen) at 55°C for two hours and DNA was contaminations, procedures for avoiding contamination were prepared using QIAamp spin columns (Qiagen) in an strictly followed. Pre- and post-PCR handling were physically Eppendorf microcentrifuge. Stool samples were concentrated seperated and performed in different rooms and positive by a water-ether sedimentation technique (Van Gool et al. displacement tips were used for all manipulations. Negative 1994b) and other body fluids by centrifugation for 10 minutes controls containing reaction mixtures without DNA were at 2,000 g. Sediments were incubated in digestion buffer with always run. 400 µg proteinase K and 0.4 U chitinase (Sigma) at 55°C for two hours with additional mechanical disruption of the spores Southern Blot Hybridization by glass beads (425-600 µm, Sigma). DNA was prepared After gel electrophoresis PCR-products were denaturated using QIAamp spin columns (Qiagen) in an Eppendorf and neutralized by soaking the gel in denaturation buffer (0.4 microcentrifuge. N NaOH, 0.6 M NaCl) and neutralization buffer (1.5 M NaCl, 0.5 M Tris, pH 7.5), for 30 min each. The separated amplified Polymerase Chain Reaction (PCR) DNA fragments were transferred to positively charged nylon General microsporidian primers.