Article Vol. 16, No. 5 369 Eur. J. Clin. Microbiol. Infect. Dis., 1997, 16:369-376 Species-Specific Identification of Microsporidia in Stool and Intestinal Biopsy Specimens by the Polymerase Chain Reaction N.R Kock 1., H. Petersen 2, T. Fenner 2, I. Sobottka 3, C. Schmetz 4, R Deplazes 5, N.J. Pieniazek 6, H. Albrecht 7, J. Schottelius I In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are re- quired. In the present study, the polymerase chain reaction (PCR) was used for species- specific detection of Encephalitozoon cunicufi, Encephalitozoon hellem, Encephafitozoon (Septata) intestinalis, and Enterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected with Enterocytozoon bieneusi (n = 9), Encephalitozoon spp. (n = 2), and Encephafitozoon intestinalis (n = 1) as well as stool spiked with spores of Encephafitozoon cunicufi and Encephalitozoon hellem and tissue cultures of Encephalitozoon cuniculi and Encephalitozoon hellem, three proce- dures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. With- out exception, the PCR assay confirmed electron microscopic detection of Enterocyto- zoon bieneusi and Encephalitozoon intestinalis in stool specimens and their correspond- ing biopsies and in spiked stool samples and tissue cultures infected with Encephalito- zoon cuniculi and Encephafitozoon hellem. Moreover, identification of Encephafito- zoon spp. could be specified as Encephalitozoon intestinalis. Whereas standard meth- ods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates species- specific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours. Microsporidia are ancient eukaryotic, obligate in- tential human pathogens since 1959 (3). As part of tracellular, spore-forming parasites infecting a the evolving pandemic of human immunodeficien- broad range of invertebrates and vertebrates, in- cy virus (HIV) infection, microsporidia have cluding primates (1). First identified in silkworms been identified with increasing frequency as (2), these protozoans have been recognized as po- causative agents of diarrhea and disseminated in- fections, mainly in AIDS patients (4). Over 400 1 Section of Parasitology, and 4Electron Microscopy Labora- cases of HIV-associated microsporidiosis have tory,Bernard Nocht Institute for TropicalMedicine, Bernard- been documented; however, only a few cases Nocht-Strage 74, D-20359 Hamburg, Germany. have been described among persons not infected 2 Institute for Clinical Pathology and Microbiology, Dres. with HIV (1, 5). Fenner and Partners, Hamburg, Germany. 3 Institute of Microbiologyand Immunology,and 7Department The six genera Encephalitozoon, Enterocyto- of Internal Medicine, University Hospital Eppendorf, Ham- zoon, Nosema (6), PIeistophora (7), Trachipleisto- burg, Germany. phora (8), and Vittaforma (9), as well as unclassi- 5 Institute of Parasitology,University of Zt~rich,Zfirich, Swit- fied microsporidian organisms (referred to by the zerland. 6 Division of Parasitic Diseases, National Center for Infectious collective term Microsporidiurn), have been asso- Diseases, Centers for Disease Control and Prevention,Atlan- ciated with human microsporidiosis. Intestinobil- ta, Georgia, USA. iary infections with Enterocytozoon bieneusi (10) 370 Eur. J. Clin. Microbiol. Infect. Dis. are the most common microsporidial diseases, at 37~ using M-199 medium with Hanks' salts and L-gluta- but disseminated infections with Encephalito- mine (Gibco BRL, Germany) supplemented with 2 to 5% heat-inactivated fetal bovine serum (Gibco BRL), nonessen- zoon cuniculi (11), Encephalitozoon hellem (12), tial amino acid solution (Sigma, Germany), and penicillin- and Encephalitozoon (Septata) intestinalis (13, streptomycin (Gibco BRL). Spores were harvested by centri- 14) are increasingly being recognized (15-17). fugation (1,500 x g for 20 min) of the cell culture supernatant, Enterocytozoon bieneusi and Encephalitozoon washed three times in 20 mM (pH 7.2) of phosphate-buffered intestinalis have been found in 10 to 40% of HIV- saline (PBS), diluted in PBS to a concentration of 106 infected patients with chronic diarrhea, mal- spores/mI, and stored at 4~ Tissue specimens were harvest- ed with a cell scraper after removing the medium, suspended absorption, and severe weight loss (18, 19). in PBS, centrifuged (600 x g for 10 min), and stored at 4~ Identification of these parasites to the species lev- Uninfected E6 cells were used as a negative control. el is of clinical importance, since epidemiological Stool Specimens. Stool specimens were obtained from HIV- data are limited and few studies have document- positive male patients with acute diarrhea caused by Entero- cytozoon bieneusi (n = 8; 1 patient was coinfected with Cam- ed the response of pathogenic microsporidia to pylobacter jejuni), Encephalitozoon spp. (n = 2), and Enceph- chemotherapeutic agents (17, 20). Despite a varie- alitozoon intestinalis (n = 1) and from an immunocompetent ty of methods for the detection of microsporidian HIV-negative female child infected with both Enterocyto- infections, definitive diagnosis depends upon ultra- zoon bieneusi and Cryptosporidium parvum (5). Microspori- structural examination using the transmission dia were detected by a fluorescent stain with Calcofluor electron microscope (TEM) (4). However, it is not White M2R (32, 33) and a modified chromotrope-based Weber stain (34). Identification to the genus level was achieved always possible to identify the species by TEM. by TEM as described previously (16). Encephalitozoon intes- Furthermore, in specimens such as stool, urine, and tinalis was determined by TEM examination of an intestinal sputum, microsporidian pathogens can only be biopsy. Furthermore, the diagnosis was confirmed by align- identified to the genus level (1). In addition, En- ment of the cloned and partially sequenced SSU rRNA gene cephalitozoon cuniculi and Encephalitozoon hel- to published sequences of this species (35). Soft stool used for spiking with spores (200 spores/g stool) of Encephalitozoon lem are morphologically indistinguishable, even in cuniculi isolate Budejovice, Encephalitozoon cuniculi isolate tissue samples (21). Since light and electron micro- CH-K-4379, and Encephalitozoon hellem isolate CH-H-3, re- scopic techniques may lack sensitivity and the use spectively, was obtained from an asymptomatic person with- of immunodiagnostic tests may be limited by the out microscopically detectable parasites. In addition, this absence of Enterocytozoon bieneusi-specific anti- stool sample was used as a negative control. For determina- tion of the influence of fixatives on DNA isolation and PCR bodies, DNA amplification methods such as the amplification, stool specimens of two patients infected with polymerase chain reaction (PCR) have been de- Enterocytozoon bieneusi and one with Encephalitozoon intes- veloped for the diagnosis of microsporidian infec- tinalis were fixed in 100% ethanol, 4% formalin, 2% glutaral- tions (22-30). Using primers targeted to the small dehyde (Piano, Germany), and 4% paraformaldehyde (Serva, subunit (SSU) rRNA gene of microsporidia, Germany), respectively. All stool samples were stored up to identification of Enterocytozoon bieneusi and one week at 4~ and then either processed immediately or frozen at -20~ until examination. Encephalitozoon intestinalis in intestinal biopsies (23, 24, 28, 30) and of Enterocytozoon bieneusi, En- Intestinal Biopsy Specimens. Biopsy specimens were obtained from the distal duodenum of two patients infected with Entero- cephalitozoon cuniculi, and Encephalitozoon intes- cytozoon bieneusi and one with Encephalitozoon intestinalis tinalis in stool specimens (22, 25, 29, 30) has been by flexible fiberglass endoscopy. One part of the biopsied ma- successfully demonstrated. The present report terial was kept in physiological NaC1 solution; the other parts describes a simple and rapid nested PCR assay for were fixed with 4% formalin and 2% glutaraldehyde, respec- species-specific detection of Enterocytozoon bie- tively. Microsporidia were detected by TEM. The diagnosis of neusi, Encephalitozoon cuniculi, Encephalito- Encephalitozoon intestinalis infection was confirmed as al- ready described. All biopsy specimens were stored up to one zoon hellem, and Encephalitozoon intestinalis in week at 4~ and then either processed immediately or frozen both tissue and stool samples. at -20~ until examination. DNA Isolation. DNA was isolated from biopsy specimens and tissue cultures using the QIAamp Tissue Kit (Qiagen, Germa- Materials and Methods ny) as described previously (23). Stool specimens (250 mg) were thoroughly diluted in 1.5 ml of TE buffer (10 mM of Tris-HC1, 1 mM of EDTA, pH 7.2), centrifuged in an Eppen- Parasite Cultures. The Vero E6 green monkey kidney cell line doff centrifuge 5415C (7,500 x g for 5 min), and resuspended as well as Encephalitozoon cuniculi isolate Budejovice were in 0.5 ml of TE buffer. Washed stool was stored up to one kindly provided by Jiri Lom and Oleg Ditrich of the Institute week at 4~ and then either processed