Species in Urine, Stool, and Nasopharyngeal Specimens

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1993, p Vol. 31, No /93/ $02.00/0 Copyright C 1993, American Society for Microbiology A New Trichrome-Blue Stain for Detection of Microsporidial Species in Urine, Stool, and Nasopharyngeal Specimens NORBERT J. RYAN,'` G. SUTHERLAND,' K. COUGHLAN,' M. GLOBAN,1 J. DOULTREE,' J. MARSHALL,' R. W. BAIRD,' J. PEDERSEN,2 AND BRIAN DWYER' Victorian Infectious Diseases Reference Laboratory, Fairfield Hospital, Yarra Bend Road, Fairfield, Victoria 3066,2 Australia Victoria 3078,1 and Melbourne Pathology, Collingwood, Received 7 April 1993/Returned for modification 29 June 1993/Accepted 31 August 1993 Detection of microsporidia in clinical specimens has relied on electron microscopy, histology, or staining. This article describes further alterations to the modified trichrome staining method which make it easier to identify microsporidial spores. The changes are a decrease in the phosphotungstic acid level and the substitution of a colorfast counterstain, aniline blue, for the fast green of the original stain. The modified stain provides good contrast between microsporidial spores and background material including human and fungal cells. Stool specimens from 139 human immunodeficiency virus-seropositive patients revealed that 5 patients were infected with Enterocytozoon bieneusi and 6 patients had larger spores. Thin-section electron microscopy of the larger spores showed a structure consistent with that of either Encephalitozoon or Septata species. Three of the patients with Encephalitozoon- or Septata-like species had disseminated infection, with spores detected in nasopharyngeal aspirates and urine samples. Microsporidia are obligate intracellular parasites characterized by production of spores which contain a coiled tubule. Infection of a host cell is initiated when appropriate stimuli cause the tubule to extrude, penetrating the host cell membrane and allowing discharge of infective sporoplasm into the cell. The subsequent replicative and spore-forming phases culminate in host-cell rupture and spore release. Microsporidia have been detected in many species including insects, fish, and mammals (6, 7). Prior to 1985, reports of microsporidial infection in humans were extremely rare, but there is emerging evidence linking these protozoa with disease of human immunodeficiency virus-seropositive patients (6). To date, two species of microsporidia have been identified in bowel infections: Enterocytozoon bieneusi, first described in 1985 (9), produces a comparatively small spore (1.5 by 0.5 jum); other reports describe larger spores (2 by 1.0,um to 2.5 by 1.0 jim) detected in jejunal biopsies (2, 16). It has been estimated that Enterocytozoon bieneusi may account for between 6.5 and 27% of previously unexplained, chronic diarrhea in AIDS patients (11, 13, 15). Although it was formerly thought that Enterocytozoon bieneusi infects only enterocytes of the small bowel (8, 15), subsequent studies indicate that it can also colonize the bile duct epithelium, giving rise to cholangitis (1, 18). Far less is known about the sequellae of infection with microsporidia of the larger spore type. However, in one study five patients with disseminated infection were described (16). All had severe diarrhea, one had microsporidial infection of the gall bladder associated with cholecystitis, and two had renal involvement. The similarity to Encephalitozoon cuniculi in spore size prompted the designation Encephalitozoon-like (16), but the researchers highlighted major differences between the two (16, 17). The culmination of their work has been the naming of a new genus and species, Septata intestinalis (3), distinguished from other members of the Microspora phylum by * Corresponding author production of a fine honeycomb-like network surrounding the developing spores within a parasitophorous vacuole (3). Despite the perceived importance of microsporidiosis in AIDS, substantive studies to determine the true prevalence of infection have been lacking. Progress has, to a large extent, been impeded by the difficulty of detecting these protozoa. Definitive diagnosis has often depended on invasive procedures, but even with access to biopsy material, there has been potential for underdiagnosis. Many of the standard histologic stains will not stain microsporidia (6). Furthermore, in the examination of histologic specimens, microsporidia can be overlooked, because they can reside in tissue without provoking an inflammatory response (20). For diagnosis of intestinal microsporidiosis, spores can be detected in feces, but their small size and similarity to some bacteria have made this a difficult task. For Enterocytozoon bieneusi, Giemsa staining of fecal extracts was proposed as one means of diagnosing infection (24), but the poor differentiation of these spores from other fecal material meant that this method did not gain popular support. In contrast, development of a modified trichrome staining method (25) constituted a significant improvement, opening the way to routine screening of much greater numbers of patients without the need for invasive procedures. In the work presented below, details are provided of a further refinement to this staining procedure, applicable to the detection of spores of both Enterocytozoon bieneusi and the larger Encephalitozoon- or Septata-like species, in feces and in body fluids. MATERIALS AND METHODS Patients. Over a 6-month period, multiple diarrheal stool specimens were received from 139 human immunodeficiency virus-seropositive patients who either attended the Outpatients clinic or were hospitalized in two AIDS-dedicated wards of Fairfield Hospital, Melbourne, Australia. In the patients with microsporidial infections, CD4 cell counts taken in the months preceding diagnosis were taken from patient histories. Specimen processing. Stool specimens were suspended in

2 VOL. 31, 1993 AN IMPROVED TRICHROME STAIN FOR MICROSPORIDIA ml portions of nutrient broth with glass beads to reach an approximate 10% solution (wtlvol). The suspension was used both for microscopy and for the inoculation of plating media for the isolation of Salmonella, Shigella, and Campylobacter spp. and Clostridium difficile. Examination for the presence of parasites was performed by microscopy of two coverslip areas (20 by 20 mm), the second with added iodine. Smears (approximately 30 by 15 mm) were also prepared from 30-,ul portions of suspension, air dried, fixed in methanol for 10 min, and stained by the cold Ziehl-Neelsen method. Stained smears were then examined for the presence of Cryptosporidium spp. and other coccidia. As an additional procedure, the modified trichrome staining method of Weber et al. (25) for microsporidia was initially incorporated in this protocol. A slight variation to this method was introduced for ease of processing: slides were prepared using 10,ul of fecal suspension without added formalin. Smears were spread to cover an area approximately 20 by 15 mm, air dried, and then fixed promptly in methanol. Microsporidial control smears were prepared in a similar manner from a formalin-preserved suspension containing Enterocytozoon bieneusi spores, kindly provided by D. Marriott, St. Vincent's Hospital, Darlinghurst, Sydney, Australia. Control slides were included with each batch for staining. Trichrome-blue stain. A number of variations to the modified trichrome stain (25) were tried in an attempt to improve the contrast between the color of spores and background staining. Optimal staining was achieved by modifying the composition of the trichrome stain solution. The solution adopted was prepared by mixing 6.0 g of chromotrope 2R (batch ; JD Bolton, Upminster, Essex, United Kingdom), 0.5 g of aniline blue CI (BDH Ltd., Poole, United Kingdom), and 0.25 g of dodecatungstophosphoric acid AR (BDH Ltd.) with 3 ml of glacial acetic acid. After the mixture was allowed to stand for 30 min, 100 ml of distilled water was added and the ph was adjusted to 2.5 with 1.0 M hydrochloric acid. The staining solution was protected from light. This trichrome-blue solution contrasts with the modified trichrome solution of Weber et al. (25) in two respects: aniline blue has been substituted for fast green and there has been a reduction in the level of phosphotungstic acid. Other steps in the staining procedure parallel those of Weber et al. (25): following fixation in methanol for 10 min, slides were stained in trichrome-blue for 90 minutes, rinsed for 10 s in acid alcohol (0.45% acetic acid in 90% ethanol) and then for 10 s in 95% ethanol. Slides were then transferred through two changes of 95% ethanol for 5 min each, transferred into 100% ethanol for 10 min, and then transferred into Histosol, a xylene substitute (Interpath Services Pty. Ltd., Melbourne, Australia), for 10 min. Slides were mounted in DePeX (BDH, Kilsyth, Australia) and examined. Thin-section electron microscopy of stool and urine samples. A 10% (wt/vol) suspension of feces was prepared in sterile water and filtered with a Parafilter of 0.5-mm mesh (Johns, Hardie Health Care Products, South Oakleigh, Australia). The homogenate was shaken with 5 ml of ether for approximately 10 s and centrifuged for 5 min at 2,500 x g. The ether overlay, fecal plug, and supernatant were discarded. The pellet was resuspended in 1 ml of sterile water and centrifuged at 13,000 x g for 2 min (14). The supernatant was discarded, and the pellet was mixed with a drop of 2% (wt/vol) agarose. The agarose-pellet mixture was centrifuged for 2 min at 13,000 x g, and the pellet was fixed in 2% (wtlvol) glutaraldehyde in 0.1 M Sorensen phosphate buffer (ph 7.3) for 1 h at 4 C. The agarose block was cut into 1-mm3 pieces and fixed for another hour. The pellets were postfixed in 1% (wtlvol) osmium tetroxide for 1 h at 4 C, dehydrated in a series of graded alcohols, cleared in propylene oxide, and embedded in LX112 resin (Ladd Research Industries Inc., Burlington, Vt.). For urine specimens, the sediment obtained by centrifuging 5 ml of urine at 1,500 x g for 10 min was resuspended in 2% (wt/vol) glutaraldehyde in 0.1 M Sorensen phosphate buffer, ph 7.3. This suspension was centrifuged for 5 min at 13,000 x g, and the pellet was mixed with a drop of 2% agarose and left at 4 C for 4 h. The pellet was cut into 1-mm3 pieces and processed as described above for feces. Ultrathin sections were cut with a diamond knife and stained with uranyl acetate (22) and lead citrate (19). Sections were examined in a Philips CM12 electron microscope. RESULTS Detection of spores in fecal suspensions. Over a 6-month period, fecal samples from 139 patients were screened for microsporidia by the trichrome-blue staining procedure. There were five patients with Enterocytozoon bieneusi detected (Fig. 1), including one case which was first diagnosed by duodenal biopsy (Warthin-Starry stain), prior to the receipt of stool specimens for testing. The spores stained pink-red and were approximately 1.5 by 0.9 pum. There were also six patients with a larger spore type which occurred in very high numbers in feces. Spore size was approximately 2 to 2.5 by 1.0 pum. These spores also stained red and had the characteristic ovoid shape and clear vacuole-like zone seen in microsporidia. Electron microscopy confirmed that these were microsporidial spores (see below). Detection of other parasites and bacterial pathogens. In this study, the following additional pathogens were detected: Clostridium difficile (8 patients), Campylobacter species (14 patients), Shigella flexneri (1 patient), hookworm (1 patient), Giardia lamblia (4 patients), Entamoeba histolytica (2 patients), and Cryptosporidium spp. (7 patients). The patient with hookworm also had a microsporidial infection (Encephalitozoon or Septata type). None of the other pathogens was detected in conjunction with microsporidial infection. Detection of spores in other sites. For one of the six patients with spores of the larger type, initial detection was made in a postbronchoscopy-induced sputum specimen. The sputum had been processed with dithiothreitol (1.0%) and stained with modified toluidine blue 0 stain for Pneumocystis cannii. The chance observation of pale, spore-like structures (stained in outline only) prompted further investigation of specimens from other sites from this patient. Spores were readily detected in the trichrome-blue-stained slides of induced sputum, feces, and nasal swabs and in cytospin deposits from nasopharyngeal aspirates (Fig. 2) and urine samples (Fig. 3) but not in bronchoalveolar lavage samples. As can be seen in Fig. 2, large numbers of spores were present in macrophage-like cells. No spores were detected in squamous or respiratory epithelium. This finding prompted the investigation of comparable specimens from other patients with the larger spores detected in feces. Three additional patients also had a similar pattern of infection (Table 1), but the other 2 patients with this parasite had already died (patients 2 and 4). Detection of spores by electron microscopy. Thin-section electron microscopy of stool and urine specimens (patient 5) confirmed the identification of microsporidia observed by the trichrome-blue stain. Microsporidial spores appeared

3 3266 RYAN ET AL. J. CLIN. MICROBIOL. i 4 4x. Ae FIG. 1. Trichrome-blue stain of fecal suspension containing Enterocytozoon bieneusi. Spores (arrow) are ellipsoidal structures staining pink-red with a small polar or central nonstaining zone. Bar = 5,um. roughly ovoid in shape, with a thick surrounding wall and coiled polar tubule (Fig. 4). In some cases, the polar tubule could be seen to have five or six turns, a finding consistent with either the genus Encephalitozoon or Septata. DISCUSSION The modified trichrome stain (25) constituted a major improvement in procedures for detection of microsporidia in t #,, I4. feces, but the researchers commented that not only microsporidial spores but also yeasts and other fecal elements may stain red. This factor coupled with the general lack of background color in this stain makes the screening of large numbers of slides very time-consuming. We therefore investigated the feasibility of using different background stains. The recipe chosen owes much to the work of Shoobridge (21), who has established a number of principles in regard to A *.*.s %.*. I^." A.-.T..f *' _... A J. 4 FIG. 2. Trichrome-blue stain of cytospin deposit of nasopharyngeal aspirate (patient 5). Numerous free spores of the larger type (Encephalitozoon- or Septata-like) are visible (arrow), and some infected macrophage-like cells are evident. Spores stain pink-red with a polar clear zone. Bar = 5,um.

4 VOL. 31, 1993 AN IMPROVED TRICHROME STAIN FOR MICROSPORIDIA _ FIG. 3. Triclirome-blue stain of a cytospin deposit of urine (patient 5). Only cell-associated spores of the larger type (Encephalitozoon- or Septata-like) are visible within macrophage-like cells. The spores stain with a polar clear zone. Bar = 5 polychrome stains. Essentially there were two differences from the method of Weber et al. (25). The phosphotungstic acid level has been reduced by approximately 65%. This has the effect of allowing greater background staining, because phosphotungstic acid appears to function as a competing "'colorless dye" (21). The second variation is in the counterstain. Fast green is quite pale and fades rapidly; this stain has been replaced by aniline blue, which in the presence of phosphotungstic acid is colorfast (21). There is an added advantage in that many of the yeast cells and pseudohyphae stain a grayish blue color. With prompt fixation of smears, the contrast between microsporidial spores and tissue and fungal cells is good and allows easy detection of this class of parasite. With experience in screening trichrome-blue-stained smears, it proved possible to recognize other parasites p.m. (although suboptimally). Cryptospondium oocysts stain either light pink or remain unstained, Giardia cysts can be readily identified, and amebic cysts are recognizable but lacking in nuclear detail. The last observation is probably indicative of inadequate preservation. However, when smears were prepared in Schaudinn's fixative or from polyvinyl alcohol-preserved specimens, results were unsatisfactory; smears stained far too strongly. This highlights the requirement that smears-preserved specimens be prepared as a very thin, dilute film. Nevertheless with some modification, this stain could perhaps evolve into a multipurpose stain suitable for detection of a range of parasites. The stain is unsuitable for use with heavily bloodstained or postmortem material because both the erythrocytes and poorly preserved tissue stain strongly with chromotrope 2R. It has been recognized that patients with Enterocytozoon bieneusi infection have low CD4 counts (11). For patients in this study, a similar trend was apparent, confirming that these pathogens are associated with marked immunosuppression. The threshold for infection by Enterocytozoon bieneusi was a CD4 count of 50, but the threshold was somewhat higher (<130) for the larger spore type. In this study, the stool specimens were from a broad cross-section of human immunodeficiency virus patients, not necessarily with advanced illness or chronic diarrhea. The prevalence of 8% microsporidial infection would presumably be higher if testing were restricted to those patients with chronic diarrhea and low CD4 counts. Further work is required to determine whether the larger microsporidia detected in this study are spores of the genus Septata or Encephalitozoon. From the varied reports of disseminated infection by microsporidia cited below, none appear to directly parallel the cases seen in our study, in the range of sites infected. In 1987, Encephalitozoon cuniculi was reported as a cause of fatal hepatitis in an AIDS patient (23); 2 years later, the same species was identified in a case of peritonitis with ascites in an AIDS patient (26). This species has previously attracted more attention as a pathogen of laboratory and domestic animals, often causing neurological symptoms and kidney damage (7). The recently identified species Encephalitozoon hellem was first detected as a cause of keratoconjunctivitis in AIDS (4, 5, 10) but has since been identified in a case of disseminated illness, found at autopsy in the urinary and respiratory tracts but not the intestinal tract (20). Additionally, a case of severe chronic sinusitis has been attributed to Encephalitozoon-like infec- TABLE 1. Clinical data on patients with microsporidial infections Patient Specimen(s)a Date of AIDS di- CD4 cell Date of microsporidial Type of microsporidia no. agnosis countlh diagnosis 1 F, U, Sp. April July 1992 Encephalitozoon- or Septata-like 2 F July August 1992 Encephalitozoon- or Septata-like 3 F, NS, U September September 1992 Encephalitozoon- or Septata-like 4 F November June 1992 Encephalitozoon- or Septata-like 5 F, NS, U, NPA May November 1992 Encephalitozoon- or Septata-like 6 F, U, NPA May November 1992 Encephalitozoon- or Septata-like 7 F January May 1992 Enterocytozoon bieneusi 8 F August June 1992 Enterocytozoon bieneusi 9 F April August 1992 Enterocytozoon bieneusi 10 F September August 1992 Enterocytozoon bieneusi 11 F June October 1992 Enterocytozoon bieneusi a Microsporidia were detected in these specimens. Abbreviations: F, feces; U, urine; Sp, sputum; NS, nasal swab; NPA, nasopharyngeal aspirate. b The CD4 cell count preceding the diagnosis of the microsporidial infection. Normal range, 550 to 880.

5 3268 RYAN ET AL. J. CLIN. MICROBIOL. a b c W.. Downloaded from 'S ".4:' :.?,14 1%,i AL V..'.1I..., Vi IL... :4 i, k..i: i4,.'k'; -%ilw, 2.-A i"?,9 W:....;2.- t:.: 'r, d / on April 27, 2018 by guest.' FIG. 4. Electron micrographs of thin sections of Encephalitozoon- or Septata-like spores from feces and urine of patient 5. (a and b) Fecal spores. Note the characteristic polar tubules in the longitudinal section (a) (arrows) and transverse section (b) (arrows). Bars = 100 nm. (c) Degenerating cell from urine showing two microsporidial spores in vacuoles (arrows). Bar = 500 nm. (d) A higher magnification of the spore marked with an asterisk in panel c. The polar tubule (arrows) is evident. Bar = 100 nm.

6 VOL. 31, 1993 AN IMPROVED TRICHROME STAIN FOR MICROSPORIDIA 3269 tion (12). The newly described species Septata intestinalis has been identified in five patients as a cause of disseminated infection involving the intestinal tract, gall bladder, and kidney (3). In contrast, in our patients there was also involvement of the respiratory tract with evidence of heavy colonization of the nasopharyngeal region. It is therefore noteworthy that several of these patients had a history of chronic sinusitis with watery nasal discharge. It is significant that four of the six patients with the larger spore type were hospitalized in one ward over a period of several weeks. These findings should be considered with those of Schwartz et al. (20), who noted "prima facie evidence for primary infection of the tracheobronchial tree" in a case of disseminated Encephalitozoon hellem infection. Taken with our results, these observations may point to nosocomial spread of microsporidial infection. The full spectrum of symptoms associated with extraintestinal spread of microsporidia remains to be elucidated. However, knowledge in this field can be improved only by extending the range of specimens routinely screened for these parasites. In this regard, the new trichrome-blue stain offers a simple method for the detection of microsporidia in a wide range of specimens. REFERENCES 1. Beaugerie, L., M. F. Teilhac, A. M. Deluol, J. Fritsch, P. M. Girard, W. Rozenbaum, Y. LeQuintree, and F. P. Chatelet Cholangiopathy associated with microsporidia infection of the common bile duct mucosa in a patient with HIV infection. Ann. Int. Med. 117: Blanshard, C., W. S. Hollister, C. S. Peacock, D. G. Torey, D. S. Ellis, E. U. Canning, and B. G. Gazzard Simultaneous infection with two types of intestinal microsporidia in a patient with AIDS. Gut 33: Cali, A., D. P. Kotler, and J. M. Orenstein Septata intestinalis N.G., N. Sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients. J. Euk. Microbiol. 40: Cali, A., D. M. Meisler, C. Y. Lowder, R. Lembach, L. Ayers, P. M. Takvorian, I. Rutherford, D. L. Longworth, J. McMahon, and R. T. Bryan Comeal microsporidioses: characterization and identification. J. Protozool. 38:215S-217S. 5. Cali, A., D. M. Meisler, I. Rutherwood, C. Y. Lowder, J. T. McMahon, D. L. Longworth, and R. T. Bryan Corneal microsporidiosis in a patient with AIDS. Am. J. Trop. Med. Hyg. 44: Cali, A., and R. L. Owen Microsporidiosis, p In A. Balows, W. J. Hausler, M. Ohashi, and A. Turano (ed.), The laboratory diagnosis of infectious diseases, vol. 1. Springer- Verlag, New York. 7. Canning, E. U., and W. S. Hollister Microsporidia of mammals-widespread pathogens or opportunistic curiosities? Parasitol. Today 3: Canning, E. U., and W. S. Hollister Enterocytozoon bieneusi (Microspora): prevalence and pathogenicity in AIDS patients. Trans. R. Soc. Trop. Med. Hyg. 84: Desportes, I., Y. Le Charpentier, A. Galien, F. Bernard, B. Cochand-Priollet, A. Laverne, P. Ravisse, and R. Modigliani Occurrence of a new microsporidian: Enterocytozoon bieneusi n.g., n.sp., in the enterocytes of a human patient with AIDS. J. Protozool. 32: Didier, E. S., P. J. Didier, D. N. Friedberg, S. M. Stenson, J. M. Orenstein, R. W. Yee, F. 0. Tio, R. M. Davis, C. Vossbrinek, N. Millichamp, and J. A. ShadducL Isolation and characterization of a new human microsporidian, Encephalitozoon hellem (n. sp.) from three AIDS patients with keratoconjunctivitis. J. Infect. Dis. 163: Eeftinck Schattenkerk, J. K. M., T. van Gool, R. J. van Ketel, J. F. W. M. Bartelsman, C. L. Kuiken, W. J. Terpstra, and P. Reiss Clinical significance of small intestinal microsporidiosis in HIV-1-infected individuals. Lancet 337: Lacey, C. J. N., A. T. M. Clarke, P. Fraser, T. Metcalfe, G. Bonsor, and A. Curry Chronic microsporidian infection of the nasal mucosae, sinuses, and conjunctivae in HIV disease. Genitourin. Med. 68: Lucas, S. B., L. Papadaki, C. Conlon, N. Sewankambo, R. Goodgame, and D. Serwadda Diagnosis of intestinal microsporidiosis in patients with AIDS. J. Clin. Pathol. 42: Lumb, R., J. Swift, C. James, K. Papanaoum, and T. Mukherjee. Identification of developmental stages of the microsporidial parasite, Enterocytozoon bieneusi in faecal samples and intestinal biopsies from an AIDS patient. Int. J. Parasitol., in press. 15. Orenstein, J. M., J. Chiang, W. Steinberg, P. D. Smith, H. Rotterdam, and D. P. Kotler Intestinal microsporidiosis as a cause of diarrhoea in human immunodeficiency virusinfected patients: a report of 20 cases. Hum. Pathol. 21: Orenstein, J. M., T. Dieterich, and D. P. Kotler Systemic dissemination by a newly recognized intestinal microsporidia species in AIDS. AIDS 6: Orenstein, J. M., M. Tenner, A. Cali, and D. P. Kotler A microsporidian previously undescribed in humans, infecting enterocytes and macrophages, and associated with diarrhea in an acquired immunodeficiency syndrome patient. Hum. Pathol. 23: Pol, S., C. A. Romana, S. Richard, P. Amouyal, I. Despotes- Livage, F. Carnot, J. F. Pays, and P. Berthelot Microsporidia infection in patients with the human immunodeficiency virus and unexplained cholangitis. N. Engl. J. Med. 328: Reynolds, E. S The use of lead citrate at high ph as an electron opaque stain in electron microscopy. J. Cell Biol. 17: Schwartz, D. A., R. T. Bryan, K. 0. Hewan-Lowe, G. S. Visvesvara, R. Weber, A. Cali, and P. Angritt Disseminated microspodidiosis (Encephalitozoon hellem) and acquired immunodeficiency syndrome. Arch. Pathol. Lab. Med. 116: Shoobridge, M. P. K A new principle in polychrome staining: a system of automated staining complementary to haematoxylin and eosin, and usable as a research tool. Stain Technol. 58: Stempak, J. G., and R. T. Ward An improved staining method for electron microscopy. J. Cell Biol. 22: Terada, S., R. Reddy, L. J. Jeffers, A. Cali, and E. R. Schiff Microsporidian hepatitis in a patient with the acquired immunodeficiency syndrome. Ann. Intern. Med. 107: Van Gool, T., W. S. Hollister, J. Eeftinck Schattenkerk, M. A. van den Bergh Weerman, W. J. Terpstra, R. J. Van Ketel, P. Reiss, and E. U. Canning Diagnosis of Enterocytozoon bieneusi microsporidiosis in AIDS patients by recovery of spores from faeces. Lancet 336: Weber, R., R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, G. S. Visvesvara, and the Enteric Opportunistic Infections Worklng Group Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. New Engl. J. Med. 326: Zender, H. Q., E. Arrigoni, J. Eckert, and Y. Kapanci A case of Encephalitozoon cuniculi peritonitis in a patient with AIDS. Am. J. Clin. Pathol. 92: