ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 24, No. 6 Copyright © 1994, Institute for Clinical Science, Inc.

Intestinal Microsporidiosis Report of Five Cases* TSIEH SUN, M.D., MARK H. KAPLAN, M.D., SAUL TEICHBERG, Ph.D., GARY WEISSMAN, M.D., THOM SMILARI, M.D., and CARLOS URMACHER, M.D. Department of Laboratories, Medicine and Pediatrics, North Shore University Hospital- Comeli University Medical College, Manhasset, NY 11030 and Departments of Pathology and Medicine, Cornell University Medical College, New York, NY

ABSTRACT Five cases of intestinal microsporidiosis are reported, including one case of a heterosexual female acquired immunodeficiency syndrome (AIDS) patient, three homosexual males, and one bisexual male AIDS patients with detailed description of their clinical course. These five cases under­ score the severity of immunodeficiency in patients with microsporidiosis. All patients had multiple opportunistic infections and a CD4 cell count below 100/|xl long before diarrhea developed. This is the first kinetic study of helper T-lymphocytes in cases of microsporidiosis. Diagnosis was made by duodenal biopsies stained with Brown and Brenn or Gram-Weigert technique (confirmed by electron microscopy) and by stool smears stained with a modified trichrome technique. However, the best preparation was plastic sections stained with toluidine blue, which demonstrated both the spores and plasmodia clearly. In our evaluation, Giemsa stain was also acceptable for identification of microsporidian spores in both intestinal biopsies and stool smears, but there was a failure to identify the organism on hematoxylin and eosin, acid-fast, periodic acid-Schiff, and Gomeri’s methenamine silver stained preparations. Therapeutic attempts using albendazole, metronidazole, octreotide, and zidovudine (AZT) failed to eradicate microsporidia in these patients.

Introduction * Address reprint requests to: Tsieh Sun, M.D., Department of Laboratories, North Shore Univer­ In industrialized countries, diarrhea sity Hospital-Comeli University Medical College, occurs in 30 to 60 percent of acquired Manhasset, NY 11030. immunodeficiency syndrome (AIDS) 521 0091-7370/94/1100-0521 $01.80 © Institute for Clinical Science, Inc. 522 SUN, KAPLAN, TEICHBERG, WEISSMAN, SMILARI, AND URMACHER patients.1 In Africa, diarrhea may be the Methods documenting feature of human immuno­ deficiency virus (HIV) infection and is The surgical specimens of cases 1, 2, called “slim disease” because of severe and 3 were fixed in formalin, embedded wasting that occurs.2 The cause of diar­ in paraffin, and stained with hematoxylin rhea in AIDS patients is multifactorial. and eosin (H&E). Special stains includ­ The most frequently identified organ­ ing Brown and Brenn, Gram-Weigert, isms are Cryptosporidium species, cyto­ Giemsa, acid-fast (AF), periodic acid- megalovirus (CMV) and Mycobacterium Schiff (PAS) and Gomori’s methenamine avium intracellulae.1 Less frequently, silver (GMS) were also performed. Salmonella, Shigella, Campylobacter Electron microscopic studies were jejuni, Giardia lamblia, Entamoeba his­ done on rehydrated, paraffin-retrieved tolytica and Strongyloides stercoralis are specimens from the intestinal biopsies of also encountered.1 Until a few years ago, patients 1, 2 and 3, using standard meth­ no known pathogen could be identified odologies. Initially, a one micron plastic in 50 percent of patients utilizing con­ section stained with toluidine blue was ventional stool studies and/or intestinal examined by light microscopy and biopsies.3,4 These patients with unex­ selected areas were then studied by elec­ plained diarrhea have been classified as tron microscopy. having AIDS enteropathy.1,5 Recently, Stool smears from cases 1, 2, 4 and 5 when intestinal biopsies from patients were stained with a modified trichrome with AIDS enteropathy were examined stain as described by Weber et al.14 A by electron microscopy, 30 percent were Giemsa stain was also performed in found to be infected with microsporidia.6 these cases. Among all AIDS patients, the incidence of microsporidiosis is now estimated to be 15 Results to 27 percent.6,7 Intestinal microsporidio­ sis is almost exclusively induced by a H is t o l o g ic F in d in g s newly discovered species, Enterocytozoon bieneusi,8 although a few cases infected by The duodenal biopsy of case 3 showed a new species, Septata intestinalis, have mild atrophy and blunting of the intesti­ recently been reported.9,10 Another newly nal villi (figure 1). The lamina propria discovered species, Encephalitozoon was infiltrated by moderate numbers of hellem, does not invade the intestine.11 plasma cells and lymphocytes. Cases 1 Five cases of intestinal microsporidio­ and 2 revealed only mild cellular infiltra­ sis are reported to exemplify the protean tion of the lamina propria. Microsporidia clinical manifestations of this infection were not identified in H&E, acid-fast, and its association with extremely low PAS, or GMS-stained sections. Brown CD4 cell counts well before the develop­ and Brenn and Gram-Weigert stains, ment of chronic diarrhea. Kinetic helper however, demonstrated clusters of T-lymphocyte study such as in this inves­ microsporidial spores inside the entero- tigation has not been reported previ­ cytes at the tip of the intestinal villi (fig­ ously. Previously reported cases of ure 2). Giemsa-stained preparations microsporidiosis were almost exclusively showed a few spores, which were not as seen in homosexual male AIDS easily recognizable as in Brown and patients6,12 with only two female patients Brenn-stained sections. Most of the para­ reported recently.13 Our case 1 may rep­ sitized enterocytes were degenerated or resent the third heterosexual female sloughed off from the villi. No organisms patient ever reported. were demonstrated in the crypts. Both INTESTINAL MICROSPORIDIOSIS 523

FIGURE 1. A duodenal biopsy of case 3, showing mild atrophy and blunting of the intestinal villi. The lamina propria is infiltrated by moderate numbers of plasma cells and lymphocytes. Hematoxylin and eosin, x 125. spores and plasmodia were clearly visi­ immature spores (figure 6). The straight ble in enterocytes when studied in tolu- section of a polar tube with connection to idine blue-stained plastic sections the anterior anchoring disc was demon­ (figure 3). strated in one sporoblast (figure 7).

E l e c t r o n M ic r o s c o p y St o o l E x a m in a t io n Electron microscopy revealed plasmo­ Microsporidial spores (figure 8) were dia, sporoblasts, and spores in all three found in all four stool specimens exam­ cases. The plasmodia included meronts, ined. They were very small (1 x 1.5 jxm), which were characterized by the pres­ requiring oil emersion (100x) for defini­ ence of multiple clefts, and sporonts, con­ tive identification. With modified tri­ taining stacks of electron dense discs (fig­ chrome stain, the organism stained red ure 4 and figure 5). Spores were and other bacteria or yeasts stain faint identified by their polar tubes (usually in green or colorless. Giemsa stain also 6 pairs), a posterior vacuole, a nucleus demonstrated the spores, but it was diffi­ and a trilaminar cell wall (figure 6). Spo­ cult to distinguish them from the sur­ roblasts were identified as unseparated rounding bacteria. SUN, KAPLAN, TEICHBERG, WEISSMAN, SMILARI, AND URMACHER

FIGURE 2. Clusters of microsporidial spores are demonstrated in the degenerated or sloughed entero- cytes (arrows) from a duodenal biopsy of case 1. Brown and Brenn, X1250.

Case Reports esophagus and stomach were normal. The duode­ num revealed a granular appearing mucosa with edematous valvulae. Aspirates were negative for Case 1 bacterial and viral pathogens. Initial studies of her intestinal biopsy for microsporidia were interpreted A 35-year-old Philippino woman developed as negative. Re-examination of the biopsy with lymphadenopathy in 1986. She was found to be Brown and Brenn stain two months later demon­ HIV positive. She contracted the disease from her strated microsporidia, which were confirmed by husband who was heterosexually infected. electron microscopy. Although she was treated with zidovudine (AZT), In April, 1992, stool examination with modified her CD4 cell count fell to less than 200 cells/jjd. In trichrome stain showed numerous microsporidial April 1991, she developed aphthous mouth sores; spores. Albendazole was started for the treatment of in May, she had oral hairy leukoplakia and oral microsporidiosis with little effect on diarrhea. In and vaginal thrush. Her CD4 cell count was now April, 1992, she developed disseminated Mycobac­ 98/|j l 1 (table 1). She was switched to Stavudime (DDC) therapy. terium avium-intracellulare (MAI), which was con­ Her CD4 cell count continued to drop, and by trolled with antibiotics. In August, 1992, she was August, 1991, it was 62 cells/(xl. In November, 1991, started on home total parenteral nutrition (TPN) she began having 4 to 5 watery bowel movements because of continued weight loss. In early 1993, the per day associated with epigastric cramps. Over patient’s general condition and diarrhea were four months, she lost 15 pounds. She was treated improved. However, the stool examination still with metronidazole and ciprofloxacin with only showed microsporidia. As of April, 1993, she had mild improvement. Routine stool cultures and para­ continued epigastric discomfort and mild diarrhea sitologic examinations were negative. A barium as she continued on TPN. She was found to have enema was normal. An upper GI with small bowel CMV retinitis at her last visit. follow-up revealed only esophageal candidiasis. In February, 1992, her CD4 cell count dropped to Case 2 22 cells/jxl. To further elucidate the etiology of her diarrhea, the patient underwent an upper endos­ A 33-year-old white homosexual man developed copy and distal duodenal aspirate and biopsy. Her chronic diarrhea in November, 1987, after returning INTESTINAL MICROSPORIDIOSIS 525

FIGURE 3. A plastic section of duodenal biopsy from case 2 showing a few clusters of microsporidial spores (open arrows) and plasmodia (arrows) in the enterocytes at the tip of an intestinal villus. Toluidine blue stain, X1250. from Mexico. He had 3 to 6 watery bowel move­ 1993, the biopsy of his intestine taken in April, ments per day. Multiple stool examinations for ova 1992, was reviewed in light of new methods of and parasites, Clostridium difficile, acid fast bacilli, staining for microsporidia, and it now showed the and bacterial pathogens were negative. Barium presence of microsporidia. Stool examination enema and upper gastrointestinal series, and upper revealed microsporidial organisms. He further dete­ and lower gastrointestinal endoscopy failed to iden­ riorated with worsening lymphoma and died in tify the cause of diarrhea. He was found to be HIV February, 1993. seropositive with a CD4 cell count of 15 cells/jo-l. Treatment with metronidazole, bismuth subsalicy­ late, AZT, diphenoxylate and atropine had only C ase 3 transient effect on diarrhea. His illness became complicated by anemia, psoriasis, esophageal thrush and staphylococcal myositis. His diarrhea A 32-year-old black homosexual man was found to persisted, and his weight fell to 145 pounds from be HIV positive in 1987. He was started on AZT in 170 pounds. 1988. His CD4 count was 342 cells/|xl. In August, By February, 1991, his diarrhea intensified, and 1990, he developed AZT associated myositis and in April, 1991, he entered a clinical trial of his CD4 count fell to 65 cells/(j.l. In December, octreotide. Colonic biopsy showed acute and 1990, he developed rectal Herpes simplex infection, chronic inflammation. Duodenal biopsy showed and syphilis. By March, 1991, he had the onset of chronic inflammation. Stains for acid fast organisms diarrhea and weight loss. His CD4 count was 33 were negative. In September, 1991, he developed cells/|xl. In September, 1991, he went to Puerto sinus pain and proptosis of his left eye. Biopsy of an Rico; on his return, he was found to have giardiasis orbital mass suggested large cell lymphoma, and he which was eliminated with metronidazole. His diar­ underwent radiation therapy to his eye with ulti­ rhea, however, persisted, and his weight fell to 126 mate control of his proptosis. His weight had fallen pounds. He had 1 to 3 bowel movements per day. In to 123 pounds. January, 1992, he underwent endoscopy, which In February, 1992, his diarrhea worsened. Upper showed parakeratosis and active inflammation in gastrointestinal series with small bowel follow-up the esophagus. Duodenal and colonic biopsies were showed small bowel dilatation. In April, 1992, negative. His CD4 count at that time was 8 cells/^l. endoscopy showed non-specific colitis. In June, By April 1992, his diarrhea remained unremitting. 1992, he was begun on azithromycin with some He transferred his care to his home in Cleveland. In weight gain. His left orbital area developed a large January, 1993, retrospective review of his biopsy opening from above his left eye into his frontal and from January, 1992, showed microsporidia. His phy­ ethmoid sinus with recurrent lymphoma. He slowly sician in Cleveland was contacted. The patient was deteriorated with continued diarrhea. In January, still experiencing florid diarrhea. 526 SUN, KAPLAN, TEICHBERG, WEISSMAN, SMILARI, AND URMACHER

FIGURE 4. Electron micrograph of duodenal biopsy from case 3 showing microsporidial plasmodia (p) in the apical supranuclear region of several enterocytes. SN = plasmodial nucleus, C = electron lucent cleft, LY = lysosome, N = enterocyte nucleus. Bar equals 1 micron, x 19,000.

C ase 4 His fever worsened, and in August, 1992, he was found to have CMV retinitis. He was started on A 45-year-old white bisexual man was tested pos­ phosphonoformate with improvement. In Decem­ itive for HIV in April, 1991, when he presented ber 1992 he developed severe headaches and the with cryptococcal meningitis which was success­ onset of a left facial palsy, diplopia with 4th and 6th fully treated. He was started on AZT, but developed nerve palsy. In November, 1992, he developed the anemia, necessitating discontinuance of the medi­ onset of diarrhea which was soon associated with cation. In October, 1991, he developed obstipation severe nausea and occasional vomiting. Stool exam­ followed by diarrhea and right upper quadrant pain. ination for microsporidia was now positive. He was He was found to have acute cholecystitis with cystic treated with albendazole without improvement. His duct obstruction and mild pancreatitis. He was weight fell to 125 pounds. He was begun on intra­ treated with antibiotics, and he improved. In venous total parenteral nutrition. His neurologic November, 1991, he had a repeat attack of cholecys­ condition worsened. As of April, 1993, his diarrhea titis. His CD4 count was 210 cells/(jLl. has remained persistent. In December 1991, he developed diarrhea and was found to have campylobacter in his stool and Clostridium difficile toxin as well. With antibiotic C ase 5 treatments, he felt better. His CD4 count fell to 44 cells/jxl. By May, 1992, he was having intermittent A 38-year-old homosexual male developed Kapo­ fever, and a blood culture was positive for CMV. si’s sarcoma of the left arm in December, 1988, INTESTINAL MICROSPORIDIOSIS 527

F ig u r e 5. Electron micrograph of duodenal biopsy from case 2 showing a microsporidial plasmodium with stacks of electron dense discs (D), electron lucent clefts (C) and nuclei (SN). The nucleus of enterocyte is at N. Bar equals 1 micron, X28,500. which was treated with interferon, resulting in com­ cisco to our hospital. He was admitted with fevers plete remission of the lesion. He was also started on and hypotension. He was suspected to have poten­ AZT which had to be discontinued because of con­ tial MAI infection and started on clarithromycin, stant intestinal upset. In November, 1991, he devel­ ethambutal, ciprofloxacin, and clofazamine. He oped progressive watery diarrhea with gradual improved but his stool examination now revealed weight loss. His CD4 cell count was 140 cells/jjd. numerous microsporidia. Examination of the stool for pathogens only revealed Endolimax nana. Cecal and gastric aspi­ rate failed to reveal pathogens. Diarrhea persisted, Discussion and colonoscopy in July, 1992, showed minimal focal colitis. His CD4 cells dropped to 73 cells/jxl The diagnosis of intestinal micro- and his diarrhea worsened. In November, 1992, he underwent endoscopy. sporidiosis was previously entirely There was focal chronic duodenitis characterized dependent on intestinal biopsy with by intramucosal infiltrates of lymphoid cells, few electron microscopic confirmation.15,16 plasma cells, and granulocytes with focal involve­ ment of glands and some areas of associated fibro­ Because most clinicians, pathologists, sis. Small intracytoplasmic bodies were seen in the and laboratory workers were unaware of surface columnar epithelium suggestive but not the existence of this disease, intestinal diagnostic of microsporidia. The proximal stomach showed marked chronic active gastritis. Colonos­ microsporidiosis was certainly under­ copy was unremarkable with only minimal colitis. diagnosed. Our five cases well exemplify His diarrhea persisted. His weight fell to 120 this situation, because the diagnosis was pounds and his diarrhea increased to 12 to 15 bowel movements per day. He began to experience right long delayed in all cases. However, since upper quadrant pain and frequent nausea. In Feb­ a convenient stool screening technique ruary, 1993, he transferred his care from San Fran­ has become available, the discovery of 528 SUN, KAPLAN, TEICHBERG, WEISSMAN, SMILARI, AND URMACHER

FIGURE 6. Electron micrograph of duodenal biopsy from case 1 showing a microsporidial spore (left) and multiple sporoblasts (right). Note the polar tubes (arrows), posterior vacuole (V), nuclei (SN) and the spore cell wall (W), consisting of an external electron dense zone and an inner lucent region. Bar equals 0.5 microns, x51,000. many more cases is expected. The modi­ was identified in an intestinal biopsy, fied trichrome stain for stool specimens subsequent stool examination was invari­ has been found reliable in making the ably positive. Therefore, stool examina­ diagnosis. Although the Giemsa stain has tion is probably as sensitive as intestinal been advocated for stool examination,16 it biopsy for the diagnosis of this disease, is less ideal than the modified trichrome and will gradually replace the invasive stain in terms of distinguishing this procedure, as occurred in cryptosporidio- protozoan from yeasts and bacteria. In sis during the last decade. tissue sections, the Brown and Brenn When biopsy is used as a last resort, or Gram-Weigert stain is helpful in small intestinal biopsy rather than revealing the organism;14 again, it is colonic or gastric biopsy should be taken, superior to the Giemsa stain. The modi­ because Enterocytozoon bieneusi has not fied trichrome stain has also been used been found in the stomach or colon. for tissue staining,10 but its merit awaits Some studies have found that jejunal further evaluation.17 biopsy has a higher positive rate than Alternatively, toluidine blue-stained duodenal biopsy,6,18 but the difference is plastic sections can effectively demon­ minimal. When a patient has protracted strate the intracellular spores as well as diarrhea, the previously “negative” the plasmodia. In our experience, this is intestinal biopsy should be reexamined the most ideal preparation for the diagno­ with a Brown and Brenn stain. In our first sis under light microscopy. Our experi­ three cases, the diagnosis of microsporidio- ence also showed that when the organism sis was established in this way. Bryan INTESTINAL MICROSPORIDIOSIS 529

F ig u r e 7. Electron micrograph of duodenal biopsy from case 2 show­ ing the straight portion of a polar tube (arrowhead), terminating in an anchor­ ing plate (arrow). Bar equals 0.5 microns, x 57,000.

and Weber17 also emphasized the need of should be concentrated on the entero- oil immersion and adequate light for cytes at the tip of the intestinal villi. The the diagnosis, in spite of the fact that organism is particularly abundant in a few experienced pathologists may be sloughing cells. The intracellular loca­ able to make the diagnosis in hema­ tion of the organism and the absence of toxylin and eosin stained sections under organisms in the crypts help to distin­ lower magnification.10 guish microsporidia from Cryptosporidia. There are no pathognomonic histologic Cryptosporidia may be found through­ features for the diagnosis of microspo- out the entire gastrointestinal tract, in the ridiosis. In fact, most biopsies show mild epithelial cells as well as the crypts. and non-specific changes, which, how­ They appear to be on the cell surface by ever, should not prevent further use of light microscopy. The distinction special stains for the diagnosis.17 When between Enterocytozoon bieneusi and searching for the organism, attention Septata intestinalis is the size of their 530 SUN, KAPLAN, TEICHBERG, WEISSMAN, SM ILARI, AND URMACHER

F ig u r e 8. A stool smear from case 5 show­ ing microsporidial spores in clusters or singles. A rod-shaped structure is visible in some organisms (arrow). Note the back­ ground bacteria stain faintly with counter stain. Modified trichrome stain, x 1250.

spores and their distribution. E. bieneusi kinetic study has not been per­ spores measure 1 to 1.5 |xm and are con­ formed.13,19 In AIDS cases, when CD4 fined to the enterocytes, whereas S. cell count is below 200/(jl1, opportunistic intestinalis spores measure 2 to 2.5 (xm infections such as pneumocystosis may and are seen in enterocytes, as well as occur. This cut-off point has been macrophages, fibroblasts and endothelial included in the new surveillance case cells in the lamina propria.9,10 definition of AIDS.20 In our cases, this Diarrhea, in cases of microsporidiosis, cut-off point was reached well before is typically watery in nature. When stool diarrhea occurred, when the CD4 cell specimen contain blood or mucus, super­ count was usually less than 100 cells/(i,l. imposed infection should be considered. A status of severe immunodeficiency in Two of our cases showed superimposed microsporidiosis is also evidenced by the infections; one patient had Campylobac­ presence of multiple complications in our ter jejuni and Clostridium difficile infec­ cases. It is surprising to see that despite tions, and another one had giardiasis. the severity of immunodeficiency, all our Therefore, even when a pathogen is patients survived for long periods of time found in stools in AIDS cases with with only gradual weight loss. This is in chronic diarrhea, microsporidiosis cannot contrast to Cryptosporidium, which pro­ be entirely excluded. duces more severe diarrhea. The most striking finding in our study Cholangitis caused by E. bieneusi has is the dynamics of CD4 cells which been reported in a few AIDS patients showed a CD4 cell count below 200/(xl recently,21,22,23,24 and microsporidia are well before the development of considered one of the common causes of microsporidiosis (table I). The extremely this condition, in addition to Cryptospo­ low CD4 cell count in microsporidiosis ridium and cytomegalovirus. Our case 4 was noted in a few studies, but a detailed showed clinical evidence of cholangitis; INTESTINAL MICROSPORIDIOSIS 531 TABLE I

T-cell Subset Studies in Five Patients

Case 1 Case 2 Case 3 Case 4 Case 5 CD4 CD8 Ratio CD4 CD8 Ratio CD4 CD8 Ratio CD4 CD8 Ratio CD4 CD8 Ratio

Pre-diarrhea Period 1987 1988 1991 541 1279 0.4 342 646 0.5 74 207 0.4 310 620 0.5 107 328 0.3 1999 304 559 0.5 331 994 0.3 203 490 0.4 210 496 0.4 1989 1989 329 10190.3 312 682 0.5 349 748 0.5 1990 452 754 0.6 435 1306 0.3 431 932 0.5 253 856 0.3 433 693 0.6 389 1188 0.3 246 1123 0.2 1990 360 768 0.5 1991 284 810 0.4 154 8960.2 130419 0.3 98 726 0.1 65 221 0.3 106 8220.1 20 412 0.05 139 1114 0.1 77 887 0.09 78 97Q 0.08 Post-diarrhea Period 1991 1988 1991 1992 1991 65 1199 0.05 15 — — 33 448 0.1 97 428 0.2 140 898 0.2 12 255 0.05 44 257 0.2 1992 1989 33 304 0.1 1992 50 594 0.05 24 588 0.04 1992 134 1157 0.1 22 685 0.03 8 1500.05 73 1356 0.05 1991 5 313 0.01 7 438 0.02 13 683 0.02 unfortunately, further investigation was produced a partial remission but did not not done to identify the etiology. The result in elimination of the parasite. preferred screening technique for chol­ Albendazole treatment showed more angitis is examination of bile smears with prominent clinical improvement and Giemsa stain for microsporidia, and Kin- induced degenerative changes in micro­ youn stain for Cryptosporidia. sporidia; however, there was no statis­ Thus far, there is no effective treatment tically significant changes in the parasite for microsporidiosis.13,16,25,26 In these load when intestinal biopsies were studies, metronidazole and octreotide compared before and after treatment.26 532 SUN, KAPLAN, TEICHBERG, WEISSMAN, SMILARI, AND URMACHER References unexplained diarrhea: Prevalence and clinical and biologic features. J Infect Dis 1993; 167: 1. Bartlett JG, Belitsos PC, Sears CL. AIDS enter­ 217-21. opathy. Clin Infect Dis 1992;15:726-35. 14. Weber R, Bryan RT, Owen RL, et al. Improved 2. Serwadda DNK, Sewankambo JW, Carswell light-microscopical detection of microsporidia AC, et al. Slim disease: a new disease in spores in stool and duodenl aspirates. New Uganda and its association with HTLV-III Engl J Med 1992;326:161-6. infection. Lancet 1985;ii:849-52. 15. Bryan RT, Cali A, Owen RL, Spencer HC. 3. Colebunders RH, Lusakumuni K, Nelson AM, Microsporidia: Opportunistic pathogens in et al. Persistent diarrhea in Zairian patients: An patients with AIDS. In: Sun T, (ed): Progress in endoscopic and histologic study. Gut 1988;29: Clinical Parasitology, Vol 2. New York, Field 1687-91. and Wood, 1991;l-26. 4. Rene EC, Marche B, Regnier B, et al. Intestinal 16. Orenstein JM. Microsporidiosis in the acquired infections in patients with acquired immunode­ immunodeficiency syndrome. J Parasitol 1991; ficiency syndrome: A prospective study in 132 77:843-64. patients. Dig Dis Sei 1989;34:773-80. 17. Bryan RT, Weber R. Microsporidia: emerging 5. Greenson JK, Belitsos P, Yardley JH, Bartlett pathogens in immunodeficient persons. Arch JG. 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