Overwhelming Watery Diarrhea Associated with a Cryptosporidium in an Immunosuppressed Patient

70:1156- 1160, 1976 Copyri!(ht © 1976 by The Willia ms & Wilkins Co.

Vol. 70, No.6

GASTROENTEROLOGY

Print ed in U.S.A.

OVERWHELMING WATERY DIARRHEA ASSOCIATED WITH A CRYPTOSPORIDIUM IN AN IMMUNOSUPPRESSED PATIENT J, L.

MEISEL, M,D ., D.

R

PERERA , M .D .,

C.

MELIGRO,

B.S"

AND

C. E.

RUBIN, M.D.

Group Health Hospital, Department of Medicine, University of Washington, and Department of Medicine, Seattle Veteran's Administration Hospital, Seattle, Washington

A 39-year-old man with severe bullous pemphigoid developed overwhelming diarrhea after 5 weeks' treatment with 150 mg of cyclophosphamide and 60 mg of prednisolone daily. Jejunal and ileal biopsies showed severe mucosal injury and tiny 2- to 4-~ organisms on the epithelial surfaces. Similar organisms were seen in smears of jejunal fluid. Electron microscopic examination of jejunal biopsies showed these spherical bodies to be trophozoites, schizonts, microgametocytes, and macrogametocytes typical of the genus Cryptosporidium. Diarrhea resolved 2 weeks after discontinuation of cycloph~­ phamide and coincided with disappearance of Cryptosporidia from the jejunal biopsies. Immunosuppression may have predisposed this patient to cryptosporidial diarrhea. Cryptosporidiosis is another infection which can be diagnosed by small bowel biopsy. When immunosuppressed patients develop severe diarrhea, opportunistic infection with this and other organisms should be considered as the possible cause. Cryptosporidia are protozoan parasites currently classified within the subphylum Sporozoa and the subclass Coccidia . They inhabit the brush border of the small intestinal mucosa in a variety of animals, but have not yet been described in man. First discovered by Tyzzer in 1907 in the gastric crypts of mice, I they have since been reported in the small intestine of guinea pigs,2 calves,3, 4 lambs,5 turkeys,6 rhesus monkeys,7 and mice. 8 Although the infection causes small intestinal mucosal abnormalities in all species, the severity of symptoms is variable. This is a report of intestinal cryptosporidiosis in an immunologically suppressed patient who developed overwhelming watery diarrhea. The jejunal and ileal mucosa showed acute inflammation, distortion of villous architecture and tiny 2- to 4-~ spherical organisms on the mucosal surface. These organisms showed morphological features consistent with Cryptosporidia by electron microscopy. Methods Jejunal biopsies were obtained using a four-hole multipurpose suction biopsy tube positioned under fluoroscopic control Received October 2, 1975. Accepted November 24, 1975. This research was supported by Research Grant AM16059 from the National Institute of Arthritis, Metabolism and Digestive Diseases. Dr. Rubin is supported by Career Research Award CA03499 from the National Institutes of Health, United States Public Health Service. The authors wish to thank Drs. H. Jervis and J. Vetterling for their helpful advice. Dr. A. Takeuchi reviewed the light and electron microscopic biopsy material and concurred with the authors' identification of Cryptosporidium. The authors are grateful for his advice and expert help. Mrs. Zoe Jonak provided excellent technical assistance . Address reprint requests to: Dr. Cyrus E. Rubin, Department of Medicine RG-20, University of Washington, Seattle, Washington 98195.

at the ligament of Treitz. 9 Ileal biopsies were obtained with a punch biopsy forceps via a pediatric sigmoidoscope inserted through the patient's ileostomy. The biopsies were oriented on plastic mesh, fixed in Bouin's solution, embedded in paraffin, and serially sectioned at 4 11- . 10 When tiny spherical particles were detected on the mucosal surface by light microscopy, the diagnosis of cryptosporidiosis was considered because of similar findings in animals, II Unfortunately, no osmic-fixed tissue was available for electron microscopy, Therefore, the remaining unsectioned, paraffin-embedded blocks of Bouin's-fixed biopsies were rehydrated, osmicated, and embedded in Epon 812 for examination with an AEI 6B electron microscope ,

Case Report A 39-year-old married college administrator was transferred to Group Health Hospital on October 17, 1974, because of 10 days of severe diarrhea requiring massive parenteral fluid replacement. At 22 years of age, this patient developed ulcerative colitis and, at 25, required a colectomy. Subsequently, his health was excellent and his ileostomy functioned well. In June, 1974, he developed a skin lesion consistent with bullous pemphigoid on skin biopsy studied by light microscopy and immunofluorescence, By August, 1974, the patient's skin had not improved despite 80 mg of oral prednisolone daily. Vincristine and cyclophosphamide 12 were, therefore, added: 0.5 mg of intravenous vincristine twice, 12 hr apart, on August 30, and 500 mg of intravenous cyclophosphamide on August 31 and September 1. When the patient's white blood count began returning toward normal on September 11, 150 mg of oral maintenance cyclophosphamide daily was begun. His skin improved dramatically. His white blood cell count remained stable at approximately 3,000 cells per cc for the remaining period of cyclophosphamide treatment, On October 4, 1974, the patient felt well enough to lay a concrete foundation for a new building on his farm. On October 5, he developed crampy abdominal pain and by October 6 had massive watery diarrhea requiring hospitalization elsewhere for

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parenteral fluid replacement. At this time he was markedly dehydrated and oliguric. His serum sodium was 125 mEq per liter, potassium 5.5 mEq per liter, bicarbonate 22 mEq per liter, chloride 94 mEq per liter, blood urea nitrogen 52 mg per dl, and hematocrit 61 %. Measured ileostomy outputs varied between 6 and 10 liters daily. During the initial 10 days of hospitalization he required more than 60 liters of parenteral fluid in addition to a substantial oral intake. He received 150 mg of intravenous cyclophosphamide on October 7, 8, and 9. On October 10, cyclophosphamide was discontinued because of the suspicion that it might be causally related to his diarrhea. High dose corticosteroids were continued parenterally throughout the period of severe diarrhea for fear of inducing an Addisonian crisis if steroids were withdrawn. Because of continued severe diarrhea, the patient was transferred to the Group Health Hospital on October 17. The patient was well hydrated, was moderately Cushingoid, and had a normal abdominal examination. There were scattered areas of skin hyperpigmentation but no bullae. Electrolytes were normal except for an elevated serum bicarbonate of 39 mEq per liter. The following tests were also normal: complete blood count, sedimentation rate, blood urea nitrogen, amylase, calcium, carotene, and gastrin. Albumin was low (1.7 g per dI). The 7.5 liters of ileostomy output during the first 24 hr contained 114 mEq per liter of sodium and 6 mEq per liter of potassium. The pH of the ileostomy drainage was 5.5 on a sucrose-containing, lactose-free diet. Basal gastric acid output was 5.3 mEq per hr. Culture of the ileostomy drainage showed only a few colonies of Candida albicans but no Shigella or Salmonella. Examination for ova and parasites by both direct and concentration methods was negative on three occasions. On October 18, proximal jejunal and distal ileal mucosal biopsies were taken. Because of the low pH of the ileostomy drainage and the histological evidence of mucosal injury, it was suspected that brush border enzymes were decreased or absent. Therefore, a diet of monosaccharides and amino acids was instituted (Vivonex). By October 20, the ileal output had begun to decrease, and on October 24 it had returned to normal. Diarrhea did not recur. Thus, diarrhea disappeared 2 weeks after discontinuance of cyclophosphamide despite continued prednisolone treatment. The only complaint that remained was epigastric pain. A duodenal ulcer was discovered by upper gastrointestinal series and the patient responded symptomatically to antacids. Because the histological appearance of the biopsy was not incompatible with that of celiac sprue and because the true diagnosis was still in question, the patient was discharged on a gluten-free diet. On October 29, jejunal biopsies were repeated and showed marked improvement. A third set of jejunal biopsies in December, 1974, was normal. Therefore, the patient was placed on a gluten-containing diet, and he remained asymptomatic. Because of the possibility of confusing the dermatological appearance of bullous pemphigoid with dermatitis herpetiformis and because celiac sprue is associated with dermatitis herpetiform is, the patient was placed on a high gluten diet in May, 1975, to bring out possible latent celiac sprue. 13 A fourth set of jejunal biopsies in June, 1975, was normal. The patient has remained well with no gastrointestinal symptoms on a normal diet. His skin disease is controlled on 5 to 10 mg of prednisolone by mouth every other day .

Intestinal Biopsies The initial jejunal and ileal biopsies were taken on October 18, 1974 (12 days after the onset of diarrhea and 8 days after the omission of cyclophosphamide). They showed a moderate to severe abnormality of villous architecture, with lengthening

of crypts and both shortening and loss of villi (fig. 1, a). There was an increased number of plasma cells, polymorphonuclear leukocytes, and lymphocytes within the lamina propria. Occasional crypt abscesses were identified. The surface epithelium was low cuboidal to columnar and infiltrated with polymorphonuclear leukocytes and lymphocytes (fig. 1, b). Spherical particles, 2 to 4 JJ. in diameter (fig. 1, b, arrows), were scattered along some epithelial surfaces of all jejunal and ileal biopsies. These particles appeared attached to the brush border of the absorptive cells of the tips and sides of some villi and to the epithelial surface of some crypts. In sections stained with hematoxylin and eosin, these tiny particles were golden brown in color. The particles did not stain with methenamine silver or periodic acid-Schiff. Fluid present in the small bowel biopsy capsule was smeared on glass slides, air dried, and stained with Giemsa. In addition to many polymorphonuclear leukocytes, similar spherical particles were seen; their diameter was slightly larger than than observed in the tissue sections, presumably because of swelling during air drying; the particles stained blue and had bright red centers. The organisms visualized by electron microscopy were similar to Cryptosporidium wrairi described by Vetterling et al. U Although there were artifacts created by Bouin's fixation , sufficient fine structural features were retained to identify various phases of the life cycle of the Cryptosporidium found in the present patient. These included a trophozoite attached to an absorptive cell (fig. 2, b), a schizont containing merozoites (fig. 2, c), a microgametocyte containing multiple condensed nuclei and membrane-bound vesicles (fig. 2, d), and a macrogametocyte containing polysaccharide granules (fig. 2, e). Details of the attachment area of the trophozoite show that the absorptive cell's microvillous membrane contributes to the membrane enveloping the trophozoite U (fig. 2, a). The second set of jejunal biopsies taken 1 week after hospitalization, when diarrhea had ceased, showed no Cryptosporidia, improved villous architecture, and fewer inflammatory cells within the epithelium and lamina propria. The third set of small bowel biopsies, obtained 1 month after discharge , was entirely normal (fig. 1, c), as were those obtained after a high gluten diet in June, 1975.

Epidemiology The patient lives in a rural area near Olympia, Washington. His farm has numerous head of beef cattle, the youngest of which were 7 to 8 months of age at the time of his diarrheal illness. His family has one German shepherd dog, but there are no other domestic animals or pets in his immediate area . Field mice and deer are commonly seen on his farm. In the 2 days before his illness, the patient had been working outside digging foundations and pouring concrete for a new barn. No members of his family developed diarrhea.

Discussion Cyclophosphamide treatment produces generalized depression of both humoral and cell-based immunity.15 Corticosteroids impair the inflammatory response as well. 16 Throughout the 5 weeks of treatment with cyclophosphamide and prednisolone this patient had no gastrointestinal symptoms whatsoever. During his overwhelming diarrhea, severe injury of jejunum and ileum occurred and the presence of Cryptosporidia was demon strated. After 2 weeks off cyclophosphamide both the organisms and the diarrhea disappeared. It is highly improbable that these doses of cyclophosphamide in-

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FIG. 1. a, Initial jejunal biopsy during diarrhea (October 18, 1974), showing lengthened crypts and only vestigial villi (H & E, x 184). b, Enlargement of insert in a showing abnormality of the surface epithelium, inflammatory cells in the lamina propria, and tiny 2- to 4-1' Cryptosporidia on the epithelial surface (arrows) (H & E, x 736). c, Normal jejunal biopsy 3 1/2 weeks after cessation of diarrhea (H & E, x 184).

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FIG. 2. Electron micrographs of various phases of the life cycle of Cryptosporidium, a, Detail of the attachment zone from 2 b suggesting that the outer membrane of the trophozoite originates from the microvillous membrane underlying the host absorptive cell ( x 48,000). b, Trophozoite with large nucleus and condensed cytoplasm. c, Schizont with merozoites. d, Microgametocyte with condensed nuclei and membrane-bound vesicles. e, Macrogametocyte with polysaccharide granules (b-e, x 22,5(0).

jured the bowel directly and caused the diarrhea. 17 It is far more likely that this patient's bowel problems were caused by an opportunistic cryptosporidial infection because of the combined immunosuppression with cyclophosphamide and corticosteroids. A variety of organisms may produce intestinal mucosal injury and malfunction in patients whose immunity is impaired. These include Giardia lamblia in patients with hypogammaglobulinemia,18 Strongyloides stercoralis hyperinfection in patients receiving high dose corticosteroid treatment,19 and cytomegalovirus in immunosuppressed hosts undergoing bone marrow or renal transplants. 20 In all likelihood other opportunistic gastrointestinal infections will be discovered in the future in

patients with impaired immune mechanisms. When a patient receiving a cytotoxic drug develops severe diarrhea or malabsorption, it is important to seek opportunistic infections which may be treatable before assuming that one is dealing with drug toxicity or some other etiology. The differential diagnosis of this patient's diarrhea includes other infectious agents, pancreatic cholera, Zollinger-Ellison syndrome, celiac sprue, and Crohn's disease. The presence of Strongyloides stercoralis, Giardia lamblia, Isospora belli, and cytomegalovirus were excluded by intestinal biopsy, jejunal aspirate, and examination of ileal contents. Toxigenic Escherichia coli and Vibrio cholerae and other organisms producing

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enterotoxins do not cause such severe intestinal injury. Ileal fluid culture ruled out Shigella and Salmonella. Invasive E. coli was not ruled out, because bioassay for invasiveness was not performed on cultures of the patient's intestinal contents during his acute illness. Zollinger-Ellison syndrome was excluded by a normal serum gastrin, lack of gastric hypersecretion, and a self-limited clinical course after 1 year's follow-up. Pancreatic cholera was considered unlikely because of a lack of hypokalemia and gastric acid hyposecretion, as well as the short duration of massive diarrhea. Vasoactive intestinal polypeptide levels were not measured. Celiac sprue was ruled out because a high gluten.diet did not injure the intestinal mucosa. The remote possibility of Crohn's disease was discounted by absence of granulomas in serial sections of multiple jejunal and ileal biopsies and by the acute, self-limited nature of the diarrheal illness. The pathogenesis of this patient's small bowel injury is not certain. The intestinal lesion was probably related to the infection with Cryptosporidia . There is no experimental work in animals which elucidates how such a superficial infection causes such profound bowel injury. However, this phenomenon is not without precedent, because G. lamblia can cause considerable damage with little if any invasion. The enormous fluid and electrolyte loss is best explained by mucosal injury and malfunction involving the whole length of the small bowel. It is also possible that a toxin liberated during schizogony might have caused a secretory state. Hypoalbuminemia probably reflected intestinal loss of proteins from the injured mucosa . Loss of intestinal disaccharidases might have contributed to diarrhea during the 1st week of illness while dissacharides were still being ingested. Cryptosporidia inhabit the microvillous border of the intestinal epithelial cells . The organisms are actually intracellular, because the outer membrane covering the organisms is of host origin. 14 By contrast, the Isospora described by Brandborg et al. 21 is intracytoplasmic. Electron microscopic evidence suggests that nutrition of the parasite is derived by endocytosis at the attachment zone between the organism and the host epithelial cell. 14 The life cycle of Cryptosporidium wrairi in the guinea pig has been described by Vetterling et al. 14.2 2 Two cycles of schizogony and a sexual cycle of gametogeny were deduced on morphological grounds. No oocysts were found in the stools by Vetterling et al. 22 C. wrairi was transmitted when guinea pigs were housed together, but was not transmitted by oral inoculation of ileal scrapings or fecal material. 22 Oral ingestion of an unknown infective stage of the organisms is probably responsible for transmission. Cryptosporidium is the second coccidia found to cause diarrhea in man , the first being I. belli. The diagnosis of cryptosporidiosis must be considered in any patient with a severe intestinal mucosal abnormality and overwhelming diarrhea not responding to a gluten-free diet. If infection with a Crytosporidium is suspected, a presumptive diagnosis can be made by careful examination of jejunal biopsies and jejunal fluid with a light micro-

scope. Cryptosporidial diarrhea may be an opportunistic infection which is only seen in immunosuppressed patients. Alternatively, Cryptosporidia may be an undescribed pathogen in which normal man is the natural host. The determination of whether or not this organism represents a separate genus must await further morphological and epidemiological studies in man. REFERENCES 1. Tyzzer EE: A sporozoan found in the peptic glands of the common mouse. Proc Soc Exp Bioi Med 5: 12- 13, 1907 2. Jervis HR, Merrill TG, Sprinz H : Coccidiosis in the Guinea pig small intestine due to a Cryptosporidium. Am J Vet Res 27:408-414, 1966 3. Panciera RJ, Thomassen RW, Garner FM: Cryptosporidial infection in a calf. Vet Pathol 8:479- 484, 1971 4. Menten DJ, Van Kruiningen HJ: Cryptosporidiosis in a calf. J Am Vet Med Assoc 165:914-917, 1974 5. Barber IK, Carbonella PL: Cryptosporidium agni sp .n . from lambs and Cryptosporidium bovis sp .n . from a calf with observations on the oocyst. Z Parasitenkd 44:289-298, 1974 6. Slavin D: Cryptosporidium meleagridis (sp. nov.) . J Comp Pathol 65:262-266, 1955 7. Kovatch RM, White JD : Cryptosporidiosis in two juvenile rhesus monkeys. Vet Pathol 9:426-440, 1972 8. Hampton JC, Rosario B: The attachment of protozoan parasites to intestinal epithelial cells of the mouse. J ParasitoI52:939-948, 1966 9. Brandborg LL, Rubin CE, Quinton WE: A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel and colon. Gastroenterology 37:1- 16, 1959 10. Perera DR, Weinstein WM, Rubin CE: Small bowel biopsy . Hum Pat hoi 6:157-217, 1975 11. Takeuchi A: Penetration of intestinal epithelium by various microorganisms. Curr Top Pathol 54:1-27, 1971 12. Krain LS, Landau JW, Newcomer VD: Cyclophosphamide in the treatment of pemphigus vulgaris and bullous pemphigoid . Arch Dermatol 106:657-661, 1972 13. Weinstein WM: Latent celiac sprue. Gastroenterology 66:489- 493, 1974 14. Vetterling JM, Takeuchi A, Madden PA: Ultrastructure of Cryptosporidium wrairi from the guinea pig. J Protozool 18:248-260, 1971 15. Gershwin ME, Goetzl EJ, Steinberg AD: Cyclophosphamide : use in practice. Ann Intern Med 80:531-540, 1974 16. Fauci AS, Dale DC. Balow JE: Glucocorticosteroid therapy: mechanisms of action and clinical considerations. Ann Intern Med. In Press 17. Wolf VM, Ley I,Klein K : Der Einfluss von Zyklophosphamid auf die Ultrastrucktur. der Diinndarm Schleimhaut. Z Gesamte Inn Med 23:727, 1968 18. Ament ME, Rubin CE: Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes. Gastroenterology 62:216-226, 1974 19. Purtilo DT, Meyers WM, Connor DH: Fatal strongyloidiasis in immunosuppressed patients. Am J Med 56:488-493, 1974 20. Rifkind D, Goodman N. Hill RB: The clinical significance of cytomegalovirus infection in renal transplant patients. Ann Intern Med 66:1116-1128, 1967 21. Brandborg LL, Goldberg SB, Breidenbach WC: Human coccidiosis-a possible cause of malabsorption. The life cycle in small bowel mucosal biopsies as a diagnostic feature. N Engl J Med 283:1306-1313, 1970 22. Vetterling JM, Jervis HR, Merrill TG, Sprinz H: Cryptosporidium wrairi sp.n. from the Guinea pig Cavia procellus, with an emendation of the genus. J Protozool 18:243- 247, 1971