Vol. 61, No.1 Printed in U.S.A.
Copyright© 1971 by The Williams & Wilkins Co.
HISTOPLASMA COLITIS: AN ELECTRON MICROSCOPIC STUDY MARY E. KIRK, M.D., JoHN LouGH, M.D., AND H. A. WARNER, M.D.
Departments of Pathology and Gastroenterology, The Montreal General Hospital, and McGill University, Montreal, Quebec, Canada
Gastrointestinal histoplasmosis may be manifest as chronic diarrhea with polypoid transformation of small and large bowel mucosa. Sequential light and electron microscopic findings on rectal biopsies obtained before, during, and 1 year after a course of amphotericin are compared. The initial biopsy showed numerous viable-appearing budding intracellular fungi. At completion of amphotericin therapy, only degenerating and dead organisms were present. After a 1-year clinical remission, no organisms could be demonstrated with light microscopy, although electron microscopy revealed remnants of fungi cell wall still present within macrophages. Histoplasmosis is endemic in many parts of North America, and several recent reports have described the clinical'- 3 and radiological4· 5 features of this disease when it is localized in the gastrointestinal tract. Although the response of histoplasmosis to amphotericin B (Fungizone, E.R. Squibb Company, Ltd.) is well documented, 6 • 7 there have been no previous reports concerning the effect of this antifungal antibiotic on the morphology of the organism. Light and electron microscopic observations are reported here, for the first time, on sequential rectal biopsies obtained before, during, and after therapy with amphotericin B for intestinal histoplasmosis.
weakness, weight loss, and abdominal pain. Bowel movements had been abnormal since the age of 15, and he had been hospitalized on several occasions for investigation of chronic diarrhea. Five years earlier, chest X-rays showed an infiltrate confined to the left upper lobe of the lung, and acid-fast bacilli were cultured from his sputum. Barium studies of the gastrointestinal tract showed pseudopolypoid lesions in the ileum, cecum, and ascending colon; these lesions were interpreted as possibly of tuberculous etiology. Therapy for tuberculosis was given for 11 months, but his diarrhea persisted. Subsequent investigations included numerous cultures for bacteria, many studies of gut function, and biopsies of jejunum and rectum; the etiology of his diarrhea remained undetermined. After 2 years of invalidism at home, he sought further opinion because of the appearance of gross blood in the stool. Bowel movements were watery, averaged 15 per day, and aggravated his rather constant lower abdominal pain. His weight had decreased from 150 to 98lbs. On examination, he appeared chronically ill. He was wasted, afebrile, and normotensive. His diarrhea amounted to 4 to 7liters per day. Physical findings were confined to the skin, mucosae, and abdomen. He showed licheniform lesions of the skin of the dorsum of the hands, a striking angular stomatitis, white plaques and pseudopolypoid mammillations of the tongue and oral mucosa, and clusters of perianal verrucae. There was moderate right lower quadrant tenderness, but no palpable mass. Sigmoidoscopy
Case Report A 48-year-old farmer and construction worker was admitted to the Montreal General Hospital because of life-threatening bloody diarrhea, Received June 17, 1970. Accepted February 18, 1971. Address requests for reprint to: Dr. Mary E. Kirk, Pathology Department, The Montreal General Hospital, 1650 Cedar Avenue, Montreal 109, Quebec, Canada. This study was supported in part by Medical Research Council Research Grant 2617. The authors express their appreciation to Miss Patricia Spicer for her valuable technical assistance, and to Mrs. E. Barrie for typing the manuscript.
revealed mucosal pseudopolyps extending from the anal verge to 24 em , and there was a striking hyperemia with contact bleeding. In the barium enema, pseudopolyps were visualized throughout the colon and in the terminal ileum (fig. 1). Chest X -rays showed fibrocalcific densities in the left apex, associated with apical pleural thickening; the findings were interpreted as consistent with old granulomatous disease. Abnormal laboratory findings included: hemoglobin, 11.4 g per 100 ml; persistent occult blood in the stool; serum albumin, 2.9 g per 100 ml; a light increase in fecal loss of albumin measured by the radioactive chromium method; and a diffuse hyperglobulinemia on serum protein electrophoresis. Immunoelectrophoresis showed a slight increase in lgG to 1800 and 2400 mg per 100 mi. Candida albicans was isolated from the stools and from scrapings of buccal mucosa, repeated cultures of stool and rectal mucosa were negative for Histoplasma, and no organisms were identified in a bone marrow aspirate. Complement fixation tests and skin tests with histoplasmin, coccidioidin, and blastomycin were negative. An old tuberculin skin test was positive at a dilution of 1: 10,000. Microorganisms morphologically consistent with Histoplasma capsulatum were present in a rectal biopsy. On the basis of the clinical picture and the tissue diagnosis of histoplasmosis, intravenous therapy with amphotericin B was begun. Clinically, there was prompt improvement with a marked decrease in the frequency of bowel movements within 48 hr. A barium enema, performed after 3 weeks of therapy, showed an apparently normal distal half of the colon, but
FIG. 1. Barium series showing polypoid pattern in distal small bowel mucosa.
persistence of pseudopolyps in the terminal ileum. There were two complications during amphotericin therapy: a transient febrile reaction and an episode of moderate hypokalemia. At completion of amphotericin therapy (2 g over 3 months) he was clinically well. Cultures of stool and rectal mucosa were negative for fungi. He had normal bowel movements, and minimal disease on barium enema. Weight on discharge was 122 lb. One year later, he remained well. Sigmoidoscopic examination was normal and tissue cultures were again negative. The dinitrochlorobenzene skin test was attenuated and ~quivocal. A normal antibody response occurred after tetanus toxoid was given. He remained unresponsive to intradermal histoplasmin.
Histology The three biopsies for electron microscopy were fixed in cacodylate-buffered glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon. Thin sections were stained with lead citrate and uranyl acetate. In the initial rectal biopsy, taken before amphotericin therapy, there was superficial ulceration of the epithelium associated with acute and chronic inflammation and edema of the submucosa. In the lamina propria, between the colonic glands, there were numerous histiocytes which measured up to 40 J.L in diameter and had eccentric, oval, indented nuclei. The cytoplasm was abundant and indistinctly vacuolated. In occasional histiocytes, rounded intracytoplasmic structures were defined by a faintly basophilic rim (fig. 2). With Gomori's methenamine-silver (Grocott) stain, 8 countless spherical 1- to 5-J.L organisms were seen within the histiocytes (fig. 3). Periodic acidSchiff (PAS) clearly stained the outer rim of these organisms, and some had a positively staining central or eccentric dot. Mucicarmine stains were negative. At high magnification (figs. 4 and 5), it was confirmed that all organisms were within histiocytes. Intact organisms were round or ovoid and lay within cytoplasmic spaces which had poorly defined and often discontinuous membranes. The cytoplasm of the organisms was finely granular and contained several typical mitochondria, scattered dense bodies, and irregular vacuoles sometimes rimmed with electron-
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FIG . 2. Rectal mucosa, before amphotericin. Histiocytes in lamina propria have a foamy vacuolated cytoplasm containing numerous faintly outlined fungi (arrows) . Inflammatory reaction is marked (hematoxylin and eosin, X 400).
FIG. 3. Rectal mucosa, before amphotericin. Abundant fungi, many budding, distended histiocytes in the lamina propria (Grocott stain, X 400).
dense, coarsely granular material. Nuclei were usually eccentric, and were without distinguishing characteristics. A well defined plasma membrane was applied to the inner surface of a nonlaminated electronlucent cell wall. Tubulovesicular structures and interconnecting structures were not identified. There were many budding
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forms of the organism. At the interface between budding organisms, the cell wall was thickened and faintly granular. In addition, numerous larger round structures interpreted as degenerating organisms were present; these showed defects in their plasma membranes and had a coarsely and finely granular cytoplasm devoid of organelles. Crescent-shaped structures were abundant ; they lacked a plasma membrane and the cytoplasmic constituents appeared to have been lost, leaving a collapsed cell wall. These structures were interpreted as severely degenerated or dead fungi. Amorphous debris, probably within lysosomes, was often seen adjacent to the cell wall remnants. The second biopsy was taken at the completion of the course of amphotericin. With light microscopy, the mucosa was intact and there was only mild chronic inflammation. Foamy macrophages were plentiful in the lamina propria, but they were now more prominent in the submucosa. With Grocott and PAS strains, spherical organisms could still be seen near the mucosal surface but there were many more pale-staining crescentic forms in both lamina propria and submucosa. With the electron microscope (figs. 6 and 7), most organisms showed no internal structure; nuclei, dense bodies, mitochondria, and plasma membrane were absent. Crescentic structures were abundant, and all lay within well defined cytoplasmic vacuoles. Rare uncollapsed round organisms contained a granular and floccular material devoid of internal structure. Irregular osmiophilic bodies in the macrophage cytoplasm were sometimes closely associated with cell wall remnants. There were no apparent morphological effects of amphotericin on the host macrophages. In the third biopsy 1 year later, the rectal mucosa appeared normal and no organisms could be demonstrated with Grocott or PAS stains. With the electron microscope (fig. 8), occasional macrophages were distended by globular aggregates with an electron density similar to that of the cell wall of intact organisms. Pretreatment biopsies of the perianal skin showed condylomata accuminata with a dense surface contamination by Candida.
Flc. 4. Histoplasma capsulatum within a macrophage in the lamina propria of the rectum. Both viable and markedly degenerate forms are seen and some appear to be within lysosomes (arrows); colonic epithelial cell (E) (uranyl acetate-lead citrate, X 11,300).
A biopsy of skin frofu the dorsum of the hand was interpreted as nonspecific dermatitis, consistent with acanthosis nigricans.
Discussion Gastrointestinal histoplasmosis was suggested by the finding of numerous foamy histiocytes in the lamina propria in the initial rectal biopsy. Histiocytes with similar morphology are encountered in a variety of circumstances rangingfrom the uncommon metabolic and storage diseases to the common and clinically insignificant muciphages. 9 · 1 0 In this patient, careful search of routine histological preparations revealed occasional histiocytes containing intracytoplasmic rounded structures suggestive of fungi. PAS and Grocott stains confirmed this impression. The failure to isolate H. capsulatum from pretreatment stool and tissue cultures is attributed to overgrowth of Candida which contaminated the superficial layers of the perianal condylomata. C. albicans is also frequently present as normal fecal myco-
flora. 11 As demonstrated by Kapica et al. 12 growing colonies of C. albicans quickly acidify the glucose-containing Sabouraud's medium, and growth of H .. capsulatum is completely inhibited when the pH falls below 4. As anticipated, no fungi could be isolated after amphotericin therapy was begun. In spite of the failure to isolate H. capsulatum from this patient, the diagnosis of histoplasma colitis is regarded as secure on the basis of the highly characteristic morphology of this organism. 13 Morphologically similar intracellular parasites such as Toxoplasma and Leishmania have been excluded on the basis of special strains. Torulopsis glabrata is frequently present in the feces and closely resembles Histoplasma in tissue sections 14 ; however, nucleoid structures were identified with ease in the present case. Negative stains for mucin exclude Cryptococcus. Small intracellular forms of Blastomyces dermatitidis were considered but are regarded as unlikely because all the fungi examined had only a single nucleus. With the electron microscope, the organisms resemble those
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FIG. 5. Budding Histoplasma. The cytoplasmic vacuole is ill-defined and interrupted (arrows); cell wall, CW; plasma membrane, P; nucleus, N (uranyl acetate-lead citrate, X 19,200).
previously described in human histoplasmosis, 1 5 in experimental splenic infection, 16 and in hamster peritoneal macrophages. 17 Correlation of light and electron microscopic features indicates that the PAS-positive chromatin dot or nucleoid represents the nucleus of the fungus. It has been suggested that the cytoplasmic clearing or halo frequently present around this fungus represents shrinkage of the fungal protoplasm away from the rigid cell wall 18 ; our observations, however, would favor that this characteristic artifact represents retraction of macrophage cytoplasm away from the outer margin of the fungus cell wall. At completion of amphotericin therapy, fungi could
still be demonstrated in paraffin sections with special stains but not with hematoxylin and eosin; electron microscopy revealed extreme degeneration and degradation of the remaining organisms. One year later, no stainable organisms were present, and no intact organisms were demonstrated ultrastructurally, although the histiocytes contained large amounts of material which was interpreted as cell wall remnants. Histoplasmosis is essentially a pulmonary infection with disease occurring after inhalation of organisms. Subsequent dissemination may occur, with seeding of extrapulmonic sites. Involvement of the small bowel and colon is common in disseminated histo-
FIG . 6. After amphotericin. Many degenerate forms are concentrated in macrophage cytoplasm. The plasma membrane is absent and organelle structure is replaced by the pale floccular material (uranyl acetate-lead citrate, X 13,000).
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FIG . 7. After amphotericin a crescentic degenerating organism is shown adjacent to the macrophage nucleus and enclosed in a cytoplasmic vacuole. There is no recognizable membrane structure inside the cell wall (uranyl acetate-lead citrate, X 22,000).
plasmosis 19 and, as in the present case, gastrointestinal symptoms may dominate the clinical picture. Although occasional clinical case reports indicate that primary infection of the gastrointestinal tract may occur, experimental evidence in hamsters suggests that this is an uncommon portal of entry for the fungus. 20 Most gastrointestinal lesions result from hematogenous dissemination from the lungs; the pulmonary lesions may be small, clinically asymptomatic, and difficult to identify even at postmortem examination. Negative histoplasmin skin tests are frequentz 1 and negative complement fixation tests are not unusual in progressive disseminated histoplasmosis. Of 24 cases reviewed by Reddy et al., 6 16 had negative skin tests and 7 had negative complement fixation tests. Although there is no clear cut explanation for this phenomenon, this nonreactivity is usually attributed to the effect of overwhelming infection in patients with some abnormality of immunological responsiveness.
Amphotericin is a polyene antibiotic which readily binds with the sterols present in the plasma membrane of sensitive fungi. 22 The bound antibiotic alters cellular permeability and interferes with the ability of the plasma membrane to act as a restraining barrier, resulting in the loss of cytoplasmic constituents. 23 The biopsy findings in this patient suggest that amphotericin results in an increased capacity for the macrophages of the lamina propria to destroy the fungi, degrade the cytoplasmic organelles, and concentrate the cell wall material. The organisms appeared to lose their plasma membrane and organelle structure initially, leaving a finely granular matrix confined by the still intact cell wall. Subsequently, there was progressive collapse of the organisms, at first into crescentic forms and, later, into irregularly rounded masses. The cell wall material remained as faintly osmiophilic residual bodies which then appeared to be transported by the macrophages into the submucosa. The eventual disposition of this residual material remains undetermined.
FIG. 8. Rectal biopsy, 1 year after cessation of amphotericin. A macrophageis shown.distended by irregular bodies containing cell wall material from Histoplasma (uranyl acetate-lead citrate, X 11,000).
The structural integrity of the cell wall of H. capsulatum is largely due to chitin, and, since human cells lack chitinase, this material is perhaps never completely eliminated. 24 Upon completion of a 3-month course of amphotericin, no intact fungi were seen. From the clinical history it is likely that this man had been infected by Histoplasma for some years, and, as anticipated, both viable and degenerate forms of the organism was identified in the pretreatment biopsy. There were no morphological differences in the manner of fungus degradation before and after therapy. The factors which predisposed to chronic histoplasmosis in this patient are not known. He had both tuberculosis and moniliasis in addition to histoplasmosis, and it is possible that he has some immunological defect. Newberry et al. 25 studied the immunologi-
cal status of 5 patients with chronic systemic histoplasmosis; only 2 had complement-fixing antibodies but all had depressed lymphocyte transformation. This patient had a good antibody response to tetanus toxoid, indicating intact humoral immunity. Although his weak response to dichloronitrobenzene and the negative histoplasmin skin test suggest that he may have a selective defect in cell-mediated delayed hypersensitivity, his reactivity to dilute tuberculin is evidence against this hypothesis. REFERENCES 1. Sturim HS, Kouchoukos NT , Ahlvin RC: Gastro-
intestinal manifestations of disseminated histoplasmosis. Amer J Surg 110:435-440, 1965 2. Bank S, Trey C, Gans I, et al: Histoplasmosis of the small bowel with "giant" intestinal villi and secondary protein-losing enteropathy. Amer J Med 39:492-501, 1965
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3. Boone WT, Allison F: Histoplasmosis. Amer J Med 46:818-826, 1969 4. Dietz MW: ileocecal histoplasmosis. Radiology 91:285- 289, 1968 5. Perez CA, Sturim HS, Kouchoukos NT, et a!: Some clinical and radiographic features of gastrointestinal histoplasmosis. Radiology 86:482487, 1966 6. Reddy P, Gorelick DF, Brasher CA, et al: Progressive disseminated histoplasmosis as seen in adults. Amer J Med 48:629-636, 1970 7. Parker JD , Sarosi GA, Doto IL, et al: Treatment of chronic pulmonary histoplasmosis. New Eng J Med 283:225-229, 1970 8. Grocott RG: A stain for fungi in tissue sections and smears. Amer J Clin Path 25:975-979, 1955 9. Azzopardi JG, Evans DJ: Mucoprotein-containing histiocytes (muciphages) in the rectum. J Clin Path 19:368-374, 1966 10. Gonzalez-Licea A, Yardley JH: Whipple's disease in the rectum . Amer J Path 52:1191- 1206, 1968 11. Cohen R, Roth FJ, Delgado E, eta!: Fungal flora of the normal human small and large intestine. New Eng J Med 280:638- 641, 1969 12. Kapica L, Shaw CE, Bartlett GW: Inhibition of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar media. J Bact 95:2171-2176, 1968 13. Binford CH: Histoplasmosis. Tissue reactions and morphologic variations of the fungus. Amer J Clin Path 25:25-36, 1955 14. Oldfield FSJ, Kapica L, Pirozynski WJ: Pulmonary infection due to Torulopsis glabrata. Canad Med Ass J 98:165-168, 1968 15. Dumont A, Piche C: Electron microscopic study
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of human histoplasmosis. Arch Path (Chicago) 87:168-178, 1969 Edwards GA, Edwards MR, Hazen EL: Electron microscopic study of histoplasma in mouse spleen. J Path Bact 77:429-438, 1958 Dumont A, Robert A: Electron microscopic study of phagocytosis of Histoplasma capsulatum by hamster peritoneal macrophages. Lab Invest 23: 278-286, 1970 Emmons CW, Binford CH, Utz JD: Medical Mycology. Philadelphia, Lea and Febiger, 1963 Schulz DM : Histoplasmosis: A statistical morphologic study. Amer J Clin Path 24:11-26, 1954 Salfelder K , Sethi KK: Experimental intestinal histoplasmosis of hamsters. Mycopathologia 32: 153- 162, 1967 Schwarz J , Furcolow ML: Some epidemiologic factors and diagnostic tests in blastomycosis, coccidioidomycosis and histoplasmosis. Amer J Clin Path 25:261-265, 1955 Lampen JO: Amphotericin Band other polyenic antifungal antibiotics. Amer J Clin Path 52:138146, 1969 Kinsky SC: Membrane sterols and selective tox· icity of polyene antifungal antibiotics, Antimi· crobial Agents and chemotherapy. Edited by JC Sylvester. Ann Arbor, American Society for Microbiology, 1963, p 387-394 Domer JE, Chandler JW, Chin TDY, eta!: Comparative study of the cell walls of the yeastlike and mycelial phases of Histoplasma capsulatum. J Bact 94:466-474, 1967 Newberry WM, Chandler JW, Chin TDY, eta!: Depressed lymphocyte transformation in chronic histoplasmosis. J Immun 100:436- 443, 1968