Cytomegalovirus Infection of the Gastrointestinal Tract in A Patient with Late Onset Immunodeficiency Syndrome

73:1397-1403, 1977 Copyright © 1977 by the American Gastroenterological Association

Vol. 73, No.6 Printed in U.S.A .

GASTROENTEROLOGY

CASE REPORTS CYTOMEGALOVIRUS INFECTION OF THE GASTROINTESTINAL TRACT IN A PATIENT WITH LATE ONSET IMMUNODEFICIENCY SYNDROME HUGH J. FREEMAN, M.D., THEODOR K. SHNITKA, M.D., JAMES R. A. PIERCEY, M.D., AND WILFRED M. WEINSTEIN, M.D. Departments of Medicine (Gastroenterology) and Pathology , University of Alberta, Edmonton, Alberta, Canada

An adult with the late onset immunodeficiency syndrome developed intractable diarrhea. Widespread cytomegalovirus (CMV) infection of the gastrointestinal tract was detected antemortem with detailed morphological studies and viral culture. The CMV-type cells were especially numerous in his severely ulcerated colon. Electron microscopy of infected cells in rectal biopsy material revealed the characteristic features of CMV infection. It is likely that the CMV infection contributed to the symptom complex and the mucosal injury. Unusual opportunistic infections as a cause of diarrhea should be considered in patients with late onset immunodeficiency, especially if Giardiasis is ruled out. Diarrhea and malabsorption occur commonly in patients with late onset immunodeficiency.'· 2 In some patients treatable associations may be found, the foremost being Giardiasis.'· 2 In other patients no overt cause of the gastrointestinal symptoms can be identified although symptomatic relief therapy with empiric therapy has been reported. 3-5 We report a patient with late onset immunodeficiency syndrome who developed intractable diarrhea. Before his death he was found to have cytomegalovirus (CMV) infection, widespread throughout the gastrointestinal tract. The diagnosis was confirmed with detailed morphological studies and viral culture. The findings strongly suggest that the CMV infection was a major contributor to the patient's symptoms and mucosal damage, especially in the colon. In this report we highlight the morphological features of CMV infection in the mucosa of the gastrointestinal tract.

Methods

Schilling's test with and without intrinsic factor was done as described previously." For fecal fat determinations/ stools were collected for the last 3 days of a 5-day 100-g of fat per day intake period. Immunoglobulins (IgG, IgA, lgM) were determined by radial immunodiffusion.~· 11 Serum samples for viral serology were assayed for complement-fixing antibodies"' against the following antigens: cytomegalovirus, Herpesvirus ·hominis, adenovirus, influenza A and B, Mycoplasma pneumoniae, and varicella-zoster. Biopsy tissue for viral culture was homogenized in phosphatebuffered saline and centrifuged at 3000 rpm for 1 hr before innoculation in duplicate into monolayer cultures of human foreskin fibroblasts, primary and continuous line human amnion cells, and African green monkey kidney cells. All cell cultures were maintained in media supplemented with 2% heat-inactivated fetal calf serum and tubes were incubated at 3TC in roller drums. Cultures which developed cytopathic changes were processed for virus identification using standard techniques. 11 These include electron microscopic examination of disrupted cells in preparations negatively stained with 2% Received March 21, 1977. Accepted June 7, 1977. phosphotungstic acid. Dr. Freeman was supported by a Fellowship from the Medical Biopsies obtained through the panendoscope or the colonascope were taken with the ACMI 7035-B biopsy forcep (AmerResearch Council of Canada. Address requests for reprints to: Wilfred M. Weinstein, M.D., idm Cytoscope Makers, Inc., Pelham Manor, N. Y.). Small Department of Medicine, Division of Gastroenterology, 9-112 Clini- intestinal suction biopsies were taken under fluoroscopic concal Sciences Building, University of Alberta, Edmonton, Alberta, trol with a multipurpose or hydraulic biopsy tube (Quinton Canada, T6G 2G3. Instruments, Seattle, Wash.). For light microscopy, Bouin's The authors thank Dr. Gerald Sinclair for providing the infor- fixative (gastrointestinal tract and liver) or 10% formalin (all mation pertaining to the patient's early course, Dr. Claudia Strehlke other tissues) were used. Gastrointestinal mucosal biopsies for referring the patient, Colleen Murdoch for the histological were processed using the special techniques described previpreparations, Sharon E. McCann for electron microscopic prepara- ously.12 Abnormalities in small intestinal villous architecture tions, Eric Beamont for the photomicrography, Dr. R. W. Sherban- were graded as previously described . 12 For electron microsiuk for performing the colonoscopy, Dr. Harvey Rabin for his copy, tissues were fixed in buffered 5% osmium tetroxide or assistance with the patient's management, Dr. Henry Pabst for 4% glutaraldehyde and embedded in Epon 812, as previously performing some of the immunological investigations, and Dee S. described. 1:1 Autopsy biopsies for light microscopy from the White for secretarial assistance. gastrointestinal tract were taken every 10 to 15 em from the 1397

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esophagus, stomach (lesser and greater curvatures), small intestine, and colon.

Case Report The patient's illness first became manifest at age 10 with the development of recurrent scalp, skin, and respiratory tract infections. Splenomegaly was first detected in 1952, at age 29. The diagnosis of hypogammaglobulinemia was first made in 1959 at age 36 and thereafter he received monthly injections of 10 ml of y-globulin . He first developed diarrhea in 1956 at age 33, and steatorrhea (fecal fat 15 g per day) was documented in 1962. Subsequent trials of pancreatic enzymes, a gluten-free diet, folic acid, and vitamin B, 2 all failed to change the pattern of the diarrhea . However, he often experienced a transient improvement in the diarrhea when he took antibiotics for respiratory tract infections. We first saw the patient in September 1973. He was thin and appeared chronically fatigued. Rhonchi were audible over both lung bases. The spleen was palpable 4 em below the left costal margin . Pertinent laboratory data were: normal hemoglobin, white blood cell count, serum folic acid, and serum albumin; serum iron 50 p.g per dl (normal 70 to 170), total iron binding capacity 332 p.g per dl (normal 250 to 400); serum globulins 1.6 g per dl; IgG 200 mg per dl (normal 600 to 1500), IgA 0 mg per dl (normal 40 to 300), and IgM 13 mg per dl (normal 70 to 300); serum alkaline phosphatase 233 IU per liter (normal < 90); fecal fat 12 g per day (normal < 7); Schilling's test 14% (normal > 8%). Barium X-ray revealed a "malabsorption pattern" throughout the small intestine. The barium enema X-ray was normal. Giardia lamblia trophozoites were found in samples of intestinal juice and the cysts were found in stool specimens. Bacterial cultures of intestinal juice revealed 7.8 x 10" aerobes per ml, and 2.9 x 10" anerobes per ml. Fourteen small intestinal biopsies were obtained from sites in the duodenum and proximal 50 em of jejunum. They were variable in the degree of villous abnormality with severe (fig. lA ) and moderate (fig. lB ) lesions. There were decreased numbers of plasma cells in the lamina propria and an apparent increase in the number of mucosal lymphoid aggregates.

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The mucosal changes in the small intestine were those of the "mixed lesion" pattern described previously in the late onset immunodeficiency syndrome.'" 14 A 10-day course of metronidazole 250 mg three times daily was given because of the finding of giardiasis. However there was no weight gain and the di·a rrhea continued. On reevaluation 2 months .later there was no evidence of Giardia lamblia in intestinal ]uice aspirates, small intestinal biopsy sections, or stool specimens. Nine mucosal biopsies from the duodenum and proximal jejunum revealed persistent moderate and severe abnormalities. The fecal fat excretion had normalized at 4 g per day. Over the next 3 months the diarrhea worsened with 4 to 6 nonbloody liquid stools per day. In February 1974 a sigmoidoscopy revealed mucosal friability to the 10-cm level and normal-appearing mucosa proximally. One of two biopsies from the 7-cm level revealed a mucosal ulcer (fig. 2A). We initially overlooked the scattered cytomegalic cells with the characteristic CMV-type inclusions in the exudate of the ulcer base (fig. 2B). The second biopsy exhibited only mild changes with slight mucus depletion of rectal crypts and scattered polymorphonuclear leukocytes (fig. 2C). There were also reduced numbers of plasma cells, as expected. Barium enema revealed absent haustral markings and a deficient mucosal fold pattern compatible with a "pancolitis." Salazopyrin, 1 g four times a day was instituted. Because the diarrhea continued, trials of a lactose-free diet, tetracycline, and Lomotil were added 1 without success. In September 1974, gastric antral and small intestinal biopsies were obtained. Again, the presence of CMV-type cells was initially overlooked. There was focal nonerosive gastritis and scattered cytomegalic cells with inclusions characteristic of CMV infection in all four antral biopsies (fig. 3). These CMV-type cells were also found in 3 of the 12 small intestinal biopsies (fig. 4) . The patient was readmitted in July 1975 because of increasing fatigue and anorexia. Immunoglobulin quantitation revealed absent IgA, IgM 9 mg per dl, and IgG 128 mg per dl. Lympocyte transformation in vitro was markedly reduced in response to phytohemagglutinin, concanavalin A, and poke-

FIG. 1. A, small intestinal biopsy taken 10 ern beyond the region of the duodenojejunal junction. Villi are absent (H & E , x 120). B, small intestinal biopsy taken from the third portion of the duodenum at the same time as A. Shortened villi are present and there are fewer cells in the lamina propria than in A (H & E , x 120).

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FIG. 3. A, antral biopsy showing increased numbers of darkstaining inflammatory cells. The antral (pyloric) glands are reduced in numbers. One deep mucosal gland (arrow) contains a cytomegalovirus (CMV)-type cell (H & E, x 75). B, higher magnification to show the cytomegalic cell with its prominent intranuclear inclusion in the antral (pyloric) gland (H & E, x 480). FIG . 2. A, rectal biopsy with ulceration in the right half of the picture (H & E, x 75) . B, higher magnification of the rectal ulcer base showing a cytomegalic cell (arrow) with a prominent intranuclear inclusion (H & E, x 1200) . C, rectal biopsy taken at the same time as A shows only a mild abnormality consisting primarily of moderate gland mucus depletion (H & E, x 75).

weed mitogen.'"·"; Skin testing for delayed hypersensitivity was negative to purified protein derivatives, mumps, streptokinase-streptodornase, and Candida. Sensitivity to dinitrochlorobenzene17 could not be elicited after repeated applications. T cell rosettes•x were reduced at 47% (normal 55 to

75%). Schilling's test with intrinsic factor was 5%, the fecal fat excretion was 4 g per day, and stool weights averaged 600 g per day. Stool cultures for bacterial and fungal pathogens were negative and Giardia were not detected. A biopsy from the duodenal bulb showed typical CMV-type cells (fig. 5). Sigmoidoscopy and pancolonoscopy revealed discrete ulcerations and small pseudopolyps throughout the colon. Biopsy of a pseudopolyp revealed a solid mass of granulation tissue containing many CMV-type cells. Biopsies of ulcerated as well as nonulcerated mucosa contained CMV-type cells but

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they were more numerous in the former. In the nonulcerated areas there was again only mild mucosal inflammation. Electron microscopy of a rectal biopsy revealed CMV-infected macrophages in the lamina propria containing characteristic skein-like nuclear inclusions consisting of electrondense material, capsids with "empty cores," and capsids with "dense cores" (fig. 6). There was no condensation or margination of the nuclear chromatin. Within the cytoplasm Golgi elements were hypertrophied and these were surrounded by many enveloped CMV capsids and membrane-bound dense bodies (fig. 6) . No multivesicular bodies were seen.

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FIG. 5. A, mucosal biopsy from the duodenal bulb. A single cytomegalovirus (CMV)-type cell (arrow) is present in the Brunner's gland region (H & E , x 120). B, higher magnification to show the cytomegalic cell with its dark-staining intranuclear inclusion (H & E, X 480).

Liver function tests revealed: serum alkaline phosphatase 328 IU per liter; 5'-nucleotidase 52 IU per liter (normal < 14); albumin 'I 6 g per dl; normal SGOT, and bilirubin. A percutaneous needle liver biopsy revealed dense lymphocytic portal tract infiltrates but no CMV-type cells were seen. Cytomegalovirus was cultured from liver biopsy tissue and urine. Cultures were negative for cytomegalovirus from throat

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FIG. 6. Cytomegalovirus (CMV)-infected cell in the lamina propria of the rectum (biopsy specimen). A, survey electron micrograph of intranuclear and intracytoplasmic viral inclusions. The features that distinguish CMV from other herpes-type virus infections are skeinlike intranuclear inclusions, lack of margination of chromatin, and the presence within the cytoplasm of collections of coated capsids, homogeneous "dense bodies" (DB) and hypertrophied Golgi cisternae (uranyl acetate and lead citrate, x 13,500). B, the intranuclear inclusion is composed of a finely granular matrix, and double-shelled uncoated capsids, one of which contains an electron-dense nucleoid (arrow) ( x 65,000) . C, the intracytoplasmic inclusion is composed of coated capsids and homogeneous dense bodies (DB) ( x 65,000).

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swabs, peripheral blood buffy coat, bone marrow , stools, small intestinal juice and mucosae of stomach, small intestine, and colon. Viral serology, as expected, was negative. Over a 4-week period there was a marked deterioration with increased diarrhea and an agonal course complicated by bacterial sepsis, pancytopenia, and disseminated intravascular coagulation. The most striking autopsy findings were in the large intestine. There were deep serpiginous mucosal ulcers and pseudopolyps. Many CMV-type cells were found in both ulcerated and nonulcerated mucosa. Small ulcerations were present in the distal esophagus and scattered CMV-type cells were found in the superficial submucosa. In the stomach CMV-type cells were found in the bases of a number of erosions and in the intervening mucosa. Despite the autolysis of the small intestinal mucosa , CMV-type cells could be identified in epithelial cell remnants, in endothelial cells, and in Brunner's glands. In the liver there were mild lymphocytic infiltrates in portal tracts but no cytomegalic cells. The intra- and extrahepa tic bile ducts were normal. In one adrenal gland there were cytomegalic ganglion cells containing prominent intranuclear inclusions compatible with CMV infection. Cytomegalic cells were not identified in sections of spleen, lymph nodes, lung, heart, pancreas, and bone marrow. Autopsy viral cultures were positive for cytomegalovirus from an area of hemorrhagic-appearing small intestinal mucosa, liver, and lung. Cultures from other sites in the gastrointestinal tract were negative.

/

Discussion Our patient's early course was quite typical for the late onset immunodeficiency syndrome. He had recurrent skin and respiratory tract infections, and then developed diarrhea with steatorrhea. The steatorrhea cleared with treatment of the giardiasis but the diarrhea persisted and became more severe. The development of pancolitis was a major factor in the terminal fulminant course. Symptomatic colitis has been reported 19 • 20 in patients with the late onset immunodeficiency syndrome but it appears to be uncommon. The colitis in this patient differed clearly from idiopathic ulcerative colitis in that it was patchy in distribution and variable in severity. There were discrete ulcerations but only mildly abnormal intervening mucosa. This differs from the diffuse change found in untreated symptomatic patients with idiopathic ulcerative colitis. Many cells of the CMVtype were found in the granulation tissue of ulcers and in pseudopolyps, whereas they were less numerous in intervening mucosa. The electron microscopy studies of the rectal biopsies were of special interest. The features ascribed to CMV infection in vitro 2 1• 22 were seen in the rectal biopsy material. Cytomegalovirus infection induces certain unique morphological changes in cells which are not seen with other herpes-type viruses2 L 22 in vitro. It appears possible to distinguish the lesions caused by CMV at an ultrastructural level on the basis of the reticular appearance of the intranuclear inclusions, an absence of margination of nuclear chromatin, and the presence of characteristic cytoplasmic dense bodies. 21 • 22 The latter do not appear to be lysosomes.2 1 Herpesvirus on the other hand produces marked condensation and margination of the nuclear chromatin and extensive

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membrane change, but not a skein-like nuclear inclusion or cytoplasmic dense bodies. 21 CMV-associated colitis has been reported previously. Some patients had what appeared to be idiopathic ulcerative colitis2a-zs and others had more atypical 23• 2" · 27 forms of colitis. One case is of special interest because a thymoma coexisted.27 Thymoma may be associated with immunoglobulin deficiency. We found morphological evidence of CMV infection in the stomach and small intestine before the patient died. In the stomach there was a focal nonerosive gastritis and in the small intestine there were villous architectural abnormalities that persisted after eradication of the giardiasis. We do not know whether the CMV caused or aggravated these mucosal changes. Similarly, CMV was cultured from liver biopsy tissue but it is impossible to know whether the elevated serum alkaline phosphatase and the portal tract infiltrates were attributable to chronic CMV infection. The histological detection of CMV-type cells in mucosal biopsies is not difficult but does require a special awareness and the examination of multiple serial sections. The characteristic cells are most numerous in areas of ulceration, especially in ulcer bases. In the small intestinal mucosa the CMV-type cells appeared to be more numerous in Brunner's glands than in other areas. Almost all regions of the gastrointestinal tract are now accessible to biopsy with the recent availability of the newer endoscopic instruments. When opportunistic infection of the gastrointestinal tract is suspected, mucosal biopsies should be taken for culture as an adjunct to conventional diagnostic histology and serology. In our patient the antemortem and postmortem colon biopsies were negative for CMV on culture. This was surprising because the colon exhibited the most striking mucosal abnormalities, and we had electron microscopic evidence of active CMV infection. Perhaps this discrepancy was caused by a sampling problem28 or to storage of some of the specimens overnight. 29 Recovery of CMV is best from fresh tissue that is processed immediately. Storage of specimens at 4°C before innoculation into cell cultures reduces the recovery of CMV to 58%, and conventional freezing of tissue to -20°C for 4 to 7 days reduces recovery of CMV to 25%.29 Viral culture is approximately 6 times more sensitive than histological examination in the detection of CMV. 30 However, the major difficulty with viral isolation is the time required, i.e., from 7 to 25 days. 3 1 One method which has potential for providing a rapid and accurate diagnosis is immunofluorescent staining with specific anti-CMV sera. 32 This is the first report of widespread CMV infeCtion of the gastrointestinal tract in the late onset immunodeficiency syndrome. Impaired cellular immunity was documented late in his course but it may have been secondary to malnutrition or to overwhelming viral infection. In opportunistic infections with ubiquitous organisms such as CMV it is difficult to prove that a given organism is responsible for symptoms and pathological changes. This "proof," as in giardiasis for example, usually rests on a response to therapy. Unfortu-

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nately, definitive therapy for CMV is not yet available. Thus, it is likely, but not proved, that the widespread CMV infection in the gastrointestinal tract of our patient contributed to the symptom complex and the mucosal injury. Unusual opportunistic infections as a cause of diarrhea should be considered in patients with immunodeficiency syndromes, especially if giardiasis is ruled out. This assumes special importance when empirical therapy with corticosteroids is being considered for enteritis or colitis. Serological tests often cannot be used as a diagnostic aid in these patients so one must rely on careful morphological evaluation and on cultures. REFERENCES 1. Ament ME, Ochs HD, Davis SD: Structure and function of the gastrointestinal tract in primary immunodeficiency syndromes; a study of 39 patients. Medicine 52:227-248, 1973 2. Hermans PE, Diaz-Buxo JA, Stobo JD: Idiopathic late-onset immunoglobulin deficiency. Am J Med 61:221-237, 1976 3. Binder HJ, Reynolds RD: Control of diarrhea in secondary hypogammaglobulinemia by fresh plasma infusions. N Eng! J Med 277:802-803, 1967 4. Gleich GJ, Hofmann AF: Use of cholestyramine to control diarrhea associated with acquired hypogammaglobulinemia. Am J Med 51:281-286, 1971 5. Diaz-Buxo JA, Hermans PE, Huizenga KA: Gastrointestinal dysfunction in immunoglobulin deficiency. Effect of corticosteroids and tetracycline . JAMA 233:1189-1191 , 1975 6. Schilling RF: Intrinsic factor studies. II. Effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B, 2 • J Lab Clin Med 42:860-866, 1953 7. van de Kamer JH, ten Bokkel Huinink H, Weijers HA: Rapid method for the determination of fat in feces. J Bioi Chern 177: 347-355, 1949 8. Fahey JL, McKelvey EM: Quantitative determination of serum immunoglobulins in antibody-agar plates. J Immunol 94:84-90, 1965 9. Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-254, 1965 10. Sever JL: Application of microtechnique to viral serological investigations. J Immunol 88:320-329, 1962 11. Lennette EH: General principles underlying laboratory diagnosis of viral and rickettsial infections, chap 1. In Diagnostic Procedures for Viral and Rickettsial Infections . Fourth edition. Edited by EH Lennette, NJ Schmidt. New York, American Public Health Association, Inc, 1969, p 1-65 12. Perera DR, Weinstein WM, Rubin CE: Small intestinal biopsy . Hum Pathol6:157-217, 1975 13. Luft JH: Improvements in epoxy resin embedding methods. J

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Biophys Biochem Cytol 9:409-414, 1961 14. Ament ME, Rubin CE: Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes . Gastroenterology 62:216-226, 1972 15. Foad BSI, Adams LE, Yamauchi Y, et al: Phytomitogen responses of peripheral blood lymphocytes in young and older subjects. Clin Exp Immunol17:657-664, 1974 16. Hagen C, Froland A: Analysis of the variation in lymphocyte response to PHA in normal subjects. Acta Pathol Microbial Scand [B] 81:253-258 , 1973 17. Eilber FR, Mizze JA , Morton DL: Sequential evaluation of general immune competence in cancer patients: correlation with clinical course. Cancer 35:660-665, 1975 18. Weiner MS, Bianco C, Nussenzweig V: Enha nced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes. Blood 42:939-946, 1973 19. Kirk BW, Freedman SO: Hypogammaglobulinemia, thymoma and ulcerative colitis. Can Med Assoc J 96:1272-1277, 1967 20. Kopp WL, Trier JS, Stiehm ER, et a!: "Acquired" agammaglobulinemia with defective delayed hypersensitivity. Ann Intern Med 69:309-317, 1968 21. Smith JD , de Harven E: Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J Virol 12:919-930, 1973 22. Michelson-Fiske S, Arnoult J, Febvre H: Cytomegalovirus infection on human lung epithelial cells in vitro. Intervirology 5:354363 , 1975 23. Wong TW, Warner NE: Cytomegalic inclusion disease in adults. Arch Pa thol 74:403-422 , 1962 24. Farmer GW, Vincent MM, Fuccillo DA, et al: Viral investigations in ulcerative colitis and regional enteritis. Gastroenterology 65:8-18, 1973 25. Keren DF, Milligan FD , Strandberg JD, et al: Intercurrent cytomegalovirus colitis in a patient with ulcerative colitis. Johns Hopkins Med J 136:178-182, 1975 26. Tamura H: Acute ulcerative colitis associated with cytomegalic inclusion virus. Arch Pathol 96:164-167, 1973 27. Dent DM, Duys PJ, Bird AR, et al: Cytomegalic virus infection of bowel in adults. S Afr Med J 49:669-672, 1975 28. Macasaet FF, Holley KE, Smith TF, et al: Cytomegalovirus studies of autopsy tissue. II. Incidence of inclusion bodies and related pathologic data . Am J Clin Pathol 63:859-865, 1975 29. Macasaet FF, Smith TF, Holley KE: Effect of storage on recovery of cytomegalovirus from necropsy tissue. J Clin Pathol 29:10771080, 1976 30. Smith TF, Holley KE, Keys TF, et al: Cytomegalovirus studies on autopsy tissue. Am J Clin Pathol 63:854-858, 1975 31. Abdallah PS, Ma rk JBD , Merigan TC: Diagnosis of cytomegalovirus pneumonia in compromised hosts. Am J Med 61:326-332, 1976 32. Huang YT, Huang ES , Pagano JS: Antisera to human cytomegalovirus prepared in the guinea pig. Specific immunofluorescence and complement fixation tests. J Immunol 112:528-532, 1974