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Legionella infection of the colon presenting as acute attack of ulcerative colitis
GASTROENTEROLOGY 1989:97:751-5
CASE
REPORTS
Legionella Infection of the Colon Presenting as Acute Attack of Ulcerative Colitis T. SCHMIDT, A. PFEIFFER, W. EHRET, E. KEIDITSCH, G. RUCKDESCHEL, and H. KAESS II.Medical Department and Institute of Pathology, Krankenhaus Miinchen-Bogenhausen, and Max v. Pettenkofer Institut, Ludwig-Maximilian-Universitlt, Germany
A 4%yr-old woman with long-standing ulcerative colitis of the descending colon, sigmoid, and rectum presented with bloody diarrhea, tenesmus, and high fever. Endoscopic findings were compatible with an acute attack of ulcerative colitis, which proved to be resistant to systemic corticosteroid treatment. In the presence of an acute abdomen with ascites and double-contoured colonic wall, hemicolectomy was performed. Postoperatively, high temperature, hyponatremia, and elevated liver enzyme levels persisted. Pleural effusions developed. Antibodies to Legionella pneumophila serogroup 3 were detected in the serum. Erythromycin therapy induced rapid improvement. In a massive submucosal edema of of the same the affected colon, L. pneumophila serogroup was demonstrated by direct immunofluorescence staining.
I
n fatal cases of Legionella pneumonia, the bacteria have been demonstrated in numerous extrapulmonary tissues (l-8),indicating the systemic character of this infection. Although diarrhea may be a prominent symptom early in the course of Legionnaires’ disease (9,10), gastrointestinal tract lesions, associated with tissue demonstration of legionellae, have rarely been described (11-13). These single cases include inflammation and hemorrhagic necrosis of the terminal ileum (12,13), as well as necrotizing inflammation of the cecum (ll), in patients with severe pneumonia. This paper describes a nonpneumonic L. pneumophila infection of the colon mimicking a severe exacerbation of long-standing ulcerative colitis.
Munich, Federal Republic of
She described 5-6 bowel movements associated with tenesmus and bouts of fever up to a temperature of 39.7”C. In the patient’s history, ulcerative colitis involving the rectum, the sigmoid, and the descending colon had been diagnosed in 1972. In 1980 and 1984, acute attacks had been treated with systemic corticosteroid administration. Since 1984, no activity of the ulcerative colitis had been observed under continuous treatment with 3 g of sulfasalazine daily. Physical examination revealed a severely ill woman (162 cm, 66 kg). Rectal temperature was 39.9”C. Abdominal palpation provoked tenderness in the upper and lower left quadrant. On rectal examination, pus and blood were found. Laboratory studies disclosed the following pathologic values: erythrocyte sedimentation rate, 18 mm/h; hemoglobin, 11.7 gidl: white blood cell count, 10.5 x log/L; C-reactive protein, 6.3 mg/dl [normal, cl.2 mg/dl); total serum protein, 5.9 g/d1 (normal, 6.1-8.2 g/dl); sodium, 131 mmol/L (normal, 135-150 mmol/L); iron, 10 pg/dl (normal, 40-140 pgldl); aspartate aminotransaminase, 23 IU/L (normal, cl9 IUIL); y-glutamyl transpeptidase, 33 IU/L (normal, <24 IUIL). Stool analyses were negative for Salmonella, Shigella, Aeromonas hydrophila, Campylobacter jejuni, Yersinia, Clostridium dificile, ova, and parasites. Serologic tests for yersiniosis, amebiasis, and the Gruber-Widal reaction were also negative, as were cultures of venous blood, urine, and sputum for bacteria. Abdominal ultrasound examination and computed tomography scan revealed several small gallstones. Chest x-ray, plain films of the abdomen, esophagogastroduodenoscopy, electrocardiogram, and echocardiogram were nondiagnostic. Thus, the diagnosis of an acute exacerbation of ulcerative colitis was made. Under treatment with 40 mg of
Case Report Abbreviation
A 42-yr-old woman was hospitalized with bloody and watery diarrhea that had started 2 days previously.
used in this paper: sg, serogroup.
0 1989 by the American
Gastroenterological
0016~5085/89/$3.50
Association
752
SCHMIDT ET AL.
GASTROENTEROLOGY Vol. 97, No. 3
Figusre 1. Microscopic section of the colon wall (H&E, x100) demonstrating ulcerations of the mucosa (arrowhead) and small c:rypt abscesses (small arrowheads). Note the atypical massive edema of the submucosa (bracket).
methylprednisolone daily and total parenteral nutrition, no improvement occurred. Colonoscopy, performed 4 days later, showed confluent shallow ulcerations continuously extending from the anal canal to the left flexure. During the following day, abdominal distention, spiking fever up to 40.4”C, hypotension, oliguria, and metabolic acidosis (pH 7.19; base excess, -12 mmol/L) developed. The following laboratory data were obtained: white blood cell count, 16.5 x log/L; sodium, 129 mmol/L (despite a daily intravenous sodium load of 180 mmol); bilirubin, 1.5 mg/dl; aspartate aminotransaminase, 313 III/L; alanine aminotransferase, 281 IU/L; y-glutamyl transpeptidase, 553 III/L; and alkaline phosphatase, 1227 III/L. Corticosteroids were discontinued and an antibiotic treatment with 6 g of cefotaxime, 1.5 g of metronidazole, and 240 mg of tobramycin daily was started. The following day, an acute abdomen was present. Abdominal computed tomography scan now revealed ascites, and the wall of the descending colon, sigmoid, and rectum seemed to be markedly thickened and double-contoured. Laparotomy was performed. The ascending colon was distended and the descending hemicolon showed congestion and loss of haustrations. Neither a perforation nor an intraabdominal abscess was found. Routine cultures of the ascitic fluid remained sterile. Preserving the distal rectum, hemicolectomy with a temporary stoma of the transverse colon was performed. Histopathologic examination confirmed the diagnosis of ulcerative colitis with confluent mucosal ulcerations and small crypt abscesses, but in addition, an atypical massive edema of the submucosa was present (Figure 1). Patchy
perivascular infiltrates, mainly consisting of lymphocytes, extended from the mucosal layer to the muscularis propria and the submucosa. The deeper layers of the bowel wall were not affected by inflammatory changes. Nonspecific inflammatory reactions were found in >70 enlarged mesenterial lymph nodes. Postoperatively, high temperature persisted despite the antibiotic treatment. Development of pleural effusions, unchanged hyponatremia, and increase in liver enzymes prompted the search for a Legionella infection. When the serologic studies were pointing to an infection with L. pneumophila serogroup 3, a trial of erythromycin (2 g daily) was started. Complete recovery of the patient and a normalization of serum sodium and liver enzymes were observed within 14 days of therapy. Titer levels of indirect immunofluorescence assay and enzyme-linked immunosorbent assay significantly decreased during the postoperative period (Figure 2). The serologic results of immunofluorescence assay and enzyme-linked immunosorbent assay were confirmed by immunoblot technique with membrane antigens of the serogroups l-12 of L. pneumophila. The patient’s serum exclusively reacted with the major membrane protein of L. pneumophila serogroup (sg) 3. Histopathologic workup of the resected formalin-fixed colon using modified Dieterle silver impregnation staining (14) was performed 4 wk after the operation. It demonstrated numerous short, black pleomorphic rods of extracellular localization in the submucosa of the diseased colon (Figure 3). These bacilli were identified as L. pneumophila serogroup 3 by direct immunofluorescence staining (Figure 4). Examination by conventional histologic
Legionella INFECTION IN ULCERATIVE COLITIS
September 1989
1:1024
1:256
1:64
1:16 0
7
14
,flw/w n
62
l77
d.YS
Figure 2. Course of indirect immunofluorescence test (immunofluorescence assay, w) and enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, *----*I titer levels reactive on L. pneumophila sg 3 in the patient’s serum before, during, and after erythromytin treatment.
methods, Dieterle staining, and immunofluorescence did not reveal any pathologic findings in the disease-free margins of the resected colon.
Methods The following serogroups of L. pneumophila were investigated: Philadelphia 1 (sg l), Togus 1 (sg 2), Bloomington 2 (sg 3), Los Angeles 1 (sg 4), Dallas 1-E (sg 5),
753
Chicago 2 (sg 6), Chicago 8 (sg 7), Concord 3 (sg 8), In-23-61-C2 (sg 9), Leiden 1 (sg lo), 797-PA-H (sg ll), and 570-Co-H (sg 12). In addition, the following strains of other Legionella species were tested: L. micdodei (Tatlock, ATCC 33204), both serogroups of L. bozemanii (Wiga and Toronto 3, ATCC 33265), L. dumoffi (NY 23, ATCC 33279), L. gormonii (LS 13, ATCC 33279), L. jordanis (BL-540, ATCC 33623), both serogroups of L. Iongbeachae (Long Beach 4, ATCC 33462 and Tucker 1, ATCC 33484) and L. feeleii (Wo-33CC3, ATCC 35072 and 691-Wi-H, ATCC 35849), L. hackeliae (Lansing 2, ATCC 35250), and L. wadsworthii (al/716 A, ATCC 33877). The immunofluorescence assay of the patient’s sera was performed by using formalin-inactivated suspensions of the antigens according to the technique of Wilkinson et al. (15). Enzyme-linked immunosorbent assay was conducted separately for each antigen on polyvinylchloride microtiter plates (Dynatech, Baton Rouge, La.), layered with ultrasonicates of the bacterial strains mentioned above. Peroxidase, conjugated with goat-antihuman immunoglobulin (Medac), was used for detection. The optical density was measured at 490 nm by using an enzyme-linked immunosorbent assay reader (Dynatech). Phosphatebuffered saline instead of patient’s serum was used as a negative control, whereas a patient’s serum with an immunofluorescence assay titer of 1:1024 was used as a positive control for the sg 1 of L. pneumophila. All other antigens were controlled with corresponding rabbit hyperimmune sera, detected with antirabbit immunoglobulin-peroxidase conjugates. According to the results determined from sera of blood donors and patients with respiratory infections
Figure 3. Microscopic section of colonic submucosa (modified Dieterle staining, x1000) demonstrating numerous short black rods, 1-3 Frn in length and up to 1 pm in width, of extracellular localization.
754
Figure
SCHMIDT ET AL.
4.
GASTROENTEROLOGYVol.
Microscopic section of colonic submucosa (direct immunofluorescence staining, x800). pneumophila sg 3 with fluorescein isothiocyanate-labeled monospecific antiserum.
caused by other microbial pathogens, an optical density of 0.5 was used as the cutoff value. Immunoblot was performed according to the technique of Ehret and Ruckdeschel (16,17). For tissue demonstration of L. pneumophila, direct fluorescent antibody staining was applied. For this purpose, rabbit antiserum was labeled with fluorescein isothiocyanate according to Cherry and McKinney (18) and was made monospecific for sg 3 by absorption. A negative control was performed by substituting labeled nonimmune rabbit serum for the anti-Legionella antibody. In the current literature the specificity of direct fluorescent antibody staining for legionellae is reported to be >99% (19).
Discussion Legionella pneumophila infections may present with protean clinical features including pulmonary, renal, neurologic, and gastrointestinal symptoms (20). Legionellae have been demonstrated in various extrapulmonary tissues, as in blood (1,2), thoracic (3-5,7) and extrathoracic lymph nodes (6,7), heart (8), kidneys (6,7), liver (5,6), spleen (5-8), and bone marrow (7). Although 50% of patients with Legionnaires’ disease (9,lO) and 15% of patients with Pontiac fever (21) complain of diarrhea, reports on gastrointestinal tract lesions due to direct bacterial infection are rare. In 1982, Fogliani et al. (11) described a case of necrotizing inflammation and several perforations of the cecum in Legionnaires’ disease. By direct immu-
97,
No. 3
Tissue demonstration (arrow) of L.
nofluorescence staining, L. pneumophila sg 1 was demonstrated in the diseased tissue. Dournon et al. (12) reported on a similar case of Legionnaires’ disease with inflammation of the terminal ileum, associated with hemorrhagic peritonitis. The authors identified L. pneumophila sg 1 in the peritoneal exudate, in the ileum wall, and in the intraluminal mucus of the small bowel. In 1986, Ozoux et al. (13) described a patient suffering from a Legionella pneumonia that was associated with hemorrhagic necrosis of the terminal ileum and enlargement of mesenteral lymph nodes related to a local infection with L. pneumophila sg 4. Legionella pneumophila sg 3, detected in our patient, only causes 2%-4% of Legionella pneumonias (22). It has been demonstrated in hepatic and splenic tissue (5). To our knowledge, this is the first observation of an L. pneumophila infection of the colon in preexisting ulcerative colitis. The clinical features and the endoscopic findings suggested an acute exacerbation of ulcerative colitis. At the patient’s admission, no clinical or radiologic signs of pulmonary involvement were present. Only the occurrence of pleural effusions, associated with persisting hyponatremia and further increase of serum liver enzymes,
prompted us to look for a Legionella infection. The histologic features of the resected colon were compatible with ulcerative colitis (23), but in addition, an intense
edema
of the submucosa
was found.
September
1989
The submucosal edema was limited to those colonic areas affected by inflammatory mucosal changes. In the normal part of the resected colon, neither inflammatory mucosal changes, nor submucosal edema, nor legionellae were present. The demonstration of L. pneumophila sg 3 in the colonic submucosa by highly specific immunologic methods, and the clinical course, characterized by resistance to corticosteroids and improvement under erythromycin therapy, suggest that this acute attack of a long-standing ulcerative colitis was related to a L. pneumophila infection. Whether Legionella infections of the colon could be implicated in some cases of severe and corticosteroid-resistant attacks of ulcerative colitis should undergo further investigation.
References 1.
2.
3.
4.
5.
6.
7. 8.
9.
10.
Edelstein PH, Meyer RD, Finegold SM. Isolation of Legionella pneumophila from blood. Lancet 1979$:750-l. Macrae AD, Greaves PW, Platts P. Isolation of Legionella pneumophila from blood culture. Br Med J [Clin Res] 1979; 2:1189-90. Weisenburger DD, Helms CM, Viner JP, et al. Demonstration of Legionnaires’ disease bacillus in hilar lymph nodes (lett). Arch Path01 Lab Med 1979;103:153. Hicklin MD, Thomason BM, Chandler FW, Blackman JA. Pathogenesis of acute Legionnaires’ disease pneumonia: immunofluorescent microscopic study. Am J Clin Path01 1980; 72:480-7. Watts JC, Hicklin MD, Thomason BM, et al. Fatal pneumonia caused by Legionella pneumophila, serogroup 3: demonstration of the bacilli in extrathoracic organs. Ann Intern Med 1980;92:186-8. Evans CP, Winn WW. Extrathoracic localisation of Legionella pneumophila in Legionnaires’ pneumonia. Am J Clin Path01 1981;73:813-5. Weisenburger DD, Rappaport H, Ahluwalia MS, Melvani R, Renner ED. Legionnaires’ disease. Am J Med 1980;69:476-82. White HJ, Felton WW, Sun CN. Extrapulmonary histopathologic manifestations of Legionnaires’ disease: evidence for myocarditis and bacteriemia. Arch Path01 Lab Med 1980; 104:287-g. Tsai TF, Finn DR, Plikaytis BD, et al. Legionnaires’ disease: clinical features of the epidemic in Philadelphia. Ann Intern Med 1979;90:509-17. Broome CV, Goings SA, Thacker SB, et al. The Vermont
Legionella INFECTION IN ULCERATIVE COLITIS
11.
12. 13.
14. 15.
16.
17.
18.
19. 20. 21.
22.
23.
755
epidemic of Legionnaires’ disease. Ann Intern Med 1979; 90:573-7. Fogliani J, Domenget JF, Hohn B, Merignargues G, Bornstein N. Maladie des legionnaires avec localisation digestive. Une observation. Nouv Presse Med 1982;11:2699-702. Dournon E, Bure A, Kemeny JL, Pourriat JL, Valeyre D. Legionella pneumophila peritonitis (lett). Lancet 1982;i:1363. Ozoux JP, Calan L, Portier G, Legue E, Brizon J. Enterite necrosante et maladie des legionnaires. Presse Med 1986; 15:75. Van Orden AE, Greer PW. Modification of the Dieterle spirochete stain. J Histotechnol 1977;1:51-3. Wilkinson HW, Fikes BF, Cruce DD. Indirect immunofluorescence test for serodiagnosis of Legionnaires’ disease: evidence for serogroup diversity of Legionnaires’ disease bacterial antigens and for multiple specificity of human antibodies. J Clin Microbial 1979;9:379-83. Ehret W, Ruckdeschel G. Species specific membrane proteins of Legionellaceae. Zentralb Bakteriol Mikrobiol Hyg [A] 1983; a255:33-8. Ehret W, Ruckdeschel G. Membrane proteins of Legionellaceae. I. Membrane proteins of different strains and serogroups of Legionella pneumophila. Zentralb Bakteriol Mikrobiol Hyg [A] 1985;a259:433-55. Cherry WB, McKinney RM. Detection in clinical specimens by direct immunofluorescence. In: Jones GL, Hebert GA, eds. “Legionnaires’,” the disease, the bacterium and methodology. Atlanta: Centers for Disease Control, 1978:147-54. Edelstein PH. Culture diagnosis of Legionella infections. Zentralb Bakteriol Mikrobiol Hyg [A] 1983;96-101. Blackman JA, Chandler FW, Cherry WB, et al. Legionellasis. Am J Path01 1981;103:429-65. Glick TH, Cregg MB, Berman B, Mallison G, Rhodes WW Jr, Kasanoff I. Pontiac fever: an epidemic of unknown etiology in a health department: I. Clinical and epidemiological aspects. Am J Epidemiol 1978;107:149-60. Reingold AL, Thomason BM, Brake BJ, Thaker L, Wilkinson HW, Kuritsky JN. Legionella pneumonia in the United States: the distribution of serogroups and species causing human illness. J Infect Dis 1984;149:819-23. Hamilton SR, Morson BC. Ulcerative colitis. Pathology. In: Berk JE, ed. Bockus gastroenterology. 4th ed. Volume 4. Philadelphia: WB Saunders, 1985:2141-l.
Received June 1, 1988. Accepted February 13, 1989. Address requests for reprints to: Dr. Thomas Schmidt. II Medical Department, Krankenhaus Munchen-Bogenhausen, Englschalkingerstrasse 77, 8000 Munich 81, Federal Republic of Germany. The authors thank B. Birkner (endoscopist), W. Heitland (surgeon), and H. Ingrisch (radiologist) for their collaboration. They also thank J. Aronbayev for reviewing the manuscript.
CASE
REPORTS
Legionella Infection of the Colon Presenting as Acute Attack of Ulcerative Colitis T. SCHMIDT, A. PFEIFFER, W. EHRET, E. KEIDITSCH, G. RUCKDESCHEL, and H. KAESS II.Medical Department and Institute of Pathology, Krankenhaus Miinchen-Bogenhausen, and Max v. Pettenkofer Institut, Ludwig-Maximilian-Universitlt, Germany
A 4%yr-old woman with long-standing ulcerative colitis of the descending colon, sigmoid, and rectum presented with bloody diarrhea, tenesmus, and high fever. Endoscopic findings were compatible with an acute attack of ulcerative colitis, which proved to be resistant to systemic corticosteroid treatment. In the presence of an acute abdomen with ascites and double-contoured colonic wall, hemicolectomy was performed. Postoperatively, high temperature, hyponatremia, and elevated liver enzyme levels persisted. Pleural effusions developed. Antibodies to Legionella pneumophila serogroup 3 were detected in the serum. Erythromycin therapy induced rapid improvement. In a massive submucosal edema of of the same the affected colon, L. pneumophila serogroup was demonstrated by direct immunofluorescence staining.
I
n fatal cases of Legionella pneumonia, the bacteria have been demonstrated in numerous extrapulmonary tissues (l-8),indicating the systemic character of this infection. Although diarrhea may be a prominent symptom early in the course of Legionnaires’ disease (9,10), gastrointestinal tract lesions, associated with tissue demonstration of legionellae, have rarely been described (11-13). These single cases include inflammation and hemorrhagic necrosis of the terminal ileum (12,13), as well as necrotizing inflammation of the cecum (ll), in patients with severe pneumonia. This paper describes a nonpneumonic L. pneumophila infection of the colon mimicking a severe exacerbation of long-standing ulcerative colitis.
Munich, Federal Republic of
She described 5-6 bowel movements associated with tenesmus and bouts of fever up to a temperature of 39.7”C. In the patient’s history, ulcerative colitis involving the rectum, the sigmoid, and the descending colon had been diagnosed in 1972. In 1980 and 1984, acute attacks had been treated with systemic corticosteroid administration. Since 1984, no activity of the ulcerative colitis had been observed under continuous treatment with 3 g of sulfasalazine daily. Physical examination revealed a severely ill woman (162 cm, 66 kg). Rectal temperature was 39.9”C. Abdominal palpation provoked tenderness in the upper and lower left quadrant. On rectal examination, pus and blood were found. Laboratory studies disclosed the following pathologic values: erythrocyte sedimentation rate, 18 mm/h; hemoglobin, 11.7 gidl: white blood cell count, 10.5 x log/L; C-reactive protein, 6.3 mg/dl [normal, cl.2 mg/dl); total serum protein, 5.9 g/d1 (normal, 6.1-8.2 g/dl); sodium, 131 mmol/L (normal, 135-150 mmol/L); iron, 10 pg/dl (normal, 40-140 pgldl); aspartate aminotransaminase, 23 IU/L (normal, cl9 IUIL); y-glutamyl transpeptidase, 33 IU/L (normal, <24 IUIL). Stool analyses were negative for Salmonella, Shigella, Aeromonas hydrophila, Campylobacter jejuni, Yersinia, Clostridium dificile, ova, and parasites. Serologic tests for yersiniosis, amebiasis, and the Gruber-Widal reaction were also negative, as were cultures of venous blood, urine, and sputum for bacteria. Abdominal ultrasound examination and computed tomography scan revealed several small gallstones. Chest x-ray, plain films of the abdomen, esophagogastroduodenoscopy, electrocardiogram, and echocardiogram were nondiagnostic. Thus, the diagnosis of an acute exacerbation of ulcerative colitis was made. Under treatment with 40 mg of
Case Report Abbreviation
A 42-yr-old woman was hospitalized with bloody and watery diarrhea that had started 2 days previously.
used in this paper: sg, serogroup.
0 1989 by the American
Gastroenterological
0016~5085/89/$3.50
Association
752
SCHMIDT ET AL.
GASTROENTEROLOGY Vol. 97, No. 3
Figusre 1. Microscopic section of the colon wall (H&E, x100) demonstrating ulcerations of the mucosa (arrowhead) and small c:rypt abscesses (small arrowheads). Note the atypical massive edema of the submucosa (bracket).
methylprednisolone daily and total parenteral nutrition, no improvement occurred. Colonoscopy, performed 4 days later, showed confluent shallow ulcerations continuously extending from the anal canal to the left flexure. During the following day, abdominal distention, spiking fever up to 40.4”C, hypotension, oliguria, and metabolic acidosis (pH 7.19; base excess, -12 mmol/L) developed. The following laboratory data were obtained: white blood cell count, 16.5 x log/L; sodium, 129 mmol/L (despite a daily intravenous sodium load of 180 mmol); bilirubin, 1.5 mg/dl; aspartate aminotransaminase, 313 III/L; alanine aminotransferase, 281 IU/L; y-glutamyl transpeptidase, 553 III/L; and alkaline phosphatase, 1227 III/L. Corticosteroids were discontinued and an antibiotic treatment with 6 g of cefotaxime, 1.5 g of metronidazole, and 240 mg of tobramycin daily was started. The following day, an acute abdomen was present. Abdominal computed tomography scan now revealed ascites, and the wall of the descending colon, sigmoid, and rectum seemed to be markedly thickened and double-contoured. Laparotomy was performed. The ascending colon was distended and the descending hemicolon showed congestion and loss of haustrations. Neither a perforation nor an intraabdominal abscess was found. Routine cultures of the ascitic fluid remained sterile. Preserving the distal rectum, hemicolectomy with a temporary stoma of the transverse colon was performed. Histopathologic examination confirmed the diagnosis of ulcerative colitis with confluent mucosal ulcerations and small crypt abscesses, but in addition, an atypical massive edema of the submucosa was present (Figure 1). Patchy
perivascular infiltrates, mainly consisting of lymphocytes, extended from the mucosal layer to the muscularis propria and the submucosa. The deeper layers of the bowel wall were not affected by inflammatory changes. Nonspecific inflammatory reactions were found in >70 enlarged mesenterial lymph nodes. Postoperatively, high temperature persisted despite the antibiotic treatment. Development of pleural effusions, unchanged hyponatremia, and increase in liver enzymes prompted the search for a Legionella infection. When the serologic studies were pointing to an infection with L. pneumophila serogroup 3, a trial of erythromycin (2 g daily) was started. Complete recovery of the patient and a normalization of serum sodium and liver enzymes were observed within 14 days of therapy. Titer levels of indirect immunofluorescence assay and enzyme-linked immunosorbent assay significantly decreased during the postoperative period (Figure 2). The serologic results of immunofluorescence assay and enzyme-linked immunosorbent assay were confirmed by immunoblot technique with membrane antigens of the serogroups l-12 of L. pneumophila. The patient’s serum exclusively reacted with the major membrane protein of L. pneumophila serogroup (sg) 3. Histopathologic workup of the resected formalin-fixed colon using modified Dieterle silver impregnation staining (14) was performed 4 wk after the operation. It demonstrated numerous short, black pleomorphic rods of extracellular localization in the submucosa of the diseased colon (Figure 3). These bacilli were identified as L. pneumophila serogroup 3 by direct immunofluorescence staining (Figure 4). Examination by conventional histologic
Legionella INFECTION IN ULCERATIVE COLITIS
September 1989
1:1024
1:256
1:64
1:16 0
7
14
,flw/w n
62
l77
d.YS
Figure 2. Course of indirect immunofluorescence test (immunofluorescence assay, w) and enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, *----*I titer levels reactive on L. pneumophila sg 3 in the patient’s serum before, during, and after erythromytin treatment.
methods, Dieterle staining, and immunofluorescence did not reveal any pathologic findings in the disease-free margins of the resected colon.
Methods The following serogroups of L. pneumophila were investigated: Philadelphia 1 (sg l), Togus 1 (sg 2), Bloomington 2 (sg 3), Los Angeles 1 (sg 4), Dallas 1-E (sg 5),
753
Chicago 2 (sg 6), Chicago 8 (sg 7), Concord 3 (sg 8), In-23-61-C2 (sg 9), Leiden 1 (sg lo), 797-PA-H (sg ll), and 570-Co-H (sg 12). In addition, the following strains of other Legionella species were tested: L. micdodei (Tatlock, ATCC 33204), both serogroups of L. bozemanii (Wiga and Toronto 3, ATCC 33265), L. dumoffi (NY 23, ATCC 33279), L. gormonii (LS 13, ATCC 33279), L. jordanis (BL-540, ATCC 33623), both serogroups of L. Iongbeachae (Long Beach 4, ATCC 33462 and Tucker 1, ATCC 33484) and L. feeleii (Wo-33CC3, ATCC 35072 and 691-Wi-H, ATCC 35849), L. hackeliae (Lansing 2, ATCC 35250), and L. wadsworthii (al/716 A, ATCC 33877). The immunofluorescence assay of the patient’s sera was performed by using formalin-inactivated suspensions of the antigens according to the technique of Wilkinson et al. (15). Enzyme-linked immunosorbent assay was conducted separately for each antigen on polyvinylchloride microtiter plates (Dynatech, Baton Rouge, La.), layered with ultrasonicates of the bacterial strains mentioned above. Peroxidase, conjugated with goat-antihuman immunoglobulin (Medac), was used for detection. The optical density was measured at 490 nm by using an enzyme-linked immunosorbent assay reader (Dynatech). Phosphatebuffered saline instead of patient’s serum was used as a negative control, whereas a patient’s serum with an immunofluorescence assay titer of 1:1024 was used as a positive control for the sg 1 of L. pneumophila. All other antigens were controlled with corresponding rabbit hyperimmune sera, detected with antirabbit immunoglobulin-peroxidase conjugates. According to the results determined from sera of blood donors and patients with respiratory infections
Figure 3. Microscopic section of colonic submucosa (modified Dieterle staining, x1000) demonstrating numerous short black rods, 1-3 Frn in length and up to 1 pm in width, of extracellular localization.
754
Figure
SCHMIDT ET AL.
4.
GASTROENTEROLOGYVol.
Microscopic section of colonic submucosa (direct immunofluorescence staining, x800). pneumophila sg 3 with fluorescein isothiocyanate-labeled monospecific antiserum.
caused by other microbial pathogens, an optical density of 0.5 was used as the cutoff value. Immunoblot was performed according to the technique of Ehret and Ruckdeschel (16,17). For tissue demonstration of L. pneumophila, direct fluorescent antibody staining was applied. For this purpose, rabbit antiserum was labeled with fluorescein isothiocyanate according to Cherry and McKinney (18) and was made monospecific for sg 3 by absorption. A negative control was performed by substituting labeled nonimmune rabbit serum for the anti-Legionella antibody. In the current literature the specificity of direct fluorescent antibody staining for legionellae is reported to be >99% (19).
Discussion Legionella pneumophila infections may present with protean clinical features including pulmonary, renal, neurologic, and gastrointestinal symptoms (20). Legionellae have been demonstrated in various extrapulmonary tissues, as in blood (1,2), thoracic (3-5,7) and extrathoracic lymph nodes (6,7), heart (8), kidneys (6,7), liver (5,6), spleen (5-8), and bone marrow (7). Although 50% of patients with Legionnaires’ disease (9,lO) and 15% of patients with Pontiac fever (21) complain of diarrhea, reports on gastrointestinal tract lesions due to direct bacterial infection are rare. In 1982, Fogliani et al. (11) described a case of necrotizing inflammation and several perforations of the cecum in Legionnaires’ disease. By direct immu-
97,
No. 3
Tissue demonstration (arrow) of L.
nofluorescence staining, L. pneumophila sg 1 was demonstrated in the diseased tissue. Dournon et al. (12) reported on a similar case of Legionnaires’ disease with inflammation of the terminal ileum, associated with hemorrhagic peritonitis. The authors identified L. pneumophila sg 1 in the peritoneal exudate, in the ileum wall, and in the intraluminal mucus of the small bowel. In 1986, Ozoux et al. (13) described a patient suffering from a Legionella pneumonia that was associated with hemorrhagic necrosis of the terminal ileum and enlargement of mesenteral lymph nodes related to a local infection with L. pneumophila sg 4. Legionella pneumophila sg 3, detected in our patient, only causes 2%-4% of Legionella pneumonias (22). It has been demonstrated in hepatic and splenic tissue (5). To our knowledge, this is the first observation of an L. pneumophila infection of the colon in preexisting ulcerative colitis. The clinical features and the endoscopic findings suggested an acute exacerbation of ulcerative colitis. At the patient’s admission, no clinical or radiologic signs of pulmonary involvement were present. Only the occurrence of pleural effusions, associated with persisting hyponatremia and further increase of serum liver enzymes,
prompted us to look for a Legionella infection. The histologic features of the resected colon were compatible with ulcerative colitis (23), but in addition, an intense
edema
of the submucosa
was found.
September
1989
The submucosal edema was limited to those colonic areas affected by inflammatory mucosal changes. In the normal part of the resected colon, neither inflammatory mucosal changes, nor submucosal edema, nor legionellae were present. The demonstration of L. pneumophila sg 3 in the colonic submucosa by highly specific immunologic methods, and the clinical course, characterized by resistance to corticosteroids and improvement under erythromycin therapy, suggest that this acute attack of a long-standing ulcerative colitis was related to a L. pneumophila infection. Whether Legionella infections of the colon could be implicated in some cases of severe and corticosteroid-resistant attacks of ulcerative colitis should undergo further investigation.
References 1.
2.
3.
4.
5.
6.
7. 8.
9.
10.
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Received June 1, 1988. Accepted February 13, 1989. Address requests for reprints to: Dr. Thomas Schmidt. II Medical Department, Krankenhaus Munchen-Bogenhausen, Englschalkingerstrasse 77, 8000 Munich 81, Federal Republic of Germany. The authors thank B. Birkner (endoscopist), W. Heitland (surgeon), and H. Ingrisch (radiologist) for their collaboration. They also thank J. Aronbayev for reviewing the manuscript.