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Fecal carriage of Streptococcus bovis and colorectal adenomas
GASTROENTEROLOGY
Fecal Carriage of Sfreptococcus Colorectal Adenomas
1991;101:721-725
bovis and
ROBERT DUBROW, STEPHEN EDBERG, ELIZABETH WIKFORS, DEBORAH CALLAN, FRANK TRONCALE, RONALD VENDER, MYRON BRAND, and ROCKFORD YAPP Departments of Epidemiology and Public Health, Laboratory University School of Medicine, New Haven, Connecticut
An association between fecal carriage of Streptococcus bovis and colorectal carcinoma has been reported. A relationship between S. bovis and colorectal adenomas has also been suggested. In the present study, the relationship between S. bovis and adenomas was investigated. Two of 18 current adenoma patients (11%) were found to have S. bovis in their stool compared with 12 of 84 controls (14%). Six of 38 patients who had ever had a diagnosis of adenoma (16%) had S. bovis compared with 8 of 64 controls (13%). These results rule out a strong association between fecal carriage of S. bovis and adenomas; however, because of the relatively small sample size, a weak to moderate association can not be excluded.
here have been frequent reports in the literature of an association between Streptococcus bovis endocarditis/‘bacteremia and colorectal carcinoma (1). In 1977, Klein et al. reported an association between fecal carriage of S. bovis and colorectal carcinoma (2). The reason for the reported association between fecal carriage of S. bovis and colorectal cancer is not known. S. bovis may play a role in the etiology of colorectal cancer. Alternatively, fecal carriage of S. bovis may be an epiphenomenon promoted in some way by the presence of a carcinoma in the large bowel. In either case, exploration of the relationship between fecal carriage of S. bovis and adenomas, the presumed precursor lesion of colorectal cancer (3), is in order. An association between colonic polyps and S. bovis endocarditis has been reported (4). With respect to fecal carriage of S. bovis, Burns et al. observed a significantly higher S. bovis fecal carriage rate among a combined group of patients with tubulovillous adenomas, villous adenomas, and colorectal carcinomas when compared with controls (5).However, the
T
Medicine,
and Medicine,
Yale
S. bovis carriage rate among adenoma patients only (tubular, tubulovillous, and villous) was not statistically different from the carriage rate among controls. There was a discrepancy in the magnitude of the carriage rate between the studies of Klein et al. and Burns et al. Fifty-six percent of the patients with colorectal cancer vs. 10% of the controls in the study by Klein et al. had S. bovis in their stool compared with 11.1% of the patients with colorectal cancer and 2.3% of the controls in the study by Burns et al. This discrepancy is most likely explained by methodological differences between the two studies. Klein et al. collected stool from rectal swabs. All patients in the study of Burns et al. had a colonoscopic examination; stool was collected from the tip of the colonoscope on completion of the examination. Possibly the bowel preparation for colonoscopy was responsible for the lower fecal carriage rate in the study by Burns et al. Differences in the specimen-processing scheme between the two studies may also have contributed. Because of the uncertain relationship between S. bovis and adenomas, the present study examined the fecal carriage rate of S. bovis in patients with adenomas using stool samples collected from rectal swabs. Methods Study Subjects This study was approved by the Yale University School of Medicine Human Investigation Committee. One hundred sixty-six patients were enrolled in the study. Ninety-six percent were outpatients seen at a large gastroenterology practice; the remainder were outpatients seen at a gastroenterology clinic. Medical records of the
o 1991 by the American Gastroenterological 0016-5085/91/$3.00
Association
722
DUBROW ET AL.
study subjects were reviewed after diagnostic workups were completed. Study subjects were classified into the following diagnostic groups according to their current diagnosis: adenomatous polyp, undetermined polyp, colorectal cancer, inflammatory bowel disease, controls with a colon examination (colonoscopy, sigmoidoscopy, and/or barium enema), and controls without a colon examination. The controls with a colon examination served as the primary control group. Of the 84 controls with a colon exam, 36 were examined by colonoscopy, 47 by flexible sigmoidoscopy, and 1 by air contrast barium enema. Boardcertified gastroenterologists with appointments at Yale University School of Medicine performed or supervised all endoscopic examinations. Thirty-three of the 36 colonoscopic examinations were complete examinations with good visualization of the entire colon; 1 was complete with a suboptimal prep; 1 was complete with suboptimal visualization of the splenic and hepatic flexures; and 1 did not get past the hepatic flexure. Of the 47 controls with a colon examination who were examined by flexible sigmoidoscopy, the sigmoidoscope reached 60 cm in 32; 55 cm in 2; 50 cm in 1;45 cm in 1; 40 cm in 10; and 30 cm in 1. Three of the sigmoidoscoped controls were also examined by barium enema (2 air contrast and 1 single contrast). The findings on the a4 controls with a colon examination were the following: normal or minor abnormalities (diverticulosis, internal hemorrhoids), 69 controls; hyperplastic polyps, 12 controls; prominent arteriovenous malformation at base of cecum, 1 control; radiation proctitis, 1 control: and changes suggestive of ischemic colitis, 1 control. Because every subject in this group did not undergo a complete colon examination, this group may contain some patients with adenomas. The controls without a colon examination served as a secondary control group. They included patients with diagnoses of normal (7 patients), irritable bowel syndrome (17), liver disease (4), hiatal hernia (3), peptic ulcer disease (3), symptoms secondary to medications (3), malabsorption disorders (21, gallbladder disease (2), internal hemorrhoids (2), and miscellaneous (8). Because this control group did not undergo colon examinations, it likely contains persons with adenomas. Patients who had antibiotic treatment < 1 month before specimen collection were excluded from the study, as were patients who had a barium enema, colonoscopy, or sigmoidoscopy examination < 1 week before specimen collection. A patient with metastatic gastric carcinoma was also excluded. All current adenomatous polyps were excised by endoscopy and pathologically confirmed. A patient with an adenoma who was also diagnosed with a rectal carcinoid tumor and a patient with an adenoma and a history of colorectal cancer were included in the current adenoma group. Study subjects were also classified according to whether they ever had a diagnosis of adenomatous polyp, undetermined polyp, colorectal cancer, or inflammatory bowel disease, based on review of their medical records. Subjects with both colorectal cancer and adenomas were classified.in the “ever-cancer” group.
GASTROENTEROLOGY
Vol. 101, No. 3
Fecal Specimens For subjects undergoing colon examination, fecal specimens were obtained before bowel preparation or no less than 1 week after the examination. Specimens were obtained from patients with polyps and patients with cancer before removal of polyps or cancer from the colon. Specimens were collected on sterile rayon-tipped swabs and immediately placed in modified Stuart’s bacterial transport medium (Marion Scientific Culturette Collection and Transport System; Marion Scientific, Kansas City, MO). Specimens were stored at 4°C until processed. Under these storage conditions, S. bovis remained viable for at least 1 week. Seventy-five percent of the specimens were processed within 2 days and the remainder within 4 days.
Iden tifca tion of S. bovis Presumptive colonies of S. bovis were isolated by plating onto a semiselective agar medium containing raffinose, ammonium sulfate, arginine, yeast extract, sodium glycerophosphate, sodium and potassium chloride, phosphate salts, magnesium sulfate, sodium deoxycholate, azide, amphotericin B, metronidazole, and bromcresol purple. S. bovis uses raffinose as its carbon source and usually forms yellow colonies (bromcresol purple turns yellow in the presence of acid) on this medium. Some enterococci also grow on raffinose. These organisms metabolize arginine as well, producing base that neutralizes the acid, generally resulting in the formation of clear colonies. The rectal swab was vortexed in a tube with sterile distilled water. Several dilutions were prepared, and diluents were passed through an exclusion filter. The filter was placed on the surface of the selective medium, which was incubated at 35°C under anaerobic conditions. A parallel internal control was run, in which a known amount of S. bovis was added to an aliquot of the first diluent. After incubation for 48-96 hours, representative yellow colonies, if present, were selected for definitive identification. In approximately 20% of the specimens, no yellow colonies were observed in the internal control. When this occurred, a sample of clear colonies was picked for definitive identification. The tests used for definitive identification were colony morphology, gram stain, catalase test, bile-esculin reaction, growth in 6.5% NaCl, starch hydrolysis, Group D Streptex (Wellcome Diagnostics, Dartford, England), and minitek system screen (esculin, lactose, sucrose, arabinose, mannitol, raffinose, arginine, dextrose, inulin, and sorbitol; Minitek Differentiation System, BBL Microbiology Systems, Becton, Dickinson and Co., Cockeyesville, MD) (6,7). The isolates were identified based on the results of all of the tests performed. About half of the specimens with yellow colonies proved to contain S. bovis. Identification of S. bovis was performed blindly with respect to the clinical diagnosis of the patient. Statistical Methods The statistical significance of the differences among proportions was measured with the x2 test. For 2 x 2 tables,
September
STREPTOCOCCUS
1991
exact 95% confidence limits for odds ratios were calculated
as described by Breslow and Day (8). Odds ratios adjusted for age and sex were calculated using the Mantel-Haenszel method (9). Results The fecal carriage rate of S. bovis is presented by current diagnosis in Table 1. The fecal carriage rate in the two control groups was similar (14% and 16%); the carriage rate among adenoma patients was slightly lower (11%). There was no statistically significant difference in fecal carriage rate among the diagnostic groups (x’ = 1.94, P = 0.86). The odds ratio for S. bovis carriage between the adenoma group and the controls with a colon exam was 0.75 (exact 95% confidence limits = 0.07 and 3.92). The odds ratio for S. bovis carriage between the adenoma group and the combined two control groups was 0.72 (exact 95% confidence limits = 0.07 and 3.46). The S. bovis carriage rate by type of tidenoma was tubvlar (O/11), tubulovillous (2/6), and villous (O/l). Table 2 presents the fecal carriage rate of S. bovis by “ever-diagnosis” diagnostic group. The carriage rate in the ever-adenoma group (16%) was slightly higher than the carriage rate in the control groups (13% and 15%). However, no statistically significant difference in the fecal carriage rate of S. bovis among the diagnostic groups was observed (x’ = 2.64, P = 0.76). The odds ratio for S. bovis carriage between the “ever-adenoma” group and the controls with a colon exam was 1.31 (exact 95% confidence limits = 0.34 and 4.75). The odds ratio for S. bovis carriage between the ever-adenoma group and the combined control groups was 1.22 (exact 95% confidence limits = 0.35 and 3.74). The ever-adenoma subjects were classified according to whether they ever had a diagnosis of a tubulovillous or villous adenoma. The S. bovis carriage rate among ever-tubulovillous adenoma subjects was 5/17 (29%); the rate was 0/2 among ever-villous adenoma subjects and l/19 (5%) among adenoma subjects with
Table 1. Fecal Carriage of S. bovis by Diagnostic Group (Current Diagnosis)
Study Controls
group
with colon exam”
Controls without colon exam Adenoma Undetermined polyp Inflammatory bowel disease Colon cancer
No.
% Male
Mean age No. with S. bovis (%) WI 12 (14)
84
51
60 (17)
51
55
58 (19)
8
(161
18
78
66 (10)
2
4 7 2
25 86 50
58 (51 53 (18) 57 (12)
2 (291
“Of the controls, 4 of 33 (12%) with examination had S. bovis in their stools.
a complete
(111 1 (251
0 (0)
colonoscopic
BOWS AND COLORECTAL
ADENOMAS
723
Table 2. Fecal Carriage of S. bovis by Diagnostic Group (Ever-Diagnosis) Mean age Study
group
Controls with colon exam Controls without colon exam Adenoma
Undetermined Inflammatory
No.
% Male
(SE1
No. with S. bovis (%)
64
50
57 (18)
8 (13)
41
56
55 (19)
6
38
71
68 (10)
6
6
17
64 (11)
2 (331
8 9
75 44
53 (17)
2
69 (11)
l(11)
polyp bowel
disease
Colon cancer
(151 (161 (251
tubular adenomas only. The odds ratio for S. bovis carriage between the ever-tubulovillous adenoma group and the controls with a colon exam was 2.92 (exact 95% confidence limits = 0.63 and 12.20). The odds ratio for S. bovis carriage between the evertubulovillous adenoma group and the combined control group was 2.71 (exact 95% confidence limits = 0.64 and 9.88). Table 3 presents the fecal carriage rate of S. bovis by sex and age. Women had a higher S. bovis carriage rate (21%) than men (11%) (x’ = 3.07, P = 0.08). Subjects age 2 65 years of age had a higher carriage rate (21%) than subjects <65 years of age (10%) (x’ = 3.94, P = 0.05). Classifying subjects by sex and age showed that both differences were accounted for by a higher rate of fecal carriage among women 2 65 years of age (35%) than the other sex-age groups (lo%-11%) (x’ = 12.5, P = 0.006). Comparing the current-adenoma group with the controls with a colon exam, the odds ratio of S. bovis carriage, adjusted for sex and age, was 0.77. The adjusted odds ratio comparing the ever-adenoma group with the controls with a colon exam was 1.42. Neither of these odds ratios differed substantially from the unadjusted odds ratios, indicating the absence of confounding by sex and age. Fifty-eight of the study subjects were known to be taking medieations for gastrointestinal problems (e.g., laxatives, bulking agents, antispasmodics, stool softeners, antacids, and histamine H, receptor antagonists). To test if fecal carriage of S. bovis is altered by these medications, the rate of fecal carriage of S. bovis was compared between subjects taking gastrointestinal medications and subjects not taking these medications. The fecal carriage rate was 16% among subjects
Table 3. Fecal Carriage of S. bovis b_ySex and Age Sex
Age
Male (%)
Female
(%)
Total (%)
<65
5/48 (10)
265
5/45
(11)
11/31
(35)
16/76
(21)
10/93
(11)
15/73
(21)
25/166
(15)
Total
4/42 (10)
s/90 (10)
724
DUBROW
ET AL.
taking the medications taking the medications, medications.
GASTROENTEROLOGY
and 15% among subjects not indicating no effect of these
Discussion
The main finding of the present study was an overall lack of association between fecal carriage of S. bovis and adenomas. No such association was observed when subjects were classified according to the presence of adenomas in their large bowel at the time of fecal collection or according to whether they have ever been diagnosed with adenomas. However, because of the small size of the study, the 95% confidence limits for the odds ratios for S. bovis carriage between current-adenoma cases and controls with a colon exam [the primary control group) (0.07 and 3.92) and between ever-adenoma cases and controls with a colon examination (0.34 and 4.75), do not rule out the possibility of a small to moderate association between S. bovis carriage and adenomas. Villous adenomas are at higher risk for malignant conversion than tubular adenomas, tubulovillous adenomas being at intermediate risk (3). Burns et al. observed a nonstatistically significant trend of increasing S. bovis carriage rate with increasing malignant potential (5). The present study was too small to make statistically meaningful conclusions about the subcategories of adenomas. However, it was interesting that the rates for both current tubulovillous adenomas (2/6) and evertubulovillous adenomas (5/17) showed nonstatistitally significant elevations. Because of the very small number of current cases of villous adenomas (1) and current cases of colorectal cancers (2), it was not possible to comment on the trend of S. bovis carriage with increasing malignant potential of current neoplasms. The pattern of S. bovis carriage rate with increasing malignant potential in the “ever-neoplasms” classification was tubular adenomas (l/19, 5%), tubulovillous adenomas (5/17, 29%), villous adenomas (O/2, O%), and carcinomas (l/9, 11%) compared with the controls with a colon exam (8/64, 13%). Again, because of small numbers in the villous adenoma and carcinoma categories, it was not possible to draw conclusions about the trend of S. bovis carriage with increasing malignant potential in the ever-neoplasms classification. The lack of association between S. bovis carriage and “ever-carcinoma” subjects does not contradict the Klein et al. study, which reported an elevated rate of S. bovis carriage among current colorectal cancer cases, but not among persons with a history of colorectal cancer. Most adenomas do not progress to carcinomas (3).
Vol. 101, No. 3
The overall nonassociation between fecal carriage of S. bovis and adenomas observed in this study suggests that if S. bovis plays a role in the etiology of colorectal cancer, it is in the progression of adenomas to carcinomas and not in the benign growth and development of adenomas. On the other hand, if the association between fecal carriage of S. bovis and colorectal cancer is an epiphenomenon, the nonassociation with adenomas suggests that although malignancy in the colon may promote the growth of S. bovis, the presence of benign adenomas does not provide an abnormally favorable milieu for S. bovis growth. Two final points should be made. First, the fecal carriage rate of S. bovis among controls in the present study (15%) was comparable with a number of other investigations (5%-16%) (2,10-12) but was 6.4 times higher than that in the study of Burns et al. (2.3%). This was most likely caused by differences in specimen collection or processing. Second, the elevated rate of fecal carriage of S. bovis among older women observed in this study has not been reported previously. Only one other study examined the relationship between age and fecal carriage of S. bovis, reporting no relationship (2); no other studies have reported on the relationship with sex. If the results of the present study are confirmed, age and sex will need to be taken into account as possible confounding variables in future S. bovis studies. In the present study, age and sex did not prove to be confounders. References 1. Noti C, Frossard E, Klaiber C. Endokarditis oder Bakteriamie durch Streptococcus botis und kolorektale Neoplasien. Schweiz Med Wochenschr 1988;118:991-1000. 2. Klein RS, Recco RA, Catalan0 MT, Edberg SC, Casey JI, Steigbigel NH. Association of Streptococcus bovis with carcinoma of the colon. N Engl J Med 1977;297:800-802. 3. Muto T, Bussey HJR, Morson BC. The evolution of cancer of the colon and rectum. Cancer 1975;36:2251-2270. 4. Leport C, Bure A, Leport J. Vilde JL. Incidence of colonic lesions in Streptococcus bovis and enterococcal endocarditis. Lancet 1987;1:748. R, Lauter CB. The association of 5. Burns CA, McCaughey Streptococcus bovis fecal carriage and colon neoplasia: possible relationship with polyps and their premalignant potential. Am J Gastroenterol 1985;80:42-46. 6. Facklam RR. Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl Microbial 1972;23:1131-1139. 7. Gross KC, Houghton MP, Senterfit LB. Presumptive speciation of Streptococcus bovis and other group D streptococci from human sources by using arginine and pyruvate tests. J Clin Microbial 1975;1:54-60. methods in cancer research. 8. Breslow NE, Day NE. Statistical Volume 1. The analysis of case-control studies. Lyon, France: International Agency for Research on Cancer, 1980. 9. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Nat1 Cancer Inst 1959;22:719-748.
September
1991
10. Sabbaj J, Sutter VL, Finegold SM. Comparison of selective media for isolation of presumptive group D streptococci from human feces. Appl Microbial 1971;22:1008-1011. 11. Noble CJ. Carriage of group D streptococci in the human bowel. JClinPathol1978;31:1182-1186. 12. Klein RS, Warman SW, Knackmuhs GG, Edberg SC, Steigbigel NH. Lack of association of Streptococcus bovis with noncolonic gastrointestinal carcinoma. Am J Gastroenterol 1987;82:540543.
Received Address Department
June 21,199O. Accepted January 29,199l. requests for reprints to: Robert Dubrow, M.D., Ph.D., of Epidemiology and Public Health, Yale University
S7’REpTOCOCCUS
BOVIS AND COLORECTAL
ADENOMAS
725
School of Medicine, P.O. Box 3333, New Haven, Connecticut 06510. This work was supported by the National Cancer Institute (POl-CA42201, Cancer Prevention Research Unit for Connecticut at Yale University). Robert Dubrow received support from the New Haven Foundation’s Hull Cancer Research Award and from a National Cancer Institute Preventive Oncology Academic Award (K07-CA01463). The authors thank Drs. Stephen Stein, Rosemarie Fisher, John Dobbins, Raymond
Suzanne Ippolito,
Lagarde, Howard Spiro, Christopher Baker, Sherwin Nuland, and Henry Binder for help in
recruiting assistance: ment.
patients into the study: Leonard Yannielli for technical and Dr. Dwight Janerich for his support and encourage-
Fecal Carriage of Sfreptococcus Colorectal Adenomas
1991;101:721-725
bovis and
ROBERT DUBROW, STEPHEN EDBERG, ELIZABETH WIKFORS, DEBORAH CALLAN, FRANK TRONCALE, RONALD VENDER, MYRON BRAND, and ROCKFORD YAPP Departments of Epidemiology and Public Health, Laboratory University School of Medicine, New Haven, Connecticut
An association between fecal carriage of Streptococcus bovis and colorectal carcinoma has been reported. A relationship between S. bovis and colorectal adenomas has also been suggested. In the present study, the relationship between S. bovis and adenomas was investigated. Two of 18 current adenoma patients (11%) were found to have S. bovis in their stool compared with 12 of 84 controls (14%). Six of 38 patients who had ever had a diagnosis of adenoma (16%) had S. bovis compared with 8 of 64 controls (13%). These results rule out a strong association between fecal carriage of S. bovis and adenomas; however, because of the relatively small sample size, a weak to moderate association can not be excluded.
here have been frequent reports in the literature of an association between Streptococcus bovis endocarditis/‘bacteremia and colorectal carcinoma (1). In 1977, Klein et al. reported an association between fecal carriage of S. bovis and colorectal carcinoma (2). The reason for the reported association between fecal carriage of S. bovis and colorectal cancer is not known. S. bovis may play a role in the etiology of colorectal cancer. Alternatively, fecal carriage of S. bovis may be an epiphenomenon promoted in some way by the presence of a carcinoma in the large bowel. In either case, exploration of the relationship between fecal carriage of S. bovis and adenomas, the presumed precursor lesion of colorectal cancer (3), is in order. An association between colonic polyps and S. bovis endocarditis has been reported (4). With respect to fecal carriage of S. bovis, Burns et al. observed a significantly higher S. bovis fecal carriage rate among a combined group of patients with tubulovillous adenomas, villous adenomas, and colorectal carcinomas when compared with controls (5).However, the
T
Medicine,
and Medicine,
Yale
S. bovis carriage rate among adenoma patients only (tubular, tubulovillous, and villous) was not statistically different from the carriage rate among controls. There was a discrepancy in the magnitude of the carriage rate between the studies of Klein et al. and Burns et al. Fifty-six percent of the patients with colorectal cancer vs. 10% of the controls in the study by Klein et al. had S. bovis in their stool compared with 11.1% of the patients with colorectal cancer and 2.3% of the controls in the study by Burns et al. This discrepancy is most likely explained by methodological differences between the two studies. Klein et al. collected stool from rectal swabs. All patients in the study of Burns et al. had a colonoscopic examination; stool was collected from the tip of the colonoscope on completion of the examination. Possibly the bowel preparation for colonoscopy was responsible for the lower fecal carriage rate in the study by Burns et al. Differences in the specimen-processing scheme between the two studies may also have contributed. Because of the uncertain relationship between S. bovis and adenomas, the present study examined the fecal carriage rate of S. bovis in patients with adenomas using stool samples collected from rectal swabs. Methods Study Subjects This study was approved by the Yale University School of Medicine Human Investigation Committee. One hundred sixty-six patients were enrolled in the study. Ninety-six percent were outpatients seen at a large gastroenterology practice; the remainder were outpatients seen at a gastroenterology clinic. Medical records of the
o 1991 by the American Gastroenterological 0016-5085/91/$3.00
Association
722
DUBROW ET AL.
study subjects were reviewed after diagnostic workups were completed. Study subjects were classified into the following diagnostic groups according to their current diagnosis: adenomatous polyp, undetermined polyp, colorectal cancer, inflammatory bowel disease, controls with a colon examination (colonoscopy, sigmoidoscopy, and/or barium enema), and controls without a colon examination. The controls with a colon examination served as the primary control group. Of the 84 controls with a colon exam, 36 were examined by colonoscopy, 47 by flexible sigmoidoscopy, and 1 by air contrast barium enema. Boardcertified gastroenterologists with appointments at Yale University School of Medicine performed or supervised all endoscopic examinations. Thirty-three of the 36 colonoscopic examinations were complete examinations with good visualization of the entire colon; 1 was complete with a suboptimal prep; 1 was complete with suboptimal visualization of the splenic and hepatic flexures; and 1 did not get past the hepatic flexure. Of the 47 controls with a colon examination who were examined by flexible sigmoidoscopy, the sigmoidoscope reached 60 cm in 32; 55 cm in 2; 50 cm in 1;45 cm in 1; 40 cm in 10; and 30 cm in 1. Three of the sigmoidoscoped controls were also examined by barium enema (2 air contrast and 1 single contrast). The findings on the a4 controls with a colon examination were the following: normal or minor abnormalities (diverticulosis, internal hemorrhoids), 69 controls; hyperplastic polyps, 12 controls; prominent arteriovenous malformation at base of cecum, 1 control; radiation proctitis, 1 control: and changes suggestive of ischemic colitis, 1 control. Because every subject in this group did not undergo a complete colon examination, this group may contain some patients with adenomas. The controls without a colon examination served as a secondary control group. They included patients with diagnoses of normal (7 patients), irritable bowel syndrome (17), liver disease (4), hiatal hernia (3), peptic ulcer disease (3), symptoms secondary to medications (3), malabsorption disorders (21, gallbladder disease (2), internal hemorrhoids (2), and miscellaneous (8). Because this control group did not undergo colon examinations, it likely contains persons with adenomas. Patients who had antibiotic treatment < 1 month before specimen collection were excluded from the study, as were patients who had a barium enema, colonoscopy, or sigmoidoscopy examination < 1 week before specimen collection. A patient with metastatic gastric carcinoma was also excluded. All current adenomatous polyps were excised by endoscopy and pathologically confirmed. A patient with an adenoma who was also diagnosed with a rectal carcinoid tumor and a patient with an adenoma and a history of colorectal cancer were included in the current adenoma group. Study subjects were also classified according to whether they ever had a diagnosis of adenomatous polyp, undetermined polyp, colorectal cancer, or inflammatory bowel disease, based on review of their medical records. Subjects with both colorectal cancer and adenomas were classified.in the “ever-cancer” group.
GASTROENTEROLOGY
Vol. 101, No. 3
Fecal Specimens For subjects undergoing colon examination, fecal specimens were obtained before bowel preparation or no less than 1 week after the examination. Specimens were obtained from patients with polyps and patients with cancer before removal of polyps or cancer from the colon. Specimens were collected on sterile rayon-tipped swabs and immediately placed in modified Stuart’s bacterial transport medium (Marion Scientific Culturette Collection and Transport System; Marion Scientific, Kansas City, MO). Specimens were stored at 4°C until processed. Under these storage conditions, S. bovis remained viable for at least 1 week. Seventy-five percent of the specimens were processed within 2 days and the remainder within 4 days.
Iden tifca tion of S. bovis Presumptive colonies of S. bovis were isolated by plating onto a semiselective agar medium containing raffinose, ammonium sulfate, arginine, yeast extract, sodium glycerophosphate, sodium and potassium chloride, phosphate salts, magnesium sulfate, sodium deoxycholate, azide, amphotericin B, metronidazole, and bromcresol purple. S. bovis uses raffinose as its carbon source and usually forms yellow colonies (bromcresol purple turns yellow in the presence of acid) on this medium. Some enterococci also grow on raffinose. These organisms metabolize arginine as well, producing base that neutralizes the acid, generally resulting in the formation of clear colonies. The rectal swab was vortexed in a tube with sterile distilled water. Several dilutions were prepared, and diluents were passed through an exclusion filter. The filter was placed on the surface of the selective medium, which was incubated at 35°C under anaerobic conditions. A parallel internal control was run, in which a known amount of S. bovis was added to an aliquot of the first diluent. After incubation for 48-96 hours, representative yellow colonies, if present, were selected for definitive identification. In approximately 20% of the specimens, no yellow colonies were observed in the internal control. When this occurred, a sample of clear colonies was picked for definitive identification. The tests used for definitive identification were colony morphology, gram stain, catalase test, bile-esculin reaction, growth in 6.5% NaCl, starch hydrolysis, Group D Streptex (Wellcome Diagnostics, Dartford, England), and minitek system screen (esculin, lactose, sucrose, arabinose, mannitol, raffinose, arginine, dextrose, inulin, and sorbitol; Minitek Differentiation System, BBL Microbiology Systems, Becton, Dickinson and Co., Cockeyesville, MD) (6,7). The isolates were identified based on the results of all of the tests performed. About half of the specimens with yellow colonies proved to contain S. bovis. Identification of S. bovis was performed blindly with respect to the clinical diagnosis of the patient. Statistical Methods The statistical significance of the differences among proportions was measured with the x2 test. For 2 x 2 tables,
September
STREPTOCOCCUS
1991
exact 95% confidence limits for odds ratios were calculated
as described by Breslow and Day (8). Odds ratios adjusted for age and sex were calculated using the Mantel-Haenszel method (9). Results The fecal carriage rate of S. bovis is presented by current diagnosis in Table 1. The fecal carriage rate in the two control groups was similar (14% and 16%); the carriage rate among adenoma patients was slightly lower (11%). There was no statistically significant difference in fecal carriage rate among the diagnostic groups (x’ = 1.94, P = 0.86). The odds ratio for S. bovis carriage between the adenoma group and the controls with a colon exam was 0.75 (exact 95% confidence limits = 0.07 and 3.92). The odds ratio for S. bovis carriage between the adenoma group and the combined two control groups was 0.72 (exact 95% confidence limits = 0.07 and 3.46). The S. bovis carriage rate by type of tidenoma was tubvlar (O/11), tubulovillous (2/6), and villous (O/l). Table 2 presents the fecal carriage rate of S. bovis by “ever-diagnosis” diagnostic group. The carriage rate in the ever-adenoma group (16%) was slightly higher than the carriage rate in the control groups (13% and 15%). However, no statistically significant difference in the fecal carriage rate of S. bovis among the diagnostic groups was observed (x’ = 2.64, P = 0.76). The odds ratio for S. bovis carriage between the “ever-adenoma” group and the controls with a colon exam was 1.31 (exact 95% confidence limits = 0.34 and 4.75). The odds ratio for S. bovis carriage between the ever-adenoma group and the combined control groups was 1.22 (exact 95% confidence limits = 0.35 and 3.74). The ever-adenoma subjects were classified according to whether they ever had a diagnosis of a tubulovillous or villous adenoma. The S. bovis carriage rate among ever-tubulovillous adenoma subjects was 5/17 (29%); the rate was 0/2 among ever-villous adenoma subjects and l/19 (5%) among adenoma subjects with
Table 1. Fecal Carriage of S. bovis by Diagnostic Group (Current Diagnosis)
Study Controls
group
with colon exam”
Controls without colon exam Adenoma Undetermined polyp Inflammatory bowel disease Colon cancer
No.
% Male
Mean age No. with S. bovis (%) WI 12 (14)
84
51
60 (17)
51
55
58 (19)
8
(161
18
78
66 (10)
2
4 7 2
25 86 50
58 (51 53 (18) 57 (12)
2 (291
“Of the controls, 4 of 33 (12%) with examination had S. bovis in their stools.
a complete
(111 1 (251
0 (0)
colonoscopic
BOWS AND COLORECTAL
ADENOMAS
723
Table 2. Fecal Carriage of S. bovis by Diagnostic Group (Ever-Diagnosis) Mean age Study
group
Controls with colon exam Controls without colon exam Adenoma
Undetermined Inflammatory
No.
% Male
(SE1
No. with S. bovis (%)
64
50
57 (18)
8 (13)
41
56
55 (19)
6
38
71
68 (10)
6
6
17
64 (11)
2 (331
8 9
75 44
53 (17)
2
69 (11)
l(11)
polyp bowel
disease
Colon cancer
(151 (161 (251
tubular adenomas only. The odds ratio for S. bovis carriage between the ever-tubulovillous adenoma group and the controls with a colon exam was 2.92 (exact 95% confidence limits = 0.63 and 12.20). The odds ratio for S. bovis carriage between the evertubulovillous adenoma group and the combined control group was 2.71 (exact 95% confidence limits = 0.64 and 9.88). Table 3 presents the fecal carriage rate of S. bovis by sex and age. Women had a higher S. bovis carriage rate (21%) than men (11%) (x’ = 3.07, P = 0.08). Subjects age 2 65 years of age had a higher carriage rate (21%) than subjects <65 years of age (10%) (x’ = 3.94, P = 0.05). Classifying subjects by sex and age showed that both differences were accounted for by a higher rate of fecal carriage among women 2 65 years of age (35%) than the other sex-age groups (lo%-11%) (x’ = 12.5, P = 0.006). Comparing the current-adenoma group with the controls with a colon exam, the odds ratio of S. bovis carriage, adjusted for sex and age, was 0.77. The adjusted odds ratio comparing the ever-adenoma group with the controls with a colon exam was 1.42. Neither of these odds ratios differed substantially from the unadjusted odds ratios, indicating the absence of confounding by sex and age. Fifty-eight of the study subjects were known to be taking medieations for gastrointestinal problems (e.g., laxatives, bulking agents, antispasmodics, stool softeners, antacids, and histamine H, receptor antagonists). To test if fecal carriage of S. bovis is altered by these medications, the rate of fecal carriage of S. bovis was compared between subjects taking gastrointestinal medications and subjects not taking these medications. The fecal carriage rate was 16% among subjects
Table 3. Fecal Carriage of S. bovis b_ySex and Age Sex
Age
Male (%)
Female
(%)
Total (%)
<65
5/48 (10)
265
5/45
(11)
11/31
(35)
16/76
(21)
10/93
(11)
15/73
(21)
25/166
(15)
Total
4/42 (10)
s/90 (10)
724
DUBROW
ET AL.
taking the medications taking the medications, medications.
GASTROENTEROLOGY
and 15% among subjects not indicating no effect of these
Discussion
The main finding of the present study was an overall lack of association between fecal carriage of S. bovis and adenomas. No such association was observed when subjects were classified according to the presence of adenomas in their large bowel at the time of fecal collection or according to whether they have ever been diagnosed with adenomas. However, because of the small size of the study, the 95% confidence limits for the odds ratios for S. bovis carriage between current-adenoma cases and controls with a colon exam [the primary control group) (0.07 and 3.92) and between ever-adenoma cases and controls with a colon examination (0.34 and 4.75), do not rule out the possibility of a small to moderate association between S. bovis carriage and adenomas. Villous adenomas are at higher risk for malignant conversion than tubular adenomas, tubulovillous adenomas being at intermediate risk (3). Burns et al. observed a nonstatistically significant trend of increasing S. bovis carriage rate with increasing malignant potential (5). The present study was too small to make statistically meaningful conclusions about the subcategories of adenomas. However, it was interesting that the rates for both current tubulovillous adenomas (2/6) and evertubulovillous adenomas (5/17) showed nonstatistitally significant elevations. Because of the very small number of current cases of villous adenomas (1) and current cases of colorectal cancers (2), it was not possible to comment on the trend of S. bovis carriage with increasing malignant potential of current neoplasms. The pattern of S. bovis carriage rate with increasing malignant potential in the “ever-neoplasms” classification was tubular adenomas (l/19, 5%), tubulovillous adenomas (5/17, 29%), villous adenomas (O/2, O%), and carcinomas (l/9, 11%) compared with the controls with a colon exam (8/64, 13%). Again, because of small numbers in the villous adenoma and carcinoma categories, it was not possible to draw conclusions about the trend of S. bovis carriage with increasing malignant potential in the ever-neoplasms classification. The lack of association between S. bovis carriage and “ever-carcinoma” subjects does not contradict the Klein et al. study, which reported an elevated rate of S. bovis carriage among current colorectal cancer cases, but not among persons with a history of colorectal cancer. Most adenomas do not progress to carcinomas (3).
Vol. 101, No. 3
The overall nonassociation between fecal carriage of S. bovis and adenomas observed in this study suggests that if S. bovis plays a role in the etiology of colorectal cancer, it is in the progression of adenomas to carcinomas and not in the benign growth and development of adenomas. On the other hand, if the association between fecal carriage of S. bovis and colorectal cancer is an epiphenomenon, the nonassociation with adenomas suggests that although malignancy in the colon may promote the growth of S. bovis, the presence of benign adenomas does not provide an abnormally favorable milieu for S. bovis growth. Two final points should be made. First, the fecal carriage rate of S. bovis among controls in the present study (15%) was comparable with a number of other investigations (5%-16%) (2,10-12) but was 6.4 times higher than that in the study of Burns et al. (2.3%). This was most likely caused by differences in specimen collection or processing. Second, the elevated rate of fecal carriage of S. bovis among older women observed in this study has not been reported previously. Only one other study examined the relationship between age and fecal carriage of S. bovis, reporting no relationship (2); no other studies have reported on the relationship with sex. If the results of the present study are confirmed, age and sex will need to be taken into account as possible confounding variables in future S. bovis studies. In the present study, age and sex did not prove to be confounders. References 1. Noti C, Frossard E, Klaiber C. Endokarditis oder Bakteriamie durch Streptococcus botis und kolorektale Neoplasien. Schweiz Med Wochenschr 1988;118:991-1000. 2. Klein RS, Recco RA, Catalan0 MT, Edberg SC, Casey JI, Steigbigel NH. Association of Streptococcus bovis with carcinoma of the colon. N Engl J Med 1977;297:800-802. 3. Muto T, Bussey HJR, Morson BC. The evolution of cancer of the colon and rectum. Cancer 1975;36:2251-2270. 4. Leport C, Bure A, Leport J. Vilde JL. Incidence of colonic lesions in Streptococcus bovis and enterococcal endocarditis. Lancet 1987;1:748. R, Lauter CB. The association of 5. Burns CA, McCaughey Streptococcus bovis fecal carriage and colon neoplasia: possible relationship with polyps and their premalignant potential. Am J Gastroenterol 1985;80:42-46. 6. Facklam RR. Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl Microbial 1972;23:1131-1139. 7. Gross KC, Houghton MP, Senterfit LB. Presumptive speciation of Streptococcus bovis and other group D streptococci from human sources by using arginine and pyruvate tests. J Clin Microbial 1975;1:54-60. methods in cancer research. 8. Breslow NE, Day NE. Statistical Volume 1. The analysis of case-control studies. Lyon, France: International Agency for Research on Cancer, 1980. 9. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Nat1 Cancer Inst 1959;22:719-748.
September
1991
10. Sabbaj J, Sutter VL, Finegold SM. Comparison of selective media for isolation of presumptive group D streptococci from human feces. Appl Microbial 1971;22:1008-1011. 11. Noble CJ. Carriage of group D streptococci in the human bowel. JClinPathol1978;31:1182-1186. 12. Klein RS, Warman SW, Knackmuhs GG, Edberg SC, Steigbigel NH. Lack of association of Streptococcus bovis with noncolonic gastrointestinal carcinoma. Am J Gastroenterol 1987;82:540543.
Received Address Department
June 21,199O. Accepted January 29,199l. requests for reprints to: Robert Dubrow, M.D., Ph.D., of Epidemiology and Public Health, Yale University
S7’REpTOCOCCUS
BOVIS AND COLORECTAL
ADENOMAS
725
School of Medicine, P.O. Box 3333, New Haven, Connecticut 06510. This work was supported by the National Cancer Institute (POl-CA42201, Cancer Prevention Research Unit for Connecticut at Yale University). Robert Dubrow received support from the New Haven Foundation’s Hull Cancer Research Award and from a National Cancer Institute Preventive Oncology Academic Award (K07-CA01463). The authors thank Drs. Stephen Stein, Rosemarie Fisher, John Dobbins, Raymond
Suzanne Ippolito,
Lagarde, Howard Spiro, Christopher Baker, Sherwin Nuland, and Henry Binder for help in
recruiting assistance: ment.
patients into the study: Leonard Yannielli for technical and Dr. Dwight Janerich for his support and encourage-