The winner after 7 rounds—Fecal occult blood testing!

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SELECTED SUMMARIES Henry J. Binder, M.D. Selected Summaries Editor Yale University School of Medicine New Haven, Connecticut

STAFF OF CONTRIBUTORS Laurence Blendis, Tel-Aviv, Israel Robert Bresalier, Detroit, MI William L. Hasler, Ann Arbor, MI Cyrus Kapadia, New Haven, CT Saul J. Karpen, Houston, TX Ronald L. Koretz, Sylmar, CA

Kris V. Kowdley, Seattle, WA James D. Lewis, Philadelphia, PA Gary R. Lichtenstein, Philadelphia, PA Douglas B. Nelson, Minneapolis, MN Richard M. Peek, Nashville, TN G. S. Raju, Galveston, TX

THE WINNER AFTER 7 ROUNDS—FECAL OCCULT BLOOD TESTING! Jorgensen OD, Kronborg O, Fenger C (Odense University Hospital, Odense, Denmark). A randomized study of screening for colorectal cancer using faecal occult blood testing: results after 13 years and seven biennial screening rounds. Gut 2002;50: 29 –32. In 1985, 61,933 persons between the ages of 45 and 75 years, living in Funen, Denmark, were enrolled in the study. Subjects were randomized either to a colorectal cancer (CRC) screening arm with biennial Hemoccult-II (H-2) testing (n ⫽ 30,967) or to a control group (n ⫽ 30,966). Control subjects were not informed about the study to ensure that their behavior remained representative of the general population. Individuals with colorectal cancer, adenomas, or any cancer with widespread metastases were excluded. Subjects in the screening arm were sent invitations for screening and reminders by mail. Those subjects who did not participate in the previous screening round or were found to have CRC or adenomas were excluded from subsequent screening rounds. H-2 was used without rehydration, but with standard dietary restrictions. Subjects with a positive test were offered a colonoscopy. A double contrast barium enema was offered when colonoscopy was incomplete or if the patient refused colonoscopy. Information on new cases of CRC and adenomas was obtained from the Funen County Database and the Danish National Registry. This was cross-referenced with the Danish Cancer Registry. Death certificates were obtained from county public health offices and central government agencies. Death was attributed to CRC if certain preset criteria were met. Death attributable to CRC was the primary end point of the study. Of the 30,967 individuals randomized to the screening arm, 20,672 (67%) completed the first screening round and 11,058 (36%) completed all 7 screening rounds. The cumulative risk of having a positive test over the course of the study was 5.1%. Of these individuals, 94.1% had at least 1 colonoscopy. The

Don C. Rockey, Durham, NC Fergus Shanahan, Cork, Ireland Nicholas J. Shaheen, Chapel Hill, NC Brad Warner, Cincinnati, OH David C. Whitcomb, Pittsburgh, PA Stephen D. Zucker, Cincinnati, OH

cumulative proportion of the whole screening group undergoing at least 1 colonoscopy was 4.8%. At the end of 7 biennial screening rounds and 13 years of follow-up, 649 subjects in the screened group were found to have CRC, compared with 637 in the control group (NS). There were 255 deaths attributed to CRC in the screened group compared with 310 in the control group (mortality ratio, 0.82; confidence interval [CI ], 0.69 – 0.97). The investigators concluded that this difference remained significant even when deaths from complications of treatment of CRC were included, and after adjusting for age and sex (mortality ratio, 0.86; CI, 0.73–1.00), although the confidence interval included 1. Although subgroup analysis revealed that risk of death from CRC was higher in men and individuals older than 65 years of age, the protective effects of screening were independent of sex and age. Subjects who participated in all 7 screening rounds showed a 30% reduction in risk of death for CRC when compared with controls Comment. The present study by Jorgensen et al., along with 2 other large randomized trials from Minnesota and Nottingham, England demonstrate the efficacy of fecal occult blood testing as a screening tool for CRC (N Engl J Med 1993;328:1365–1371, Lancet 1996;348:1472–1477). These 3 carefully done studies provide strong direct evidence that screening with fecal occult blood testing (FOBT) reduces mortality from CRC, with reductions in CRC mortality of 15% (England), 18% (Denmark), and 33% (Minnesota). There are subtle differences in the design and results of these trials that are worth highlighting. Both European trials were population-based and may be less subject to “volunteer bias” than the Minnesota study. However, because the Minnesota trial was a randomized controlled study, the reduction in mortality seen in the screening groups relative to the controls should not have been affected. The European studies performed biennial FOBT screening, whereas the greatest reduction in CRC mortality occurred in the annual FOBT screening arm of the Minnesota study. The 21% mortality reduction observed in the biennial screening arm is similar to the European studies ( J Natl Cancer Inst 1999;91:434 – 437). All 3 trials followed different protocols for testing and interpreting H-2 slides. The British study, in an attempt to minimize false positives, sent repeat FOBT slides to subjects with up to 4 of 6 positive windows on H-2 slides. A colonoscopy was only offered to subjects if they had 5 or more positive windows at the first test, or 1 or more positive windows after repeat testing. The Danish study offered colonoscopy to all patients with 1 or more positive windows on the H-2 slides. The Minnesota study

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rehydrated a majority of H-2 slides (82.5%) before they were interpreted. Colonoscopy was offered to all subjects with 1 or more positive windows. Rehydration has important ramifications for the specificity and sensitivity of the test. Rehydration seems to substantially increase sensitivity at the expense of lower specificity (i.e., more false positives). In the Minnesota trial, rehydration increased the rate of positive tests from 2.4% to 9.8%. Another factor that is often underappreciated is the importance of dietary and medication restrictions before FOBT (Am J Gastroenterol 2000;95:3629 –3632), which can have significant effects on test performance characteristics. Macrae studied 156 healthy young volunteers under age 40 (and thus presumably without colorectal pathology) on a diet restricted in red meats, fruits, and vegetables. None of these subjects were found to have a positive nonrehydrated H-2 test. Two of the 310 hemoccult tests developed a “false-positive” result when slides were rehydrated. The rate of positive tests jumped to 6.6% when subjects were tested on an unrestricted diet with rehydrated slides (Gastroenterology 1982;82:899). There are no reported data on compliance with dietary restrictions from any of the 3 major FOBT trials. The positive predictive value of the H-2 test for detecting CRC was 2.2% in the Minnesota trial, whereas it was 12% in the British trial. The cumulative proportion of screened subjects undergoing colonoscopy in the Minnesota trial was 38% in the annually screened arm and 28% in the biennial screened arm. This resulted in a 33% reduction in the risk of death from CRC. The cumulative proportions of screened subjects undergoing colonoscopy in the British and the Danish study were 4% and 4.8%, respectively. The risk reductions for CRC were correspondingly lower, with the British study reporting an 18% risk reduction and the Danish study a 15% risk reduction. Lang and Ransohoff, using a mathematical model, have argued that much of the risk reduction reported by the Minnesota group was simply the result of chance finding of CRCs because of the high rate of colonoscopy in the screened group (JAMA 1994;271:1011–1013). However, using the same mathematical model, the authors of the Minnesota trial estimated that only 16%–25% of the observed risk reduction noted in their trial was attainable by chance alone (J Natl Cancer Inst 1997;89:1423–1428). The question of rehydrating H-2 slides remains unsettled but is no doubt central to the interpretation of this and other similar trials. The current GI Consortium guidelines for CRC screening do not have a formal recommendation regarding rehydration of FOBT slides, but simply observe that this practice increases the sensitivity of the test while lowering the specificity (Gastroenterology 1997;112:594 – 642). An overall reduction in risk of death from CRC of 15% was reported in the present study by Jorgensen et al. This is probably an underestimate of the effect of screening and should be regarded as the “minimum risk reduction” that is achievable by this intervention. We know that 33% of subjects randomized to the screening group refused to accept the first round of screening and were therefore not invited to participate in further screening. Since analysis was done as intention to screen, this would tend to minimize the effect of screening. Furthermore, the authors did not attempt to collect data on screening for CRC that may have occurred in the control group, which would also tend to underestimate the effect of screening. Finally, patients who refused any round of screening were not reinvited to participate in subsequent screening rounds; only 11,058 subjects out of the original 30,762 randomized to the screening arm actually completed all 7 screening cycles. The most important new information that has emerged from the present study is the importance of patient compliance. The reduction in risk of death from CRC was 30% in subjects

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who were deemed fully compliant per the study protocol over all 7 screening cycles when compared with the control population. Subjects who refused any screening had a significantly increased risk of CRC death when compared with those who accepted the full screening program. A trend toward increased risk was also noted in subjects accepting the first screening, but refusing subsequent screening. Although intuitive, this is the first study to show that subjects can significantly improve the efficacy of screening by complying with screening guidelines. A 30% reduction in the risk of colorectal cancer death in patients following screening guidelines is a more understandable statistic and therefore more useful to quote to a patient while discussing CRC screening. Another factor that could influence the mortality benefit from CRC screening is the technical performance of colonoscopy once a positive screening test is found. The rate of complete colonoscopy reported in this trial, ranging from 81% to 89%, was lower than is generally reported in the United States. Comparable trials have shown that complete colonoscopy can be performed in more than 95% of subjects. The present study provides long-term follow-up on the effectiveness of biennial FOBT screening in reducing CRC mortality. Even greater benefit has been shown for FOBT performed annually, and this is the current recommendation (Gastroenterology 1997;112: 594 – 642). Although there is increasing interest in using colonoscopy as a primary screening modality, the best evidence for reduction in CRC mortality belongs to FOBT, and as such it remains a viable screening modality. MANDEEP S. SAWHNEY, M.D. DOUGLAS B. NELSON, M.D.

Reply. Because FOBTs could not be obtained outside of this trial during the first screening rounds, there was no possible bias for an underestimation of the screening effect. We accept that a complete colonoscopy should be performed in more than 95% of selected patients, but we are not aware of any published screening trial similar to our own, in which such a high rate of completeness has been achieved. No more than 1 CRC was detected among persons with a positive FOBT followed by an incomplete colonoscopy minimizing this possible underestimation of the effect of screening. We agree with the recommendation for annual screening, although our results were obtained in a biennial screening program. OLE KRONBORG, M.D., FRCS

FORECAST FOR USING METABOLITE MEASUREMENTS IN THE DOSING OF AZATHIOPRINE OR 6 –MERCAPTOPURINE FOR IBD PATIENTS: “PARTLY CLOUDY” Lowry PW, Franklin CL, Weaver AL, Pike MG, Mays DC, Tremaine WJ, Lipsky JJ, Sandborn WJ (Division of Gastroenterology and Hepatology, Division of Clinical Pharmacology, and Section of Biostatistics, Mayo Clinic, Rochester, Minnesota). Measurement of thiopurine methyltransferase activity and azathioprine metabolites in patients with inflammatory bowel disease. Gut 2001;49:665– 670. The emergence of the purine analogues 6-mercaptopurine (6-MP) and azathioprine (AZA) in the induction and mainte-

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nance of remission in patients with Crohn’s disease (CD) and ulcerative colitis (UC) has raised concerns over appropriate dosing. It is now possible to measure these drugs’ metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP), as well as the thiopurine methyltransferase (TPMT) enzyme responsible for their catabolism. The association of these methodologies to patient outcomes has been the topic of hot debate in the gastroenterology community and has spurred recent studies intending to further elucidate this relationship. The most recent foray into this debate has been the Mayo Clinic’s study of their inflammatory bowel disease (IBD) population currently receiving 6-MP or AZA (Gut 2001;49:665– 670). Of 376 eligible patients, 170 (80 men; mean age, 43 years; 130 CD; 40 UC) completed questionnaires regarding demographics, IBD drug use, and the 32-question Inflammatory Bowel Disease Questionnaire (IBDQ) categorizing quality of life. Blood samples assaying CBC, absolute neutrophil count (ANC), red blood cell (RBC) TMPT enzyme activity, whole blood 6-TGN and 6-MMP concentrations, and sulfasalazine, mesalamine, and N-acetyl-5-ASA levels, were collected 1 hour after their 6-MP or AZA dose (as well as concomitant aminosalicylate dose). Patient charts were abstracted for data on demographics, IBD type, location and diagnosis date, surgical history, pretreatment RBC TMPT enzyme activity, dates and doses for initial AZA or 6-MP dosing and subsequent doseadjustments (and reasons), lowest recorded white blood cell (WBC) count at each dose, and concomitant medications. The investigators found no correlation between initial thiopurine dose and pretreatment TPMT levels. Doses were adjusted or temporarily discontinued in 19% because of a decreased WBC count. TPMT activity was normal in 83% and intermediate in 14%, whereas none were homozygous deficient. Mean pretreatment TPMT activity was higher than at the time of the study (mean 2.2 years later). The median 6-TGN and 6-MMP concentrations were 136 (range, 12–564) and 771 pmol/8 ⫻ 108 RBC (58 –23, 294), respectively, with significantly higher 6-TGN levels in patients with intermediate TPMT activity. (Readers familiar with 6-TGN dosing should note that the assay used requires multiplication by 1.6 to correlate to the more familiar commercial 6-TGN assay; so a level of 136 is comparable to a level of 218). Patients on concomitant aminosalicylates trended toward higher 6-TGN levels and had significantly lower WBC counts (median, 4.5 vs.5.4 ⫻ 109/L). However, WBC and ANC were not associated with medication dose, TPMT activity, or 6-TGN levels. There was no correlation between IBDQ scores (mean, 176; range, 75–223) and 6-TGN or 6-MMP concentrations, TPMT levels, AZA or 6-MP dose, WBC, or ANC. 6-TGN levels were similar in patients with active disease and in remission (139 vs. 131 pmol/8 ⫻ 108 RBC). The authors concluded that, in contrast to earlier studies, there was no correlation between 6-TGN levels and disease activity or remission. Possible reasons cited included differ-

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ences in study design, study size, population (adults vs. pediatric), disease activity scales used, duration of purine analogue therapy, active disease vs. remission patients, and exclusion of patients with low or intermediate TPMT activity. Comment. The confusion over the “correct” dosing of the purine analogues in patients with IBD is in part derived from their “offlabel” use for these diseases, and the lack of typical dose-ranging and pharmacokinetic studies conducted in this patient population. As a result, the dosing guidelines used in oncology patients have been adopted, or adapted, for use in IBD. Using body weight guidelines, AZA dosed at 2.0 –3.0 and 6-MP at 1.5 mg 䡠 kg⫺1 䡠 day⫺1 has been proven effective in CD (Am J Dig Dis 1975;20:721–726, N Engl J Med 1980;302:981–987) and in UC (BMJ 1974;4:627– 630, Am J Gastroenterol 1990;85:717–722). IBD specialists seasoned in the use of these drugs have advocated pushing the dose with the intent of establishing a low WBC, ANC, or absolute lymphocyte count. The commercially available measurements of 6-TGN and 6-MMP have raised the possibility of more precise, patient-specific dosing of these agents. Another contributing factor to the “metabolite controversy” has been the disparate results in various studies trying to establish a relationship between drug levels and drug efficacy in IBD patients. Although some authors have shown a correlation between 6-TGN levels and decreased disease activity or remission (Gut 1996;39:401– 406, Gastroenterology 2000;118:705–713), there is often a widerange of 6-TGN levels in both the remission and nonremission (or improved or nonimproved) groups, with considerable overlap of values. These findings were also observed by yet another group who also failed to find a correlation between 6-TGN levels and efficacy (Scand J Gastroenterol 2001;36:71–76). Lowry et al. list many possible reasons for their disparate finding of a lack of correlation between 6-TGN levels and disease activity. Foremost may be the choice of disease activity “index.” Unlike the previously cited studies that used true disease activity indices such as the Harvey-Bradshaw Index (HBI) or the Crohn’s Disease Activity Index (CDAI), the authors of this study chose the IBDQ. The IBDQ is a 32-question quality of life questionnaire that had been shown to correlate with commonly used disease activity indices, including the HBI and CDAI (Gastroenterology 1994;106:287–296). The IBDQ score is typically broken down to 4 main categories: bowel symptoms, systemic symptoms, emotional function, and social function (Gastroenterology 1989;96:804 – 810). As a result, it is not unusual for some, but not all, of the 4 categories to correlate with a particular outcome. It would be interesting if Lowry et al. were to reanalyze their data with comparisons to each of the 4 categories. It is conceivable that the scores from the “bowel,” and perhaps “systemic,” components would have a stronger correlation with 6-TGN levels than perhaps some of the other outcomes. The design of the Lowry study, whereby only patients currently receiving either 6-MP or AZA were included, selected for patients for whom the drugs were successful, because they otherwise would have abandoned therapy in the past. This contention is supported by the patients’ mean IBDQ score of 176, which is above (i.e., “better than”) the remission level of 170. By excluding drug failures, the study design may have been doomed to fail to find any association between metabolites and remission. It would be interesting if the authors analyzed the clinical course of the 734 patients who either had stopped the drug, or failed to participate in the survey, and see how well (or poorly) metabolite levels correlated with outcome.

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The other major issues addressed by the Lowry study included lack of correlation between TPMT activity and leukopenia, and the absence of patients homozygous-deficient for the TMPT enzyme (estimated to be 1:300). This may reflect physician practice to stop purine analogues in patients who develop early leukopenia or avoid these drugs altogether in the TPMT-deficient patients. The authors also showed that their previous in vitro data of the inhibitory effect of concomitant dosing of aminosalicylates upon TPMT activity (Gastroenterology 1999;116:1505–1506) can be translated into in vivo elevation of 6-TGN concentrations and lower WBC counts. The “take-home” forecast for the clinician is “partly cloudy” for the current reliance upon metabolite levels for dosing of the purine analogues. Although some retrospective studies have shown that target levels of 6-TGN correlate with efficacy as measured by conventional disease activity indices (Gut 1996;39:401– 406, Gastroenterology 2000;118:705–713), the same does not hold true when one applies “quality of life” outcomes (Gut 2001;49:665– 670). One must be aware that coadministration of an aminosalicylate may result in increased activity of the purine analogue, and that discontinuation of such an agent may result in an undesired decrease in efficacy of 6-MP or AZA. I concur with the authors for the necessity of large, prospective studies of dosing protocols for these important agents, as well as investigations into the molecular mechanisms for their actions and metabolism. RUSSELL D. COHEN, M.D.

Reply. I want to make 3 points regarding the Selected Summary of our article. First, in addition to the 2 studies cited by Cohen, at least 2 other sizable studies have failed to demonstrate a relationship between clinical efficacy and erythrocyte 6-TGN concentrations in patients with IBD treated with azathioprine or 6-mercaptopurine (Gastroenterology 1999;117:527–535, Gastroenterology 2000;118: A4202). Second, the assertion of Cohen that the IBDQ may not accurately measure disease activity in patients with IBD is incorrect. The previous studies by Cuffari (Gut 1996;39:401– 406) and Dubinsky (Gastroenterology 2000;118:705–713) that reported a relationship between 6-TGN and clinical response to 6-mercaptopurine used the Harvey Bradshaw Index (HBI) (Lancet 1980;1:514), which is in essence a simplified version of the CDAI (Gastroenterology 1976;70: 439 – 444) to measure disease activity. Although the original CDAI was rigorously developed and validated, the HBI has never been validated, and its responsiveness to change in the context of clinical trials has never been determined. In contrast, the IBDQ was rigorously developed and validated, is sensitive to change, and correlates well with the CDAI (Gastroenterology 1989;96:804 – 810, Gastroenterology 1994;106:287–296). Thus, the IBDQ is a more valid alternative to the CDAI for measuring Crohn’s disease activity than is the HBI. Third, in another study by Cuffari (Gut 2001;48:642– 646), nonresponding patients with IBD treated with very low doses of azathioprine (1.1 ⫾ 0.1 mg/kg) who did not have leukopenia and who had “subtherapeutic” 6-TGN concentrations had their azathioprine doses gradually increased to a mean of 1.5 ⫾ 0.1 mg 䡠 kg⫺1 䡠 day⫺1, with subsequent clinical response and increase in 6-TGN concentrations. It is likely that the same result could have been achieved by simply administering the doses of azathioprine proven to be efficacious for Crohn’s disease in controlled trials (2–3 mg 䡠 kg⫺1 䡠 day⫺1) (Ann Intern Med 1995;123:132–142) from the outset, without therapeutic drug monitoring. Thus, the utility of routinely measuring 6-TGN concentrations in clinical practice remains unclear (Gut 2001; 48:591–592). A randomized, double-blind, controlled trial compar-

ing standard therapy (azathioprine 2.5 mg 䡠 kg⫺1 䡠 day⫺1 with dose adjustment only for toxicity) to azathioprine adjusted to a target 6-TGN concentration in patients with Crohn’s disease is needed to answer this question. WILLIAM J. SANDBORN, M.D.

PERSISTENCE AND PATHOGENESIS: A GREAT DEFENSE FACILITATES A GOOD OFFENSE Gobert AP, McGee DJ, Akhtar M, Mendz GL, Newton JC, Cheng Y, Mobley HLT, Wilson KT (Departments of Medicine and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland; Department of Veterans Affairs, Maryland Health Care System, Baltimore, Maryland; and School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia). Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for bacterial survival. Proc Natl Acad Sci U S A 2001;98:13844 –13849. In the nearly 2 decades since Helicobacter pylori was first isolated, it has become increasingly clear that the true relationships between this organism and diseases of the upper gastrointestinal tract are highly complex. Considerable efforts have focused on delineating the precise mechanisms by which H. pylori colonization leads to peptic ulcer disease, adenocarcinoma of the distal stomach, and mucosal lymphoproliferative diseases. However, a signature feature of H. pylori–induced gastritis is its capacity to persist for decades without causing serious damage in most cases. This is in marked contrast to inflammatory reactions induced by other Gram-negative pathogens, which either resolve over a limited time-span or progress to eliminate the host. Nitric oxide (NO) is an important intracellular mediator of cell growth and apoptosis that can directly damage DNA, and levels of this molecule are enhanced in chronic inflammatory and autoimmune conditions. NO also possesses potent antimicrobial activities against both intra- and extracellular pathogens, including H. pylori (Gut 1998;42:334 –337, Infect Immun 2000;68:4378 – 4383). NO is generated from an amino acid substrate (L-arginine) by NO synthases, enzymes that exist in both constitutive and inducible forms. Inducible NO synthase (iNOS) is increased within foci of H. pylori⫹ gastritis, localizing to epithelial and endothelial cells, as well as macrophages within the lamina propria (Gastroenterology 1999; 116:1319 –1329). Up-regulation of iNOS (and hence NO) within H. pylori– colonized tissue seems counterintuitive when one considers the chronic and persistent lifestyle of this organism, necessitating the hypothesis that H. pylori may possess mechanisms to counteract the effects of NO. Arginases are enzymes that, similar to iNOS, use L-arginine as substrate (Cell Mol Life Sci 1999;55:1015–1028). However, instead of generating NO, arginases catalyze the formation of urea and L-ornithine (Cell Mol Life Sci 1999;55:1015–1028), thereby diverting L-arginine away from iNOS, which results in reduced

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amounts of NO. Because H. pylori has been previously shown to possess a gene (rocF) encoding a functional arginase ( J Bacteriol 1999;181:7314 –7322), Gobert et al. sought to determine whether rocF regulates cellular production of NO, thus contributing to the ability of H. pylori to survive within the context of enhanced gastric mucosal levels of iNOS. H. pylori wild-type and isogenic rocF mutants (containing an inactivated arginase) were cocultured with macrophages in the presence of physiologic concentrations of L-arginine substrate. Both parental and mutant strains robustly induced iNOS expression in macrophages, findings that are consistent with previous observations of increased iNOS levels within H. pylori– colonized gastric tissue (Gastroenterology 1999;116: 1319 –1329). However, only the rocF mutant strain induced significantly increased levels of NO, indicating that arginase activity in the wild-type strain was effectively siphoning Larginine away from iNOS, and these events occurred regardless of macrophage activation state. L-arginine consumption studies conducted in the absence of macrophages confirmed these findings by demonstrating that levels of L-arginine were significantly reduced by wild-type, but not rocF mutant, H. pylori strains. To delineate the bacterial components required for these effects, Gobert et al. cocultured macrophages with live or heat-killed H. pylori wild-type strains, crude water extracts containing bacterial surface proteins, cytosolic extracts, or bacteria separated from cells by a filter support. Suppression of NO release was only found with live H. pylori, in close proximity to or separated from macrophages, suggesting that viable bacteria that are not in direct contact with macrophages consume L-arginine, resulting in inhibition of iNOS-catalyzed NO production. The investigators next complemented these experiments with elegant functional studies in which they measured survival rates of wild-type or rocF mutant strains exposed to macrophages as a measure of NO-mediated killing. As expected, macrophage coculture had no effect on survival of the parental H. pylori strain; in contrast, the arginase-deficient rocF mutant had a ⬎5-log reduction in colony-forming units. To further implicate NO in H. pylori killing, peritoneal macrophages isolated from wild-type or iNOS⫺/⫺-deficient mice were cocultured with the H. pylori rocF mutant for 24 hours. Compared with the profound cidal effect of wild-type macrophages, iNOS⫺/⫺ macrophages exerted no effect on the arginase-deficient H. pylori mutant, indicating that iNOS-derived NO is likely required for these antimicrobial effects. Comment. Although chronic gastritis clearly increases the risk for peptic ulceration and noncardia gastric adenocarcinoma, only a minority of individuals carrying H. pylori ever develop these sequelae. Clinical complications of H. pylori colonization may instead be viewed as imbalances in gastric homeostasis that are disadvantageous for both the bacteria and humans, especially if death of the host occurs. In support of this, recent data indicate that H. pylori not only induces gastritis but also possesses means to subdue the host inflammatory response, a requirement seemingly inherent for an organism that persists for the lifetime of its host. H. pylori infection is associated

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with increased mucosal levels of interleukin 10 (Lab Invest 1995;73: 760 –770, Gastroenterology 1996;110:1744 –1752, Infect Immun 1997;65:4229 – 4235, Gut 1997;40:739 –744), an anti-inflammatory peptide that inhibits secretion of proinflammatory cytokines from macrophages and neutrophils. When compared with lipopolysaccharide (LPS) from the Enterobacteriaceae, H. pylori LPS is 1000-fold less active and only weakly activates macrophages (Infect Immun 1995; 63:1183–1187). Inactivation of a cag island gene (cag10) results in a paradoxical increase in proinflammatory cytokine secretion compared with levels induced by wild-type H. pylori (J Clin Pathol 1999;52: 653– 657). The failure of H. pylori to invade the mucosa may contribute to its long-term persistence. It is tempting to speculate that H. pylori can also orchestrate the host response by negatively regulating intracellular eukaryotic signaling pathways, as has recently been described in Salmonella typhimurium (Science 2000;289:1560 – 1563). Certain nonpathogenic Salmonella attenuate interleukin-8 secretion induced by pathogenic bacteria by inhibiting the ubiquitination of I␬B␣, a novel mechanism for dampening the inflammatory response (Science 2000;289:1560 –1563). Gobert et al. have now added another component to the defensive armamentarium of H. pylori by demonstrating that a bacterial arginase can regulate iNOS activity that is induced by host-pathogen interactions. The experiments from which these results stemmed were rigorously controlled, and independent assays support the overall conclusion that H. pylori arginase attenuates NO-mediated bacterial killing. It will be interesting in the future to determine whether complementation of rocF in an arginase-deficient H. pylori mutant regenerates the ability to survive in the presence of macrophagederived NO. Arginase may not only protect the bacterium but also may attenuate host mucosal damage, thereby contributing to longterm persistence of H. pylori within the gastric niche. As stated by the authors, NO is notable for its ability to induce DNA damage and lipid peroxidation, events that are usually lethal for eukaryotic cells. Although the experiments of Gobert et al. were performed in isolated bacterial:macrophage systems, their results have set the stage for important in vivo confirmations, some of which have already been completed. Studies by this same group of investigators have revealed that H. pylori rocF mutants have an attenuated ability to colonize wild-type mice (J Bacteriol 1999;181:7314 –7322) and that wildtype H. pylori can infect wild-type and iNOS⫺/⫺ mice with no differences in colonization efficiency or severity of inflammation, supporting the contention that H. pylori arginase modulates NO synthesis within inflamed tissue. A critical experiment will be to determine if successful colonization can be restored when H. pylori rocF mutants are infected into iNOS⫺/⫺ knockout mice that lack the ability to produce NO, and such studies are ongoing at the present time. It is apparent that regulation of chronic gastric inflammation by H. pylori is governed by levels of host-bacteria equilibria that are not found during cellular interactions with acute pathogens, and clinically apparent disease likely results from the cumulative effect of multiple interactions between H. pylori and its host. Gobert et al. have identified a novel mechanism through which H. pylori may persist for the virtual lifetime of its cognate host. These types of studies that focus on characterization of host-microbial interactions are critical because they allow fundamental questions to be addressed regarding the basis of host defense and microbial persistence. RICHARD M. PEEK, JR.

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CELIAC SPRUE AS A POSSIBLE CAUSE OF SYMPTOMS IN PRESUMED IRRITABLE BOWEL SYNDROME Sanders DS, Carter MJ, Hurlstone DP, Pierce A, Ward AM (Royal Hallamshire Hospital, Sheffield, England). Association of adult coeliac disease with irritable bowel syndrome: a casecontrol study in patients fulfilling Rome II criteria referred to secondary care. Lancet 2001;358:1504 –1508. Diagnostic approaches to suspected irritable bowel syndrome (IBS) rely on eliciting symptoms that satisfy specific criteria and performing limited tests to ensure that organic diseases that produce similar symptoms are not present. Delineating which tests are useful has been a matter of debate. In the present study, Sanders et al. quantified the prevalence of celiac sprue in patients satisfying criteria for IBS. The investigators hoped to gain insight into the role of celiac disease in the mimicry of IBS and to determine if serologic testing should be performed in patients with presumed IBS. Three hundred uninvestigated patients (214 women; median age, 56 years) satisfying the Rome II criteria for IBS were referred from primary care physicians from 1999 to 2000. Twenty-one percent were constipation-predominant, 28% were diarrhea-predominant, and 51% had alternating bowel habits. Initial evaluation included blood counts, sedimentation rates and C reactive proteins, thyroid chemistries, and endoscopic and/or radiographic colon examinations. To screen for celiac disease, enzyme-linked immunosorbent assays quantified immunoglobulin (Ig) A antigliadin and IgG antigliadin antibodies and indirect immunofluorescence assessed for endomysial antibodies. Endoscopic duodenal biopsies were recommended for patients with positive serologies. Results were compared with 300 age- and sex-matched asymptomatic healthy volunteers. Of patients with presumed IBS, 3 had inflammatory bowel disease, whereas 1 each had collagenous colitis and radiationinduced colitis. Other organic diseases detected during testing included ethanol-related diarrhea in 2 patients, lactase deficiency in 1, pancreatic insufficiency in 1, and amyloidosis in 1. Several findings of questionable importance were noted including diverticulosis and colon polyps. Nineteen patients were lost to follow-up. Of 223 remaining patients, 66 exhibited positive serologic tests. Eleven had endomysial antibodies and 5 had IgA antigliadin antibodies without endomysial antibodies. Only 2 healthy volunteers exhibited positive endomysial antibodies and 1 had IgA antigliadin antibodies without endomysial antibody positivity. More than 40 patients in each group exhibited isolated IgG antigliadin antibodies of doubtful importance. Duodenal biopsy specimens confirmed celiac disease in 14 patients with presumed IBS (11 endomysial antibody positive, 3 endomysial antibody negative), whereas 43 had normal histology; 9 did not follow-up or refused endoscopy. Characteristic pathology was observed in 2 patients with constipa-

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tion, 4 with diarrhea, and 8 with alternating bowel patterns. Abnormal histology was found in 2 healthy volunteers with endomysial antibodies. Compared with the control group, celiac sprue and IBS were associated with an odds ratio of 7.0 (95% confidence interval, 1.7–28.0). On the basis of these findings, the authors advocated routine testing for celiac disease in patients with presumed IBS regardless of the symptom subtype. Comment. IBS is the most common disorder treated by gastroenterologists. What constitutes an appropriate diagnostic evaluation to establish a confident diagnosis has been a matter of debate. Symptom criteria have been devised to facilitate diagnosis, including the recent Rome II criteria, but directed laboratory and structural testing is still advocated to ensure that organic disease is not the cause of symptoms mimicking IBS (Rome II: The functional gastrointestinal disorders, 2nd edition. McLean, VA: Degnon Associates, 2000:351– 432). The American Gastroenterological Association (AGA) and British Society of Gastroenterology recommend performing complete blood counts, sedimentation rates, and thyroid chemistries as well as fecal ova and parasite examinations in patients with predominant diarrhea (Gastroenterology 1997;112:2120 –2136, Gut 2000;47[suppl 2]:1–19). Endoscopy (sigmoidoscopy in low-risk young patients vs. colonoscopy in patients ⬎45 years old or with a family history of colorectal neoplasia) is promoted for all patients by the AGA and for patients referred to secondary medical centers by the British Society. In patients with diarrhea, colonic biopsies may test for microscopic colitis, whereas hydrogen breath testing or initiation of a lactose-free diet can assess for lactase deficiency . The use of these protocols has been scrutinized. Symptom criteria exhibit sensitivities for diagnosing IBS ranging from 57% to 90%, whereas specificities range from 74% to 100% (Gut 1990;31:77– 81, Am J Gastroenterol 1999;94:2912–2917). The yield of specific tests also has been quantified. In one study, blood and stool tests were normal in all individuals, whereas structural examinations detected only 1 colon cancer and 1 colitis in 196 patients with presumed IBS (Am J Gastroenterol 1994;89:176 –178). Lactose intolerance was found in 26%, which may have been an incidental observation not relevant to the presenting symptoms in many patients. A second investigation found abnormal thyroid levels in 6%, ova and parasites in 2%, and structural abnormalities in 2% (Am J Gastroenterol 1999;94:1279 –1282). Again, lactose intolerance was diagnosed in 23% of patients. These reports emphasize the reality that large numbers of normal examinations are performed to confirm the diagnosis of IBS. Should we consider celiac sprue more seriously when evaluating a patient with presumed IBS? The AGA guidelines do not recommend serologic tests for celiac disease, while the British Society suggests a yield of only 1%–2% for antibody testing (Gastroenterology 1997; 112:2120 –2136, Gut 2000;47[suppl 2]:1–19). In stating their goals for diagnostic testing, the British Society aimed for sensitivities ⬎99% in detecting serious conditions such as malignancy with lower sensitivities (⬎90%) for less morbid conditions such as lactase deficiency. Like IBS, celiac sprue can present with altered bowel habits, dyspepsia, abdominal discomfort, bloating, and milk intolerance (BMJ 1999;319:236 –239). IBS patients may report wheat intolerance (Gut 2000;47[suppl 2]:1–19). The epidemiology of celiac disease parallels IBS with both conditions predominating in women by approximately 3:1 ratios (BMJ 1999;319:236 –239). The incidence of celiac sprue peaks in the fifth decade, although many IBS patients

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experience initial symptoms in the 40s (Gastroenterology 1991;100: 998 –1005). IBS subsets that respond less well to treatment include women and those with diarrhea (Lancet 1987;1:963–965). It is conceivable that undiagnosed celiac disease might provide an explanation for some refractory cases. However, the overlap of IBS- and celiacrelated symptoms is incomplete, with pain being more prominent in IBS and diarrhea being more prevalent in symptomatic celiac disease. Furthermore, unlike celiac sprue, IBS is not associated with diabetes, thyroid disease, primary biliary cirrhosis, and Sjogren’s syndrome, nor is there a clear genetic predisposition (Am J Gastroenterol 1998;93: 1311–1317, BMJ 1999;319:236 –239). The diagnostic approach to celiac disease has been updated with the introduction of serologic tests for endomysial and tissue transglutaminase antibodies, which exhibit sensitivities of ⬎90% and specificities ⬎95% in large series (Gastroenterology 1998;115:1584 – 1594, J Pediatr Gastroenterol Nutr 2000;31:513–519, Pediatrics 2001;107:E8, Can J Gastroenterol 2000;14:668 – 671). In contrast, older tests such as antigliadin antibodies are less sensitive and usually are obtained when newer tests are negative. Because of the predictive values of these tests, some have argued that a positive serology coupled with a symptom response to gluten exclusion is sufficient for diagnosis (BMJ 1999;319:236 –239). However, others report high false-negativity rates for IgA antibody tests in IgA-deficient patients and those with partial villous atrophy (Dig Dis Sci 1999;44:2133– 2138, Scand J Gastroenterol 2000;35:181–183, Am J Gastroenterol 1999;94:888 – 894). Furthermore, 25% of untreated patients in one study exhibited negative endomysial antibodies (Dig Dis Sci 2001; 46:214 –221). Thus, a recent review concluded that duodenal histology provides the most reliable information and will continue to be obtained for suspected celiac disease (BMJ 1999;319:236 –239). Sanders et al. are the first to report positive serologies for celiac sprue in patients with presumed IBS. Fourteen of 223 (6%) patients had celiac disease on duodenal histology. Three of 11 (27%) patients with positive biopsies exhibited negative endomysial antibodies. Although provocative, the findings of the current study must be interpreted with caution. The patients studied were older (median age, 56 years) than in most community surveys of IBS symptoms, suggesting an inherent increased likelihood for organic disease in the test population (Gastroenterology 1991;100:998 –1005). The investigators did not restrict dietary gluten in those with positive biopsies, thus a causative role for celiac disease in symptom production was not proved. Furthermore, one would expect celiac sprue to be more common in patients with diarrhea. Because celiac disease was found

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nearly equally in those with constipation and diarrhea raises concern that some patients indeed did have IBS as a cause of their symptoms plus the incidental finding of silent sprue. This would be analogous to reports that lactose restriction and lactase supplements only benefit a subset of patients with IBS symptoms and documented lactase deficiency (Am J Gastroenterol 1989;84:756 –762). Finally, the association of celiac disease with IBS may relate to the high prevalence of sprue in the study locations. It would be interesting to test the reproducibility of these results in regions where celiac disease is less common. This study should be compared to a recent report by Wahnschaffe et al. in which IBS patients with diarrhea were evaluated (Gastroenterology 2001;121:1329 –1338). As noted in an accompanying editorial by Drossman, symptom criteria for IBS including the presence of abdominal pain were not used, raising concern about patient selection (Gastroenterology 2001;121:1512–1515). Nevertheless, of 102 patients, none were positive for gliadin, endomysial, or transglutaminase antibodies in contrast to the findings of the current study. A diagnosis of celiac sprue in approximately 30% of the IBS group was suggested by detection of celiac disease–associated antibodies in duodenal aspirates, increased intestinal intraepithelial lymphocytes, and positive genetic markers. Furthermore, 26 IBS patients placed on gluten-free diets for 6 months exhibited reduced stool frequency and duodenal juice IgA antibodies. These concurrent studies with different findings reach the same conclusion, that celiac disease is prevalent in patients with presumed IBS. They question if routine testing for celiac sprue should be included in the diagnostic algorithm for IBS. This issue relates to the British Society guidelines, which aim to stratify diagnostic tests on the basis of the consequences of missed organic diagnoses (Gut 2000;47[suppl 2]:1–19). As with lactase deficiency, patients with mild symptoms may suffer no ill effects from delayed diagnosis of celiac sprue. However, celiac disease has significant long-term consequences including osteoporosis and gastrointestinal neoplasms and therefore should be considered a diagnosis of intermediate severity (BMJ 1999;319:236 –239). Furthermore, both studies observed that serologic tests fail to detect many cases of celiac disease. Thus, if we are concerned about celiac disease in apparent IBS patients, should we bypass serologic testing and perform intestinal biopsies or aspirates to confirm the diagnosis? These issues await further controlled investigation. WILLIAM L. HASLER, M.D.