By Michael Pignone, M.D., M.P.H.a; Melissa Rich, M.D.b; Steven M. Teutsch, M.D., M.P.H.c; Alfred O. Berg M.D., M.P.H.d; Kathleen N. Lohr, Ph.D.e
Address correspondence to: Michael Pignone, M.D., M.P.H.; Division of General Internal Medicine; CB # 7110 UNC Hospitals; Chapel Hill, NC 27599-7110; E-mail: pignone@med.unc.edu
This article originally appeared in the Annals of Internal Medicine. Select for copyright and source information.
The summaries of the evidence briefly present evidence of effectiveness for preventive health services used in primary care clinical settings, including screening tests, counseling, and chemoprevention. They summarize the more detailed Systematic Evidence Reviews, which are used by the U.S. Preventive Services Task Force (USPSTF) to make recommendations.
Abstract
Introduction
Methods
Role of the Funding Agency
Results
Discussion
Acknowledgments
References
Notes
Author Affiliations
Copyright and Source Information
Appendix. Methods
Search Strategies
Eligibility Criteria
Table. Eligibility Critera
Tables
Table 1. Characteristics of Screening Tests for Colorectal Cancer
Table 2. Trials of Fecal Occult Blood Testing
Purpose: To conduct an updated systematic evidence review for the U.S. Preventive Services Task Force to assess the effectiveness of different colorectal cancer screening tests for adults at average risk.
Data sources: We used recent systematic reviews, the second edition of the Guide to Clinical Preventive Services, and focused searches of MEDLINE® from 1966 through September 2001. We also conducted hand searches, reviewed bibliographies, and consulted context experts to assure completeness.
Study selection: When available, we used the most recent high-quality systematic review to identify relevant articles and then supplemented that review with a MEDLINE® search for more recent articles.
Data extraction: One reviewer abstracted information from the final set of studies into evidence tables and a second reviewer checked them for accuracy. Discrepancies were resolved by consensus.
Data synthesis: Evidence from multiple well-conducted randomized trials supports the effectiveness of fecal occult blood testing (FOBT) in reducing colorectal cancer incidence and mortality rates compared with no screening for adults over age 50 who are at average risk. Data from well-conducted case-control studies support the effectiveness of sigmoidoscopy, and possibly colonoscopy, in reducing colon cancer incidence and mortality rates. A nonrandomized controlled trial examining colorectal cancer mortality rates and randomized trials examining diagnostic yield support the use of FOBT plus sigmoidoscopy. The effectiveness of barium enema is unclear. Data are insufficient to determine with confidence the most effective screening strategy.
Conclusions: Colorectal cancer screening is effective in reducing mortality rates from colorectal cancer and can decrease the incidence of disease through removal of adenomatous polyps. Several screening options seem to be effective, although data are insufficient to determine the best single screening approach.
The U.S. Preventive Services Task Force (USPSTF) last considered its recommendations regarding colorectal cancer screening in 1996(1). At that time, the available evidence included one randomized controlled trial showing that fecal occult blood testing (FOBT) reduced mortality rates(2), a case-control study showing that persons having sigmoidoscopy were less likely to die from colorectal cancer(3), and one nonrandomized controlled trial of FOBT combined with rigid sigmoidoscopy that suggested some benefit from the two tests together(4). Based on this evidence, the USPSTF gave a "B" recommendation to screening for colorectal cancer with FOBT, sigmoidoscopy, or both. The USPSTF did not recommend for or against other means of screening (digital rectal examination [DRE], double contrast barium enema [DCBE], or colonoscopy) on the grounds that evidence was insufficient. The Task Force also recommended that FOBT be performed yearly but did not specify an interval for sigmoidoscopy.
Since 1996, important new evidence has emerged regarding the effectiveness of colorectal cancer screening. The USPSTF requested that the RTI-University of North Carolina-Chapel Hill Evidence-based Practice Center prepare an updated systematic evidence review to help the USPSTF evaluate new evidence on the effectiveness of different colorectal cancer screening tests as it updated its previous recommendation.
In this review, we examine the evidence concerning the effectiveness of screening in adults over age 50 who are at average risk for colorectal cancer. We consider evidence about the effectiveness, accuracy, and adverse effects of DRE (with or without a single office-based FOBT), traditional 3-card FOBT (hereafter referred to as "FOBT"), sigmoidoscopy, FOBT with sigmoidoscopy, DCBE, and colonoscopy. Other tests or combinations of tests have not been well evaluated and are not discussed here. A more detailed report of our review can be found in Pignone et al.(5), available on the AHRQ Web site (http://www.preventiveservices.ahrq.gov). The USPSTF's colorectal cancer screening recommendations, which are based on this review, are also published in Annals of Internal Medicine(6).
We used the second edition of the Guide to Clinical Preventive Services(1), existing systematic reviews, focused MEDLINE® literature searches from 1966 through September 2000, and hand-searches of key articles to identify the relevant literature. When available, systematic reviews were used to identify older relevant studies. Literature searches were used to identify newer studies. Detailed descriptions of the literature searches can be found in the Appendix.
To identify relevant studies, one reviewer examined the abstracts of the articles identified in the initial search. A second reviewer examined the excluded articles. Disagreements about inclusion were resolved by consensus. Two reviewers examined the full text of the remaining articles to determine final eligibility.
We used evidence from randomized controlled trials or observational studies that have measured patient outcomes, particularly changes in colorectal cancer mortality rates and incidence. When such data were not available, we included indirect information on screening test accuracy. Details about study inclusion are available in the Appendix.
Included articles were quality rated using the criteria developed by the USPSTF Methods Group(7). These criteria are described in the Recommendation and Rationale statement authored by the USPSTF(6).
We used the final set of eligible articles to create evidence tables and a draft report. The draft report was extensively peer reviewed by the USPSTF, experts in the field, governmental agencies, and nongovernmental organizations.
This evidence report was funded through a contract to the RTI-University of North Carolina Evidence-based Practice Center from the Agency for Healthcare Research and Quality (AHRQ). Staff of the funding agency contributed to the study design, reviewed draft and final manuscripts, and made editing suggestions.
Our general search identified 719 articles published since 1995 on colorectal cancer screening, of which we retained 19 in our final document. Specific searches from 1966 through 2001 for articles about the accuracy of barium enema and complications of screening yielded 621 and 839 articles, respectively. After review, we retained 13 articles about barium enema, and 19 about complications of screening. We also included 15 articles identified from the previous USPSTF review or from hand searches of other articles. Table 1 summarizes our findings.
Digital Rectal Examination
Effectiveness. A case-control study from the Kaiser Permanente Medical Care Program in northern California examined the effect of screening DRE on mortality from colorectal cancer(8). The investigators identified patients aged 45 and older who died of distal rectal cancers between 1971 and 1986 and selected matched controls from their patient membership. They examined medical records to determine whether the patients who died and controls had undergone screening DREs within a year of cancer diagnosis. Investigators found no difference between groups after controlling for potential confounders, although the confidence interval was wide (odds ratio [OR], 0.96; 95 percent confidence interval [CI], 0.56, 1.7).
Accuracy. The potential sensitivity of screening DRE is low: fewer than 10 percent of colorectal cancers arise within reach of the examining finger(28). The specificity of a positive DRE has not been examined in outpatients at average risk for colorectal cancer.
In-Office Fecal Occult Blood Testing After Digital Rectal Exam
Effectiveness. No studies have examined the effect of a single in-office FOBT after DRE on colorectal cancer incidence or mortality rates.
Accuracy. A single in-office FOBT is likely to be less sensitive than the traditional three-card home-performed FOBT because only one sample is taken(9). In a large study from Japan, Yamamoto and Nakama found that the first test card detected only 58 percent of the cancers found with a three-card test(10).
A single in-office FOBT may be less specific than a properly performed three-card FOBT because the in-office test does not allow degradation of vegetable peroxidases that sometimes produce false-positive results(9). In addition, the potential trauma from the in-office examination itself may also result in lower specificity(9). Two studies of poor to fair quality that used existing data to compare retrospectively the specificity of the single in-office FOBT and the three-card home FOBT(11,12) found little difference in specificity between the two groups. However, the validity of these studies is limited because neither could ensure that similar patient samples received each test.
Fecal Occult Blood Testing
Effectiveness. In addition to an older randomized trial performed in Minnesota(2) that was available to the USPSTF in 1996, two newer randomized controlled trials (RCTs) from the United Kingdom(13) and Denmark(14) have examined the effectiveness of biennial FOBT for reducing colorectal cancer mortality. The more recent trials found reductions in mortality rates of 15 percent and 18 percent, respectively, using biennial testing. Neither trial used slides that were rehydrated prior to development (Table 2).
The Minnesota trial compared annual and biennial testing with no screening and rehydrated most test cards (83 percent). Cumulative mortality rates from colorectal cancer after 18 years of followup were 33 percent lower (95 percent CI, 17 percent, 49 percent) among persons randomized to undergo annual FOBT than in a control group that was not offered screening (absolute rates: 9.5 deaths per 1,000 participants versus 14.1 deaths per 1,000 participants; difference, 4.6 deaths per 1,000 participants)(2). Biennial screening, which did not show a reduction in mortality at 13-year followup, produced a 21 percent reduction in mortality rates at 18 years (95 percent CI, 3 percent, 38 percent).(15) The 18-year followup also showed that the incidence of colorectal cancer decreased by 20 percent (95 percent CI, 10 percent, 30 percent) and 17 percent (95 percent CI, 6 percent, 27 percent) in the groups screened annually and biennially, respectively, compared with controls(16). Differences in hydration, test frequency, duration, and effect size preclude combining the results of these trials in a meta-analysis.
Accuracy. A systematic review from 1997 found that the sensitivity of a single unrehydrated FOBT for cancer was approximately 40 percent; its specificity appears to range from 96 percent to 98 percent. Rehydration was found to increase sensitivity to between 50 percent and 60 percent but lowered specificity to 90 percent(10,18). In a recent study, Lieberman et al. found that the sensitivity of rehydrated FOBT for cancer was 50 percent (95 percent CI, 30 percent, 70 percent)(17). For advanced neoplasia (cancers and polyps that are large, villous or dysplastic), sensitivity was 24 percent (95 percent CI, 19 percent, 29 percent); specificity was 94 percent (95 percent CI, 93 percent, 95 percent).
In the annual screening arm of the 13-year Minnesota trial, which used primarily rehydrated test cards and had a high initial rate of participation (about 90 percent), 49 percent of patients who developed colorectal cancer were identified through screening; and 38 percent of all patients had had at least 1 colonoscopy(2). Biennial screening detected 39 percent of patients with cancer in the intervention group, and 28 percent of patients required colonoscopy. In contrast to the Minnesota trial, the two UK and Danish trials were population-based, 8 to 10 years in duration, used only biennial testing, and had lower rates of participation (60 percent to 70 percent of patients completed the first screening). Screening detected 27 percent of patients in the intervention group who developed colorectal cancer; only 5 percent of patients underwent colonoscopy(13,14).
Adverse Effects. FOBT itself has few adverse effects, but false-positive FOBTs lead to further tests, such as colonoscopy, during which adverse effects may occur. The specific adverse effects of colonoscopy are described below. Theoretically, a previously negative FOBT could falsely reassure patients and lead to delayed response to the development of colorectal symptoms if a cancer were to develop, but this concern has not been evaluated empirically.
Sigmoidoscopy
Effectiveness. Thiis-Evensen et al. performed a small randomized trial of sigmoidoscopy screening in Norway(18). In 1983, 799 men and women ages 50-59 drawn from a population registry were randomly assigned to receive screening flexible sigmoidoscopy (400 patients) or no screening (399 patients). Eighty-one percent of those offered flexible sigmoidoscopy accepted. All patients found to have polyps on sigmoidoscopy underwent immediate diagnostic colonoscopy and had surveillance examinations 2 and 6 years later. Over the 13 years of the trial, two colorectal cancers were diagnosed in the intervention group and 10 in the control group (relative risk [RR] for colorectal cancer, 0.2; 95 percent CI, 0.03, 0.95). One person who was assigned to the intervention group but never had the sigmoidoscopy screening examination, died from colorectal cancer; three colorectal cancer deaths occurred in the control group (RR, 0.50; 95 percent CI, 0.10, 2.72). The overall mortality rate was higher in the intervention group than in the control group (14 percent versus 9 percent; RR, 1.57; 95 percent CI, 1.03, 2.40), mostly because of an excess of cardiovascular deaths. No clear relationship emerged between the excess deaths and any complications from the procedures.
Two ongoing randomized trials using flexible sigmoidoscopy can be expected to report their initial results within 5 years. One trial is examining the effect of once-in-a-lifetime sigmoidoscopy in the United Kingdom(19); a second trial in the United States is examining sigmoidoscopy every 5 years with the assumption that patients are receiving FOBT as part of usual care(30).
Two older, well-designed case-control studies that provide other important information on the effectiveness of sigmoidoscopy screening were available to the USPSTF in 1996. Using data from the Kaiser Permanente Medical Care Program in northern California (3), Selby et al. found that 9 percent of persons who died of colorectal cancer occurring within 20 cm of the anus had previously undergone a rigid sigmoidoscopic examination, whereas 24 percent of persons who did not die of a cancer within 20 cm of the anus had received the test(3). The adjusted odds ratio of 0.41 (95 percent CI, 0.25, 0.69) suggested that sigmoidoscopy screening reduced the risk of death by 59 percent for cancers within reach of the rigid sigmoidoscope.
The investigators noted that the adjusted odds ratio for proximal colon cancer that was beyond the reach of the sigmoidoscope was 0.96(3). This finding added support to the hypothesis that the reduced risk of death from cancers within reach of the rigid sigmoidoscope could be attributed to screening rather than to confounding factors. The risk reduction associated with sigmoidoscopy screening did not diminish during the first 9 to 10 years after the test was performed. Although the Selby et al. study mostly used rigid sigmoidoscopes, in another case-control study supporting the effectiveness of sigmoidoscopy, 75 percent of the examinations were performed with a flexible instrument(31).
Accuracy. Two recent studies have examined the sensitivity of screening sigmoidoscopy for cancer or advanced adenomas in healthy patients using colonoscopy as the criterion standard. They found that sigmoidoscopy would identify 70 percent to 80 percent of patients with advanced adenomas or cancer(17,20).
Sigmoidoscopy can produce false-positive results by detecting hyperplastic polyps that do not have malignant potential or adenomatous polyps that are unlikely to become malignant during the patient's lifetime. Because studies of diagnostic accuracy cannot measure whether small or large adenomas that are identified and removed would have gone on to become cancer, it is not possible to classify such findings in terms of their accuracy in detecting cancer. In practice, most investigators consider all adenomas to be "true positives," whether or not they would ever progress to cancer. Comparison of the specificity of sigmoidoscopy with that of other screening methods, such as FOBT and barium enema, is therefore difficult.
Adverse Effects. Estimates of bowel perforations from sigmoidoscopy have generally been in the range of 1 to 2 or fewer per 10,000 examinations, particularly since the introduction of the flexible sigmoidoscope(32). Atkin et al. recently reported initial results from a sigmoidoscopy screening trial(19) in which experienced endoscopists performed sigmoidoscopy in 1,235 asymptomatic adults aged 55-64; 288 patients had polyps removed during the examination. Adverse effects, including pain, anxiety, or any degree of bleeding, were assessed by a written questionnaire immediately after the test and by a mailed questionnaire 3 months later. Of all patients, 3.2 percent (40/1,235) reported bleeding, (16/288 or 5.5 percent after polypectomy; 24/947 or 2.5 percent after diagnostic studies). One patient required hospital admission; none required a transfusion. Of all patients, 14 percent reported moderate pain and 0.4 percent reported severe pain. More than 25 percent of patients reported gas or flatus. No perforations were reported, but one patient died from peritonitis after a complicated open surgical procedure to remove a severely dysplastic adenoma. A recent study of endoscopic complications from the Mayo Clinic in Arizona identified two perforations in 49,501 sigmoidoscopy procedures(33).
Fecal Occult Blood Test and Sigmoidoscopy
Effectiveness. Currently no randomized trials with colorectal cancer mortality as an end point have compared FOBT alone or sigmoidoscopy alone with a strategy of performing both tests.
In 1992, Winawer et al. published a nonrandomized trial of more than 12,000 first-time attendees at a preventive health clinic in New York. This trial was available to the USPSTF in 1996(4). The control group received rigid sigmoidoscopy at the first visit. All study participants were invited to return for annual rigid sigmoidoscopy re-examinations. Patients in the intervention group received rigid sigmoidoscopy and were also asked to complete Hemoccult™ (Beckerman Coulter, Fullerton, California) FOBT cards. Patients with adenomas more than 3 mm on sigmoidoscopy or who had a positive FOBT underwent full colonic examination with barium enema and colonoscopy. The control group received rigid sigmoidoscopy at the first visit and participants were invited to return for annual reexamination. Few patients continued to participate after the first examination (20 percent had FOBT at year 2 and 15 percent at year 3). Incidence of colorectal cancer and mortality were assessed over a 9-year period; followup data were available for 97 percent of subjects.
Demographic and clinical data suggest that the groups were comparable, despite the absence of randomization. More cases of colorectal cancer were detected on initial examination among intervention patients than in control patients (4.5 versus 2.5 per 1,000 participants). Incidence rates (cancers detected after the initial examination) were similar between groups (0.9 per 1,000 person-years in each group). Colorectal cancer mortality was 0.36 per 1,000 patient-years in the intervention group and 0.63 per 1,000 patient-years among controls (RR, 0.56; 95 percent CI, 0.25, 1.19).
Thus, adding FOBT to rigid sigmoidoscopy appears to increase the yield of initial screening and may reduce mortality rates. Because rigid sigmoidoscopy is no longer used for screening, the generalizability of these results to the use of FOBT plus flexible sigmoidoscopy is unclear. Whether the incremental yield of combined screening will change after additional rounds of testing also remains uncertain.
Accuracy. Recent randomized trials from Europe have examined the additional diagnostic yield of performing sigmoidoscopy plus FOBT at one point in time for patients who were not already part of an ongoing screening program(21-23). In each study, adding sigmoidoscopy to FOBT increased the identification of significant adenomas or cancer by a factor of two or more. Adding FOBT to sigmoidoscopy did not appear to identify any additional significant lesions. Winawer et al., (4) however, found an increased yield from adding FOBT to rigid sigmoidoscopy. In each study, data were limited to a single round of testing. The additional yield of this strategy may be lower after the first round of testing, but the impact of this strategy on mortality rates has not been fully evaluated.
Adverse Effects. The adverse effects of FOBT plus sigmoidoscopy are equal to the adverse effects of each test alone.
Double Contrast Barium Enema
Effectiveness. We identified no published studies that examined the effectiveness of DCBE in reducing the incidence or death from colorectal cancer.
Accuracy. Several studies have examined the accuracy of DCBE for diagnosing colorectal cancer or adenomatous polyps(24,34-43). Most are of methodologically poor quality because they examined patients with symptoms or did not prospectively collect blinded data.
The National Polyp Study is a randomized trial of different intervals of surveillance after polypectomy (examinations at 1 and 3 years versus at 3 years only). In a substudy of this trial, Winawer et al. compared the accuracy of DCBE with that of colonoscopy(24). The sensitivity of DCBE for polyps smaller than 0.5 cm was 32 percent (95 percent CI, 25 percent, 39 percent); for polyps 0.6 to 1 cm, sensitivity was 53 percent (95 percent CI, 40 percent, 66 percent); for polyps larger than 1 cm, including two cases of cancerous polyps, sensitivity was 48 percent (95 percent CI, 24 percent, 67 percent). Of 470 patients in whom colonoscopy detected no polyps, DCBE was positive in 83 (specificity, 85 percent; 95 percent CI, 82 percent, 88 percent).
The Winawer et al. study examined patients who previously had colonoscopy and removal of all polyps. Their results, therefore, may have limited generalizability for screening, because screening largely involves persons who have not had a recent colonoscopic examination and polypectomy and therefore may be more likely to have large polyps or cancers. However, the low sensitivity for large polyps and cancers found in this study is cause for concern and may limit the potential effectiveness of screening with DCBE.
Adverse Effects. The estimated risk of perforation during barium enema is low. In a study by Kewenter and Brevinge, no perforations or other complications occurred among the 1,987 screening patients undergoing barium enema as part of their screening work-up(44). Blakeborough et al. surveyed UK radiologists about the complications of barium enema during a 3-year period from 1992 through 1994(25). All examinations were included, regardless of the indication for the procedure. Important complications of any type occurred in 1 in 10,000 examinations. Perforation occurred in 1 of 25,000 examinations; death occurred in 1 in 55,000 examinations, although whether all deaths were related to the procedure is not clear.
Colonoscopy
Effectiveness. The ability of colonoscopy to prevent colorectal cancer cases or mortality has not been measured in a screening trial. The National Polyp Study estimated that 76 percent to 90 percent of cancers could be prevented by regular colonoscopic surveillance examinations, based on comparison with historic controls(45). However, these results should be interpreted with caution because the comparison groups were not from the same underlying population, which could introduce bias. In addition, all trial participants had polyps detected and removed, which limits generalizability of the results to the average screening population.
Muller and Sonnenberg, in a case-control study at Veterans Affairs hospitals, found that patients diagnosed with colorectal cancer were less likely to have had previous colonoscopy. The odds ratios for disease incidence were 0.47 (95 percent CI, 0.37, 0.58) for colon cancers and 0.61 (95 percent CI, 0.48, 0.77) for rectal cancers(26). For colorectal cancer mortality, the odds ratio was also lower for patients with previous colonoscopy (OR, 0.43; 95 percent CI, 0.30, 0.63)(44).
Accuracy. Because colonoscopy is commonly used as the criterion standard examination, calculating sensitivity is difficult. Using tandem colonoscopic examinations, Rex et al. found single-test sensitivity to be 90 percent for large adenomas and 75 percent for small adenomas (less than 1 cm); sensitivity for cancers is likely to be greater than 90 percent(27).
The recent identification of flat lesions that can be missed on regular colonoscopy suggests that some histologic variants do not pass through the typical adenoma-carcinoma development sequence and thus may not be easily detectable in the precancerous phase(46). If flat lesions account for 10 percent of all adenomas, sensitivity of all endoscopic screening methods may be lower than previously thought.
The specificity of colonoscopy with biopsy is generally reported to be 99 percent or 100 percent, but this assumes that all detected adenomas represent true-positive results. As with sigmoidoscopy, most detected adenomas, especially small adenomas, will never develop into cancer. If detection of an adenoma that will not become cancer is considered a false positive result that subjects a patient to risk without benefit, then the actual specificity of colonoscopy would be much lower.
Adverse Effects. Colonoscopy, which uses sedation and requires skilled support personnel, is more expensive than other screening tests and has a higher risk of procedural complications than other screening tests, particularly when polypectomy is performed. Use of conscious sedation adds the risk of complications attributable to the sedative agent.
In our systematic review of studies examining the principal complications of colonoscopy(5), we focused on hemorrhage and perforation but noted the less frequent complications of death, infections, sedation-related events, and chemical colitis. Two recent studies examined the incidence of complications from colonoscopy performed in screening populations. In the study in Veterans Affairs medical centers by Lieberman et al., 10 of 3,121 patients (0.3 percent) had major complications during or immediately following the procedures, including 6 who had bleeding requiring hospitalization and 1 each with a stroke, myocardial infarction, Fournier gangrene, and thrombophlebitis(17). Three other patients died within 1 month, probably from causes unrelated to the procedure. In the study by Imperiale et al. of employees of a large corporation, 1,994 persons aged 50 and older underwent colonoscopy(20). One (0.05 percent) had a perforation not requiring surgery; 3 (0.15 percent) had bleeding that required emergency room visits but not admission or surgery.
Apart from these 2 screening studies, most of the studies examining colonoscopy complications are retrospective reviews of endoscopy records from U.S. university hospitals that recorded only immediate complications of the procedure and included a mixture of screening and diagnostic procedures.(17,20,33,47-61) A prospective study that also included a patient questionnaire administered 10 days after the procedure identified several additional important complications that occurred outside the hospital, suggesting that hospital record review alone may underestimate actual complication rates(47).
Despite these limitations of reporting and nonscreening study populations, these studies provide a useful approximation of the complication rates that can be expected from colonoscopy. For diagnostic procedures, perforation rates were low (0.029 percent to 0.61 percent) and bleeding was not reported in enough studies to generate an estimate of its frequency. For therapeutic procedures, complication rates were higher (perforations, 0.07 percent to 0.72 percent; bleeding, 0.2 percent to 2.67 percent). Deaths occurred infrequently (reported rates from 1 in 30,000 to 1 in 3,000 with higher rates in studies with older patients and more symptomatic patients). The rate of screening related death may be on the lower end of this range; one cost-effectiveness analysis estimated it to be 1 per 20,000 patients(29). Other clinically relevant complications were identified and reported too infrequently and measured too inconsistently to allow accurate estimation of their true incidence.