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Published online before print March 4, 2005, 10.1148/radiol.2351040310
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(Radiology 2005;235:89-96.)
© RSNA, 2005


Gastrointestinal Imaging

Differentiation of Nonperforated from Perforated Appendicitis: Accuracy of CT Diagnosis and Relationship of CT Findings to Length of Hospital Stay1

Thomas A. Foley, BS, Frank Earnest, IV, MD, Mark A. Nathan, MD, David M. Hough, MD, Henry J. Schiller, MD and Tanya L. Hoskin, MS

1 From the Mayo Clinic College of Medicine (T.A.F.) and the Departments of Radiology (F.E., M.A.N., D.M.H.), Surgery (H.J.S.), and Health Sciences Research (T.L.H.), Mayo Clinic College of Medicine, 200 First St SW, Mayo W2, Rochester, MN 55905. Received February 16, 2004; revision requested April 21; revision received May 21; accepted June 28. Address correspondence to F.E. (e-mail: earnest@mayo.edu).


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine retrospectively the sensitivity and specificity of computed tomographic (CT) signs in differentiating acute nonperforated appendicitis from perforated appendicitis and to compare CT findings with the length of hospital stay.

MATERIALS AND METHODS: Institutional Review Board approval was obtained for this study, and patient informed consent was obtained for record review for research purposes. Two radiologists were blinded to patient identification but were informed that all patients presented to the emergency department with abdominal pain and underwent appendectomy. Radiologists independently reviewed CT images of 86 consecutive patients (45 males, 41 females; mean age, 33.7 years; age range, 8.2–87.1 years) who presented to the emergency department with acute abdominal pain, who underwent CT after initial emergency department assessment, and who underwent appendectomy within the subsequent 24 hours. Individual findings and confidence level for the diagnosis of perforated appendicitis were noted. Consensus interpretation was performed with a third radiologist. The consensus CT findings were correlated with the surgical and pathologic findings by using {chi}2 or Fisher exact tests for univariate analysis and logistic regression for multiple variable analysis. Wilcoxon rank sum tests were used to assess the association between consensus CT findings and length of hospital stay.

RESULTS: Twenty-one (24%) of the 86 patients had appendiceal perforation. Extraluminal air and either moderate or severe periappendiceal inflammatory stranding were statistically significant independent predictors for perforation (P < .001). A focal defect in enhancing appendiceal wall was significantly associated with perforation (P < .001) and had a sensitivity of 58.8% and specificity of 85.7% on consensus review, with eight false-positive results. There was a strong association between the degree of periappendiceal inflammatory stranding and the length of hospital stay (P < .001).

CONCLUSION: Extraluminal air and moderate or severe periappendiceal inflammatory stranding are statistically significant independent predictors for appendiceal perforation and are associated with increased hospital stay.

© RSNA, 2005


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute appendicitis is the most common surgical condition associated with nontraumatic acute abdominal pain. There are approximately 250 000 new cases of appendicitis per year in the United States (1). Perforation occurs in 19%–35% of cases of acute appendicitis (13) and is associated with an increased rate of many complications, including wound infections, urinary retention, ileus, small-bowel obstruction, and intraperitoneal abscess. Perforated appendicitis is associated with substantial morbidity and mortality in the elderly (4).

Computed tomography (CT) has been shown to be an accurate imaging technique for the evaluation of appendicitis. The use of this modality has led to a reduction in the misdiagnosis of appendicitis (58) and has had a variable effect on perforation rates. Preoperative identification of perforated appendicitis would be helpful in determining therapy, including consideration for nonsurgical treatment (9,10), and in estimating the risk of complications.

CT findings of perforated appendicitis have been published. Studies have included patients who were examined over several years, with high proportions of patients with perforated appendicitis and abdominal abscess (9,11). The purpose of our study was to determine retrospectively the sensitivity and specificity of CT signs in differentiating acute nonperforated appendicitis from perforated appendicitis and to compare CT findings with the length of hospital stay.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients
The study population consisted of a consecutive series of 86 patients (45 males, 41 females; mean age, 33.7 years; age range, 8.2–87.1 years) with acute abdominal pain who were evaluated in the emergency department between June 1 and December 31, 2001, who underwent CT at the time of initial evaluation, and who underwent appendectomy within the subsequent 24 hours. During this same period, 52 other patients underwent appendectomy but were excluded from the study. Reasons for exclusion were appendectomy performed more than 24 hours after CT, no preoperative CT examination, or appendicitis that was not evaluated in the emergency department. Our Institutional Review Board approved our study, and patient informed consent was obtained for record review for research purposes.

CT Examination
Eighty-five CT examinations were performed by using one of two multisection CT scanners (LightSpeed QXi or LightSpeed Plus; GE Medical Systems, Milwaukee, Wis) with 5-mm reconstruction thickness at 5-mm intervals through the abdomen and pelvis. One examination was performed by using a single-section helical CT scanner (HiSpeed; GE Medical Systems) with 7-mm reconstruction thickness at 7-mm intervals. In 80 of 86 patients, bowel opacification was accomplished by administering 600 mL of 2% dilute oral meglumine diatrizoate solution (MD-Gastroview; Mallinckrodt, St Louis, Mo) after a variable delay prior to imaging. In 78 of 86 patients, intravenous contrast material enhancement was accomplished by administering 140 mL of iohexol (Omnipaque 300; Amersham Health, Princeton, NJ) with an iodine concentration of 300 mg per milliliter. Intravenous contrast material was injected at a rate of 3 mL/sec, with image acquisition performed in the portal venous phase after a 70-second scanning delay. Exposure technique was based on patient size.

Image Review
The names and identifying record numbers were electronically removed from all CT images, and research numbers were assigned for computer workstation review (GE Advantage Windows Workstation, version 4.1_03; GE Medical Systems). Two experienced abdominal radiologists (M.A.N. and D.M.H., with 6 and 10 years experience, respectively) independently reviewed images from each CT examination. Reviewers were blinded to all records (including patient identification and surgical and pathologic diagnosis) with the understanding that all of the examinations were performed in patients who came to the emergency department presenting with acute abdominal pain and who underwent subsequent appendectomy. A third radiologist (F.E., 26 years experience) then joined both of the independent reviewers to perform a blinded consensus review of images from each examination. Specific CT findings of appendicitis and perforation were evaluated and recorded. These findings included (a) visualization of the appendix, (b) appendiceal location, (c) appendiceal diameter, (d) relative appendiceal enhancement compared with adjacent bowel enhancement (ie, enhancement less than, equal to, or greater than that of the adjacent bowel or no enhancement), (e) focal defect in the enhancing appendiceal wall, (f) the presence and degree of periappendiceal inflammatory stranding, (g) the presence of discrete periappendiceal fluid and other abdominal fluid collections, (h) the presence of an abscess, (i) the presence of appendicolith(s), extraluminal air, cecal inflammation, and mesenteric lymphadenopathy, and (j) subjective assessment of bowel dilatation.

Appendiceal location was classified as abdominal if the appendix was anterior to the plane of the iliac vessels, retrocecal if the appendix was posterior or posterolateral to the cecum, or pelvic if the appendix extended posterior to the plane of the iliac vessels. The presence and degree of periappendiceal inflammatory stranding were classified as absent, mild (ie, just perceptible haziness or increased attenuation in the mesoappendix or retroperitoneal fat), moderate, or severe, with progressively more severe changes. The presence of discrete periappendiceal fluid was defined as perceptible fluid collections without enhancing appendiceal walls. The presence of an abscess was defined as a discrete fluid collection or as an air and fluid collection with an enhancing appendiceal wall. Cecal inflammation was classified as absent, mild (ie, just perceptible cecal wall thickening at the cecal apex), moderate, or severe, with progressively more severe changes. Mesenteric lymphadenopathy was defined as one or more nodes larger than 1.0 cm in short axis diameter or as a cluster of four or more nodes of any size in the right lower quadrant.

Diagnostic confidence for both appendicitis and appendiceal perforation was separately recorded as either not present, possible, probable, or definite; diagnostic confidence was based on the overall assessment of findings as recorded first by the individual reviewers and then later by the consensus panel. The findings were then combined into two categories for statistical analysis. Images were considered not to show perforation if the independent reviewers or consensus group classified the findings of perforation as "not present" or "only possibly present." Images were considered to show perforation if the independent reviewers or consensus group classified the findings of perforation as "moderately present" or "definitely present." Comments regarding other observations, including diagnostic quality, were also recorded.

Medical Records
The medical records of the patients in the study were reviewed jointly and retrospectively (T.A.F., F.E.). The prospective reports were reviewed for the diagnosis of appendicitis and perforation. The diagnosis of acute appendicitis without perforation, acute appendicitis with perforation, or no appendicitis was determined from surgical and pathology reports. The interval from CT to the beginning of the surgical procedure was determined from the time recorded on the CT images and from the time recorded on the anesthesia record. The length of hospital stay following surgery was obtained from the hospital record.

Statistical Analysis
Statistical analyses were performed by using a commercially available statistical software program (SAS, version 8.2; SAS Institute, Cary, NC). Descriptive statistics were reported as means and standard deviations, and continuous variables were reported as medians and ranges. Categoric variables were reported as frequencies and percentages.

Sensitivity and specificity for the diagnosis of perforation were estimated by using surgical findings and pathologic diagnosis as the reference standard. Estimates were reported with 95% exact binomial confidence intervals. Interobserver agreement was assessed by using simple {kappa} statistics for binary CT signs, such as presence or absence of abscess. Weighted {kappa} statistics were used to assess agreement between readers for CT variables collected by using ordinal scales. Simple and weighted {kappa} statistics were interpreted by using the following scale: fair agreement, 0.21–0.40; moderate agreement, 0.41–0.60; substantial agreement, 0.61–0.80; and almost perfect agreement, 0.81–1.00 (12). Ninety-five percent confidence intervals were reported for each estimated {kappa} value.

The association between each CT sign and perforated appendicitis was assessed by using {chi}2 or Fisher exact tests, as appropriate. For these analyses, the response was perforation (yes or no), as determined according to surgical or pathologic findings. The independent variables of interest were CT signs that were based on the consensus interpretation. A multiple logistic regression model was used to identify independent predictors of perforation. Both stepwise and backward model selection methods were applied. The final logistic regression results were reported with odds ratios and 95% confidence intervals in addition to P values.

The association between perforation and length of hospital stay, as well as between CT signs and length of hospital stay, was assessed by using Wilcoxon rank sum tests. This analysis was performed for all patients, and a separate analysis was performed for those without perforation.

The 56 patients in the study group were compared with the 52 patients who underwent appendectomy but were excluded because they did not undergo CT. These comparisons were performed by using {chi}2 tests for binary variables (ie, sex and perforation) and Wilcoxon rank sum tests for continuous variables (ie, age and length of hospital stay). A P value of less than .05 was considered to indicate a statistically significant difference for all analyses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All reviewed images were considered to be of diagnostic quality. Diagnosis, sex, and age of the 86 patients are given in Table 1. The 52 patients who were excluded from the study did not differ with respect to age (P = .15), sex (P = .96), perforation rate (P = .66), or length of hospital stay (P = .98) when compared with patients who were included in the study. The appendix was identified on CT images in 81 of 86 patients and was not identified in five patients.


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TABLE 1. Sex and Age Characteristics of Study Population

 
Review of the prospective CT diagnosis of appendicitis demonstrated a sensitivity of 92%. Sensitivity and specificity for the diagnosis of perforation by retrospective review are shown in Table 2. The sensitivity and specificity for each CT finding that helped differentiate nonperforated appendicitis or no appendicitis from perforated appendicitis are listed in Table 3. Six findings, including extraluminal air, moderate or marked inflammatory stranding, focal enhancement defect, the presence of periappendiceal fluid collections, the presence of appendicolith, and cecal inflammation, demonstrated a statistically significant association with the reference standard (P <.05) (Table 4). The stepwise and backward logistic regression models each included only extraluminal air and the presence of moderate or marked periappendiceal inflammatory stranding (both as binary variables) as significant predictors of perforation. When included together in the model, extraluminal air had an estimated odds ratio of 11.5 (95% confidence interval: 2.0, 65.7; P = .006), and inflammatory stranding had an estimated odds ratio of 5.2 (95% confidence interval: 1.5, 17.9; P = .01).


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TABLE 2. Sensitivity and Specificity Analysis for Reader Diagnosis of Appendiceal Perforation

 

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TABLE 3. Consensus Review for the Presence of CT Signs and Appendiceal Perforation

 

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TABLE 4. Association of CT Findings at Consensus Review with Appendiceal Perforation

 
Extraluminal air was the most specific sign of perforated appendicitis (Fig 1), with only two false-positive findings. One false-positive finding was likely related to partial volume averaging (Fig 2), and the other was associated with a peridiverticular abscess (Fig 3). Of the four patients who were believed to have an abscess at consensus review, one patient had an abscess on the right side of the pelvis owing to perforated diverticulitis (Fig 3), and another patient had no abscess identified at appendectomy (Fig 4).



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Figure 1. Transverse CT image of an 18-year-old man with perforated appendicitis, an extruded appendicolith (black arrow), and an abscess containing air and fluid (arrowheads). Laparoscopic appendectomy demonstrated perforated appendicitis, with a collection of purulent fluid in the pelvis. Patient was discharged after a 6-day hospital stay.

 


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Figure 2a. (a, b) Transverse CT images of a 48-year-old woman with a dilated appendix (arrowheads) extending into the pelvis, with minimal periappendiceal inflammatory fat stranding. Eccentric gas collections (arrow) on nonadjacent images were interpreted as extraluminal air. Acute appendicitis was identified at appendectomy without any evidence of perforation. Patient was discharged after a 3-day hospital stay.

 


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Figure 2b. (a, b) Transverse CT images of a 48-year-old woman with a dilated appendix (arrowheads) extending into the pelvis, with minimal periappendiceal inflammatory fat stranding. Eccentric gas collections (arrow) on nonadjacent images were interpreted as extraluminal air. Acute appendicitis was identified at appendectomy without any evidence of perforation. Patient was discharged after a 3-day hospital stay.

 


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Figure 3. Transverse CT image of a 58-year-old man with a peripherally enhancing abscess containing fluid and gas bubbles located on the right side of the pelvis (black arrowheads). There are multiple loops of dilated small bowel caused by obstruction (white arrowheads). The appendix was not identified at CT. A peridiverticular abscess was found at surgery. Appendix was normal. Patient was discharged after an 8-day hospital stay.

 


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Figure 4a. (a, b) Transverse CT images of a 42-year-old woman with a mildly distended enhancing appendix (arrow), distal appendiceal periinflammatory changes, and a small periappendiceal abscess (arrowhead). The appendix was not perforated at surgery, and no periappendiceal abscess was described. Patient was discharged after a 2-day hospital stay.

 


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Figure 4b. (a, b) Transverse CT images of a 42-year-old woman with a mildly distended enhancing appendix (arrow), distal appendiceal periinflammatory changes, and a small periappendiceal abscess (arrowhead). The appendix was not perforated at surgery, and no periappendiceal abscess was described. Patient was discharged after a 2-day hospital stay.

 
A focal defect in the enhancing appendiceal wall was found in 10 of 17 patients with perforated appendicitis (Fig 5) who underwent contrast material–enhanced CT. This finding demonstrated less specificity than extraluminal air or abscess, with a focal defect being identified on consensus review in eight patients without perforation at appendectomy (Fig 6). The mean diameter of normal and nonperforated appendices was 10.6 mm (range, 5–24 mm), and the mean diameter of perforated appendices was 12.5 mm (range, 7–17 mm). Although the difference in the mean diameters was statistically significant (P = .02), the difference was not clinically helpful in differentiating perforated appendicitis. There was no statistically significant association between appendiceal perforation and the location of the appendix, the presence of mesenteric adenopathy, relative appendiceal enhancement, or the presence of small-bowel dilatation.



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Figure 5. Transverse CT image of a 48-year-old woman with decreased focal enhancement along the lateral wall of the distended appendix and moderate periappendiceal inflammatory changes (arrow). A dilated perforated appendix was resected; purulent material and fecal spillage were noted in the right lower quadrant. The peritoneal cavity was copiously irrigated, and drains were placed. Patient was discharged after a 7-day hospital stay.

 


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Figure 6. Transverse CT image of a 16-year-old girl with decreased focal enhancement along the anterior wall (arrow) of the distended appendix and moderate periappendiceal inflammatory changes (arrowheads). Appendicitis without perforation was identified at appendectomy. Patient was discharged after a 2-day hospital stay.

 
Interobserver agreement for the diagnosis of perforation and for individual CT findings is shown in Table 5. There was moderate or substantial agreement ({kappa} = 0.40–0.80) comparing the observations of the independent observers with consensus review for most CT findings. There was less consistent interobserver agreement regarding the differentiation of fluid collection and abscess, the grading of relative appendiceal enhancement, and the identification of a focal defect in the appendiceal wall.


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TABLE 5. Agreement among Readers for the Diagnosis of Appendiceal Perforation and the Presence of CT Signs

 
The time from CT to surgery compared with the time of surgical or pathologic diagnosis of perforation is depicted in Table 6. Mean time from CT to surgery was 260 minutes for patients with perforated appendicitis (median, 232 minutes; range, 32–734 minutes) and 269 minutes for patients with nonperforated appendicitis (median, 193 minutes; range, 51–1285 minutes). The surgical or pathologic diagnosis of perforation, as well as five CT findings, was associated with a statistically significant increase in the length of hospital stay (P < .05) for the entire cohort (Table 7). The same analysis of only those patients without perforation (Table 8) demonstrated a significant association between increased hospital stay and the presence of extraluminal air, periappendiceal inflammatory stranding, and focal enhancement defect (P < .05).


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TABLE 6. Time Interval from CT Examination to Surgery as Relates to the Diagnosis of Appendiceal Perforation

 

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TABLE 7. Association of Diagnosis and CT Findings with Length of Hospital Stay

 

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TABLE 8. Association of CT Findings with Length of Hospital Stay in Patients without Appendiceal Perforation

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early observations of the use of CT suggested an important role for imaging in the diagnosis and management of appendicitis (13,14). CT is now established as the imaging modality of choice for the evaluation of acute abdominal pain as the result of suspected appendicitis in adults (6,15). The use of CT for the evaluation of suspected appendicitis has reduced the number of false-positive diagnoses of appendicitis, with decreased negative appendectomy rates, particularly in women (5,1618). The use of CT may also reduce the use of hospital resources (19).

Optimal therapy and surgical approach for patients with perforated appendicitis are less clear. Surgical morbidity and the incidence of complications are increased in patients with perforated appendicitis. Laparoscopic appendectomy becomes more difficult after perforation and may necessitate conversion to an open appendectomy procedure (10,20). The presence of perforation may also influence the surgeon’s choice of incision for those patients undergoing appendectomy. Antibiotic therapy, possibly with percutaneous abscess drainage, and interval appendectomy may be an alternative treatment for patients who have perforated appendicitis and a discrete abscess (21). These factors make the preoperative diagnosis of perforation more critical to treatment planning.

Although contrast-enhanced CT of the abdomen and pelvis has been demonstrated to improve the diagnosis of appendicitis and to facilitate the differentiation of appendicitis from other causes of acute abdominal pain (22,23), the reliability of CT signs in differentiating perforated from nonperforated appendicitis is less clear. There are few studies that have specifically focused on imaging findings and the diagnosis of perforated appendicitis (9,11). In these studies, researchers retrospectively reviewed images from selected patient examinations performed by using older CT technology and collected over several years. Findings from both studies (9,11) indicated a higher proportion of perforations than have been observed in large epidemiologic studies (1,3,24) or in our current study.

In a single-observer study, Horrow et al (11) emphasized a constellation of findings that were helpful in the diagnosis of appendiceal perforation, including the observation of a focal defect in the enhancing appendiceal wall. Definitive findings of abscess with or without extraluminal air or of an extraluminal appendicolith were specific but not sensitive. The sensitivity of these findings is further diminished as the rate of perforation and abscess has decreased in the scanned patient population at risk for appendicitis. Other findings by Horrow et al (11), such as phlegmon (defined as both periappendiceal inflammatory stranding and fluid), increased sensitivity but decreased specificity for the diagnosis of perforation. Our findings show that CT signs demonstrate only moderate sensitivity in depicting appendiceal perforation.

In the current study, findings from CT examinations performed in the emergency department in a consecutive series of patients validate the use of several signs, such as extraluminal air and abscess formation, that are specific for perforation as previously reported in other studies. These findings were observed in only 13 of 86 patients. Moderate or marked periappendiceal inflammation and a focal defect in the appendiceal wall were frequently associated with perforation, but these signs were also observed in a substantial number of patients without surgical or pathologic findings of perforation. There was less consistent agreement between the blinded observers for differentiation between periappendiceal fluid and abscess and for identification of a focal defect in the appendiceal wall. During the consensus review, we found that the identification of a focal defect in the enhancing appendiceal wall required great care to avoid misinterpretation of partial volume averaging, particularly when the appendix was parallel or oblique to the scanning plane. There was poor interobserver agreement between the two blinded observers for specifying the degree of relative appendiceal enhancement. The finding of increased relative appendiceal enhancement proved to be subjective depending on the bowel segment that was used as the reference standard, and, even after consensus review, this finding was not significantly associated with perforation.

We correlated the individual CT findings with the length of hospital stay and found a significant association between extraluminal air, periappendiceal inflammation, focal defect in the appendiceal wall, the presence of an abscess, and periappendiceal and mesenteric fluid collections and an increased hospital stay (P < .05). In patients without appendiceal perforation, the association between increased hospital stay and extraluminal air, periappendiceal inflammation, and focal defect in the enhancing appendiceal wall suggests either that CT signs of inflammation have greater relative importance as indicators of increased hospital stay or that the presence of small perforations went undetected during surgical or pathologic examination in a subset of patients with significant findings of periappendiceal inflammation.

Limitations of this study include those associated with patient selection, analysis criteria, and the reference standard. Only consecutive patients who underwent an emergency department evaluation for abdominal symptoms, a CT examination ordered from the emergency department and performed emergently, and subsequent appendectomy within a 24-hour period were included in our sample. Patients who had appendicitis with or without perforation, who underwent surgery more than 24 hours after CT, or who did not undergo emergent CT or surgery were excluded. There is an inherent verification bias in the analysis of the accuracy of prospective diagnosis of appendicitis, but the focus of our retrospective analysis was appendiceal perforation. Comparison between our patient group and the patients who did not fulfill the inclusion criteria but who had undergone appendectomy did not reveal any selection bias. Analysis criteria were more precise for appendiceal location, luminal diameter of the appendix, and mesenteric lymphadenopathy. Criteria for grading periappendiceal inflammatory changes, classifying the relative enhancement of the appendix compared with that of the adjacent bowel, or grading bowel dilatation were more subjective. The reference standard of surgical or pathologic verification of appendiceal perforation was subject to the completeness of the surgical observation and surgical notes, as well as to the thoroughness of pathologic evaluation of the appendix specimen and the completeness of the pathology report. Because there were only 21 patients with appendiceal perforation, statistical power was limited for developing a multivariate model to predict perforation on the basis of CT signs.

In conclusion, the identification of an abscess or extraluminal air is a specific but insensitive finding in patients with perforated appendicitis. The presence of moderate or marked periappendiceal inflammatory changes in the mesoappendix and adjacent retroperitoneal fat and a focal defect in the enhancing appendiceal wall are more sensitive but less specific signs. Great care must be taken to avoid the misinterpretation of partial volume averaging as a focal enhancement defect in the appendiceal wall. CT signs of substantial appendiceal inflammation are independent predictors of increased hospital stay.


    FOOTNOTES
 
Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, T.A.F., F.E.; study concepts, all authors; study design, T.A.F., F.E.; literature research, T.A.F., F.E.; clinical studies, F.E., M.A.N., D.M.H.; data acquisition, T.A.F., F.E., M.A.N., D.M.H.; data analysis/interpretation, all authors; statistical analysis, T.A.F., F.E., T.L.H.; manuscript preparation, T.A.F., F.E., T.L.H.; manuscript definition of intellectual content, editing, revision/review, and final version approval, all authors


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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