Accuracy of the stool antigen test for the diagnosis of childhood Helicobacter pylori infection

THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2003 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.

Vol. 98, No. 2, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(02)05931-2

Accuracy of the Stool Antigen Test for the Diagnosis of Childhood Helicobacter pylori Infection: A Multicenter Japanese Study Seiichi Kato, M.D., Kyoko Ozawa, M.D., Masumi Okuda, M.D., Takuji Fujisawa, M.D., Seiichi Kagimoto, M.D., Mutsuko Konno, M.D., Shunichi Maisawa, M.D., and Kazuie Iinuma, M.D. Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan; Department of Pediatrics, Wakayama Rosai Hospital, Wakayama, Japan; Fujisawa Children’s Clinic, Takamatsu, Japan; Division of Infectious Disease, Immunology, and Allergy, Saitama Children’s Medical Center, Saitama, Japan; Department of Pediatrics, Sapporo Kousei General Hospital, Sapporo, Japan; and Morioka Children’s Hospital, Morioka, Japan.

OBJECTIVE: The 13C-urea breath test (UBT) has been accepted as a reliable noninvasive test for detecting Helicobacter pylori infection. Recently, another noninvasive test, a new enzyme immunoassay for H. pylori antigens in stool, has been widely investigated for its clinical usefulness. The purpose of this multicenter study was to evaluate the diagnostic accuracy of the stool antigen test in Japanese children. METHODS: A total of 264 children (148 male and 116 female; mean age 9.2 yr, range 2–17 yr) who underwent 13 C-UBT and the stool antigen test were studied. The diagnosis in these patients was gastritis (n ⫽ 49), gastric ulcer (n ⫽ 4), duodenal ulcer (n ⫽ 24), recurrent abdominal pain (n ⫽ 43), and other conditions (n ⫽ 144). The stool antigen test was performed using the HpSA ELISA (Premier Platinum HpSA, Meridian Diagnostics). According to manufacturer’s instructions, an absorbance at 450/630 nm of ⬍0.100, ⱖ0.120, and 0.100 – 0.119 was defined as negative, positive, and indeterminate, respectively. Based on the 13C-UBT with a cutoff value of 3.5 per mil, the performance of HpSA was studied. In 21 patients who received eradication therapy, the HpSA was performed at baseline and at 1, 2, and 6 months after completion of therapy. Eradication of H. pylori was confirmed by 13C-UBT at 2 or 3 months of follow-up. RESULTS: 13C-UBT showed that 76 children were infected with H. pylori and 188 were not infected. In these same children, HpSA results were positive in 77 children, negative in 183, and indeterminate in four. The overall sensitivity, specificity, and accuracy of the test were 96.0% (95% CI ⫽ 88.6 –99.2%), 96.8% (95% CI ⫽ 94.2–99.3%), and 96.5% (95% CI ⫽ 94.3–98.8%), respectively. There were no significant differences in these results among age groups of ⱕ5, 6 –10, and ⱖ11 yr. Receiver operating characteristic curve analysis demonstrated that the best cutoff value of absorbance at 450/630 nm was 0.110. When a single cutoff value of 0.110 without indeterminate results was used, the

sensitivity, specificity, and accuracy were 96.1% (95% CI ⫽ 90.8 –99.7%), 96.3% (95% CI ⫽ 93.6 –99.0%), and 96.2% (95% CI ⫽ 93.9 –98.5%), respectively. In 19 patients in whom H. pylori was successfully eradicated, HpSA results were negative at 1 month of follow-up and remained negative through 6 months. CONCLUSIONS: The HpSA is an accurate test for the detection of H. pylori infection in all age groups of children. (Am J Gastroenterol 2003;98:296 –300. © 2003 by Am. Coll. of Gastroenterology)

INTRODUCTION Helicobacter pylori infection is associated with the pathogenesis of chronic gastritis, gastric and duodenal ulcers, and gastric cancer in a subset of infected individuals. Recently, its association with some extra-GI diseases such as iron deficiency anemia, short stature, or chronic urticaria is under investigation (1). At present, invasive biopsy tests, including histology, a urease test and biological culture, and noninvasive 13C-urea breath test (UBT) have been accepted as reliable tests for H. pylori infection. Among noninvasive tests, serology is less reliable for active H. pylori infection (2, 3). 13C-UBT has high diagnostic accuracy in children (4 – 6); however, it is not easy to perform the test in children who do not ingest 13C-urea or in whom the collection of exhaled breath is difficult because of age or because of mental or physical disturbances (7). Recently, a new enzyme immunoassay for the detection of H. pylori antigen in stool has been developed (8). In 1998, the United States Food and Drug Administration (FDA) approved the stool antigen test (HpSA) for both diagnosing H. pylori infection and monitoring eradication efficacy in adults (9). A meta-analysis in adults reported that the mean of sensitivity and specificity of the stool antigen test are 93.1% and 92.8%, respectively, and that the test is comparable to 13C-UBT (3). The European Helicobacter pylori

AJG – February, 2003

Stool Antigen Test for Childhood H. pylori

Table 1. Baseline Characteristics of Children Studied H. pylori Status Characteristic

Infected

Not Infected

Total

Number of patients Sex (male/female) Mean age (yr) Range Disease Gastritis Gastric ulcer Duodenal ulcer Recurrent abdominal pain Others‡

76 45/31* 9.7† 3–16

188 103/85* 8.9† 2–17

264 148/116 9.2 2–17

24 2 10 6 34

25 2 14 37 110

49 4 24 43 144

* p ⫽ 0.26 for male to female ratio between infected and noninfected children. † p ⫽ 0.16 for mean age between infected and noninfected children. ‡ Others include asymptomatic, healthy children, and diseases such as iron deficiency anemia or rheumatoid arthritis.

Study Group has recommended the stool antigen test and 13 C-UBT for diagnosis of the infection and eradication assessment. However, some authors have indicated a low sensitivity (10) or specificity of the test in adults (11, 12). Moreover, the test is under debate with regard to monitoring eradication efficacy (7, 8, 11–15). In the pediatric population, there are several studies on the anti–H. pylori polyclonal capture antibodies (2, 16 –20) and the monoclonal antibody (20), suggesting that the stool antigen test is a potent diagnostic tool. However, there is no agreement about its best cutoff value (12). Commercially available kits sometimes provide different diagnostic accuracies in different patient populations or geographic regions (21). Makristathis et al. have stressed the importance of local test validation (20). In addition, usefulness of the test for younger children is controversial (17–19). In this prospective study, we investigated the diagnostic accuracy of the stool antigen test in Japanese children and evaluated the usefulness of this test for H. pylori eradication.

MATERIALS AND METHODS Study Patients This multicenter open study was conducted at 12 pediatric institutions (see Acknowledgments). A total of 264 children (148 male and 116 female, mean age 9.2 yr, range 2–17 yr) who underwent 13C-UBT and stool antigen testing were prospectively studied (Table 1). Among these children, 84 underwent upper GI endoscopy because of abdominal symptoms. Besides those with gastritis, gastric ulcer, duodenal ulcer, and recurrent abdominal pain, the population included asymptomatic healthy children (n ⫽ 105) and those with various disorders such as iron deficiency anemia (n ⫽ 6) and rheumatoid arthritis (n ⫽ 4). Children who had received antibiotics, histamine-2 blockers, or proton pump inhibitors within 4 wk before the performance of H. pylori tests were excluded from this study. A total of 21 patients with a diagnosis of H. pylori– associated chronic gastritis (n ⫽ 17), gastric ulcer (n ⫽ 1),

297

and duodenal ulcer (n ⫽ 3) received eradication therapy. The eradication regimen consisted of a proton pump inhibitor (omeprazole or lansoprazole), amoxicillin, and clarithromycin (22, 23). Each investigator was free to choose the duration of therapy ranging between 7 and 14 days. Eradication of H. pylori was confirmed by 13C-UBT at 2 or 3 months after the completion of the therapy. Informed consent was obtained from the children or their parents at each institution. 13

C-Urea Breath Test The test protocol has been reported previously (4). In brief, 13 C-labeled urea with a maximal dose of 100 mg was administered without a test meal after at least 4 h fasting. The breath samples were obtained at baseline and 20 min and were analyzed by nondispersive infrared spectrophotometry (UBiT-IR 300, Otsuka Pharmaceutical, Tokyo, Japan), and values were expressed as ⌬ 13C (per mil). A cutoff value of ⌬ 13C was defined as 3.5 per mil. The sensitivity and specificity of the test against biopsy tests were 97.8% and 98.5%, respectively (4). HpSA The stool samples were obtained within 1 day before or after 13 C-UBT was done. In 21 patients who received eradication therapy, stool samples were serially collected at baseline and at 1, 2, and 6 months after the therapy. The samples were stored at ⫺20°C until the assay. As reported previously (13), the samples were measured at one center using an enzyme immunoassay kit (Premier Platinum HpSA, Meridian Diagnostics, Cincinnati, OH) in blinded fashion concerning clinical data. The results were spectrophotometrically read at dual wavelengths of 450/630 nm. According to manufacturer’s instructions, an absorbance at 450/630 nm of ⬍0.100, ⱖ0.120, and 0.100 – 0.119 was defined as negative, positive, and indeterminate, respectively. The performance of HpSA was calculated against the results of 13C-UBT. Statistical Analysis A difference in the male-to-female ratio between H. pylori– infected and noninfected children was assessed using the ␹2 test. The two-tailed t test was used to assess a difference in the mean age between both groups. Differences in the performances of the HpSA among three age groups were analyzed by ␹2 test. A difference of absorbance values at 450/630 nm between H. pylori–infected and noninfected children was examined using the Mann-Whiteney U test. An analysis with a receiver operating characteristic curve was performed, as previously reported (24). A p value of ⬍ 0.05 was regarded as statistically significant. Data are presented as mean ⫾ SD.

RESULTS Based on the results of the 13C-UBT, 76 children were found to be infected with H. pylori and 188 were not infected (Table 1). In these same children, the HpSA was positive in 77 children and negative in 183; results were indeterminate

298

Kato et al.

AJG – Vol. 98, No. 2, 2003

Figure 1. Distribution of values of absorbance (OD) at 450/630 nm in children with negative and positive results of the 13C-urea breath test (UBT). Bars represent mean values; broken lines denote indeterminate zone ranging between absorbance values of 0.100 and 0.119.

in four children (1.5%). The mean absorbance value at 450/630 nm in 13C-UBT-positive and negative groups was 0.046 ⫾ 0.039 and 1.110 ⫾ 0.827, respectively (p ⬍ 0.001) (Fig. 1). In children with a positive 13C-UBT, there was no significant correlation between values of HpSA and age (r ⫽ ⫺0.07, p ⫽ 0.53). Table 2 lists the results for overall sensitivity, specificity, positive- and negative-predictive values, and accuracy of HpSA against 13C-UBT. Among age groups of ⱕ5, 6 –10, and ⱖ11 yr, there were no statistically significant differences in these results (p values not shown). The sensitivity in patients with gastritis, gastroduodenal ulcer, and recurrent abdominal pain was 95.5% (95% CI ⫽ 77.2–99.9%), 90.0% (95% CI ⫽ 55.5–99.7%), and 83.3% (95% CI ⫽ 35.9 –99.6%), respectively, and the specificity in these patients was 92.0% (95% CI ⫽ 74.0 –99.0%), 93.8%

Figure 2. Receiver operating characteristic curve for the HpSA against the 13C-urea breath test, demonstrating absorbance at 450/ 630 nm of 0.110 as the best single cutoff value.

(95% CI ⫽ 69.8 –99.8%), and 97.3% (95% CI ⫽ 85.8 – 99.9%), respectively. An analysis with the receiver operating characteristic curve demonstrated that the best cutoff value of absorbance at 450/630 nm was 0.110 (Fig. 2). When a single cutoff value of 0.110 without indeterminate results was used, the sensitivity, specificity, positive- and negative-predictive values, and accuracy of the test were 96.1% (95% CI ⫽ 90.8 –99.7%), 96.3% (95% CI ⫽ 93.6 –99.0%), 91.3% (95% CI ⫽ 82.8 –96.4%), 98.4% (95% CI ⫽ 96.5–100%), and 96.2% (95% CI ⫽ 93.9 –98.5%), respectively. Discordant cases of 13C-UBT and HpSA, including indeterminate results, are summarized in Table 3. Among four cases with indeterminate results of HpSA, stool samples of two cases were reanalyzed; one sample (from patient 11) showed a negative result with an absorbance value of 0.061, and one sample (from patient 12) showed a positive result with an absorbance of 0.247. Two cases (patients 10 and 13) were not reanalyzed for lack of stool samples.

Table 2. Performance Results of the HpSA Test, by Age Age (yr)

No. of Patients

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

Accuracy (%)

2–5

67

6–10

80

11–17

113

Overall

260

92.9 (66.1–99.8) 90.0 (68.3–98.8) 100 (91.2–100) 96.0 (88.6–99.2)

96.2 (87.0–99.5) 98.3 (91.1–100) 95.9 (88.5–99.1) 96.8 (94.2–99.3)

86.7 (59.5–98.3) 94.7 (74.0–99.9) 93.0 (80.9–98.5) 92.2 (83.8–97.1)

98.1 (89.7–100) 96.7 (88.7–99.6) 100 (94.9–100) 98.4 (96.5–100)

95.5 (87.5–99.1) 96.3 (89.4–99.2) 97.4 (94.4–100) 96.5 (94.3–98.8)

NPV ⫽ negative predictive value; PPV ⫽ positive predictive value. Data in parentheses represent 95% CI. There were no significant differences in the results among the three age groups.

AJG – February, 2003

Stool Antigen Test for Childhood H. pylori

Table 3. Summary of Cases With Disconcordant Results of

13

C-Urea Breath Test (UBT) and HpSA

13

Patient No.*

Age (yr)/ Sex

Diagnosis

1 2 3 4 5 6 7 8 9 10 11 12 13

2/M 10/M 11/M 12/M 14/F 5/M 3/M 8/F 10/M 15/F 12/M 12/M 14/M

Recurrent abdominal pain Gastritis Gastritis Asymptomatic Juvenile rheumatoid arthritis Gastric ulcer Recurrent abdominal pain Gastritis Duodenal ulcer Gastritis Recurrent abdominal pain Duodenal ulcer Gastric ulcer

299

C-UBT (%) 1.8 2.9 0.1 2.1 2.7 2.0 9.6 4.7 3.8 0.1 0.1 21.6 3.8

HpSA (Absorbance at 450/630 nm)

Histology

Urease Test

Culture

0.125 0.137 0.152 0.307 0.354 2.147 0.042 0.068 0.068 0.104 0.110 0.111 0.111

ND ND ND ND ND ND ND ⫹ ⫺ ⫺ ⫺ ND ⫹

ND ND ND ND ND ⫺ ND ⫹ ⫺ ⫺ ⫺ ND ND

ND ND ⫹ ND ND ⫺ ND ND ⫺ ⫺ ⫺ ND ⫹

Biopsy Test

F ⫽ female; M ⫽ male; ND ⫽ not done; ⫹ ⫽ positive; ⫺ ⫽ negative. * Patients 1– 6, 7–9, and 10 –13 are cases with false-positive, false-negative, and indeterminate results, respectively.

Follow-up 13C-UBT confirmed that H. pylori was eradicated in 19 patients and was not eradicated in two. In all 19 patients with successful eradication, HpSA was negative at 1 month of follow-up and continued to be negative through 6 months (Fig. 3). In 10 patients studied, HpSA was already negative at 2 wk after the completion of therapy. On the other hand, in two patients with eradication failure, HpSA was persistently positive during the follow-up periods.

DISCUSSION This study has demonstrated excellent results of the HpSA in children in all age groups. Several studies suggested that the HpSA is a reliable method in preschool children including toddlers (18, 19), although Braden et al. reported a relatively low sensitivity (83%) in children younger than 6 yr (17). The test would cross-react with antigens of other

Figure 3. Follow-up study of the HpSA after eradication therapy in 21 patients. Closed circles represent patients with successful eradication (n ⫽ 19); open circles represent those with failed eradication (n ⫽ 2). mo ⫽ months; OD ⫽ absorbance.

Helicobacter species, such as H. heilmannii and H. pullorum (12); however, the clinical significance of these bacteria is small. On the other hand, serology has a low sensitivity in younger children, given that immunological response to H. pylori is immature (2). In addition, serum antibodies, both IgG and IgA classes, continue to be positive for several months after the organism has been eradicated (25), leading to decreased specificity. Assessment of eradication efficacy with the HpSA is controversial. Studies have shown that the test is useful for monitoring eradication in adults (7, 13–15) and in children (20, 26), although there are reports to the contrary (8, 11, 12). The HpSA may show false-negative results shortly after completion of the therapy, because of a transiently decreased bacterial load (26). Furthermore, the HpSA might detect antigen profiles of both bacillary and coccoid forms of H. pylori (20, 27). On the other hand, replacement of gastric epithelial cells takes ⬍1 wk (28). This study demonstrated that the HpSA exactly reflects H. pylori status at 1 month of follow-up (even at 2 wk), although the number of patients is small. Although it seems that success or failure of H. pylori eradication can be confirmed by the HpSA shortly after therapy, the optimal timing of test performance remains to be determined. There is no consensus of opinion as to what the cutoff value of the HpSA should be (12). In general, the test is spectrophotometrically measured with either single or dual wavelengths. Oderda et al. reported that the accuracy of the test is sufficient with direct visual reading of the microwells, without use of a plate reader (16). In our experience, however, it was not easy to judge borderline coloring by direct visual reading. In this study, indeterminate absorbance values ranging between 0.110 and 0.119, a gray zone, was set up. However, cases with a gray zone were rare in our study and previous studies (13, 18). It is thought that the HpSA can be performed with a single cutoff value, without a gray zone. However, absorbance values around the cutoff used should be interpreted with caution.

300

Kato et al.

The HpSA is inexpensive and requires no specialized test equipment. Consequently, it is possible to perform the test at any medical center. Furthermore, as stool samples can be stored frozen, the test is also suitable for epidemiological studies. As a noninvasive test, the HpSA could be used in all age groups of children.

AJG – Vol. 98, No. 2, 2003

11. 12.

ACKNOWLEDGMENTS This study was performed by the Japanese Pediatric HpSA Study Group. Participating institutions and investigators were as follows: Tohoku University Hospital (Seiichi Kato, Kyoko Ozawa, Takanori Minoura, and Kazuie Iinuma); Sapporo Kousei General Hospital (Mutsuko Konno); Iwate Prefectural Central Hospital (Daiki Abukawa); Morioka Children’s Hospital (Shunichi Maisawa); Saitama Children’s Medical Center (Seiichi Kagimoto); Juntedo University Hospital (Toshiaki Shimizu); Nippon Medical School (Takeshi Matsuhisa); Kanagawa Prefecture Midwives and Nurses Training Hospital (Shigeru Toyoda); Osaka University Hospital (Hitoshi Tajiri and Atsushi Sawada); Osaka Medical Center for Maternal and Child Health (Norikazu Yoshimura and Shinobu Ida); Wakayama Rosai Hospital (Masumi Okuda); and Fujisawa Children’s Clinic (Takuji Fujisawa). Reprint requests and correspondence: Seiichi Kato, M.D., Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Received Apr. 30, 2002; accepted Aug. 19, 2002.

13. 14.

15.

16.

17.

18.

19.

REFERENCES 1. Leontiadis GI, Sharma VK, Howden CW. Non-gastrointestinal tract associations of Helicobacter pylori infection. Arch Intern Med 1999;159:925–40. 2. Ni YH, Lin JT, Huang SF, et al. Accurate diagnosis of Helicobacter pylori infection by stool antigen test and 6 other currently available tests in children. J Pediatr 2000;136:823–7. 3. Vaira D, Vakil N. Blood, urine, stool, breath, money, and Helicobacter pylori. Gut 2001;48:287–9. 4. Kato S, Ozawa K, Konno M, et al. Diagnostic accuracy of 13 C-urea breath test for childhood Helicobacter pylori infection: A multicenter Japanese study. Am J Gastroenterol 2002; 97:1668 –73. 5. Vandenplas Y, Blecker U, Devreker T, et al. Contribution of the 13C-urea breath test to detection of Helicobacter pylori gastritis in children. Pediatrics 1992;90:608 –11. 6. Bazzoli F, Cecchini L, Corvaglia L, et al. Validation of the 13 C-urea breath test for the diagnosis of Helicobacter pylori infection in children: A multicenter study. Am J Gastroenterol 2000;95:646 –50. 7. Ishihara S, Kaji T, Kawamura A, et al. Diagnostic accuracy of a new non-invasive enzyme immunoassay for detecting Helicobacter pylori in stools after eradication therapy. Aliment Pharmacol Ther 2000;14:611–4. 8. Makristathis A, Pasching E, Schutze K, et al. Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay. J Clin Microbiol 1998;36:2772–4. 9. Vakil N, Affi A, Robinson J, et al. Prospective blinded trial of a fecal antigen test for the detection of Helicobacter pylori infection. Am J Gastroenterol 2000;95:1699 –701. 10. Puspok A, Bakos S, Oberhuber G. A new, non-invasive

20.

21. 22. 23. 24. 25.

26. 27.

28.

method for detection of Helicobacter pylori: Validity in the routine clinical setting. Eur J Gastroenterol Hepatol 1999;11: 1139 –42. Trevisani L, Sartori S, Galvani F, et al. Evaluation of a new enzyme immunoassay for detecting Helicobacter pylori in feces: A prospective pilot study. Am J Gastroenterol 1999;94:1830 –3. Forne M, Dominguez J, Fernandez-Banares F, et al. Accuracy of an enzyme immunoassay for the detection of Helicobacter pylori in stool specimens in the diagnosis of infection and posttreatment check-up. Am J Gastroenterol 2000;95:2200 –5. Vaira D, Malfertheiner P, Me´ graud F, et al. Diagnosis of Helicobacter pylori infection with a new non-invasive antigen-based assay. Lancet 1999;354:30 –3. Vaira D, Malfertheiner P, Me´ graud F, et al. Noninvasive antigen-based assay for assessing Helicobacter pylori eradication: A European multicenter study. Am J Gastroenterol 2000;95:925–9. Braden B, Teuber G, Dietrich CF, et al. Comparison of new faecal antigen test with 13C-urea breath test for detecting Helicobacter pylori infection and monitoring eradication treatment: Prospective clinical evaluation. Br Med J 2000;320:148. Oderda G, Rapa A, Ronchi B, et al. Detection of Helicobacter pylori in stool specimens by non-invasive antigen enzyme immunoassay in children: Multicenter Italian study. Br Med J 2000;320:347–8. Braden B, Posselt HG, Ahrens P, et al. New immunoassay in stool provides an accurate noninvasive diagnostic method for Helicobacter pylori screening in children. Pediatrics 2000; 106:115–7. Konstantopoulos N, Russmann H, Tasch C, et al. Evaluation of the Helicobacter pylori stool antigen test (HpSA) for detection of Helicobacter pylori infection in children. Am J Gastroenterol 2001;96:677–83. van Doorn DJ, Bosman DK, van Hoff BW, et al. Helicobacter pylori stool antigen test: A reliable non-invasive test for the diagnosis of Helicobacter pylori infection in children. Eur J Gastroenterol Hepatol 2001;13:1061–5. Makristathis A, Barousch W, Pasching E, et al. Two enzyme immunoassays and PCR for detection of Helicobacter pylori in stool specimens from pediatric patients before and after eradication therapy. J Clin Microbiol 2000;38:3710 –4. Ohkura R, Miwa H, Murai T, et al. Usefulness of a novel enzyme immunoassay for the detection of Helicobacter pylori in feces. Scand J Gastroenterol 2000;35:49 –53. Kato S, Takeyama J, Ebina K, Naganuma H. Omeprazolebased dual and triple regimens for Helicobacter pylori eradication in children. Pediatrics 1997;100:E3. Kato S, Ritsuno H, Ohnuma K, et al. Safety and efficacy of one-week triple therapy for eradicating Helicobacter pylori in children. Helicobacter 1998;3:278 –82. Swets JA. Measuring the accuracy of diagnostic systems. Science 1988;240:1285–93. Kato S, Furuyama N, Ozawa K, et al. Long-term follow-up study of serum immunoglobulin G and immunoglobulin A antibodies after Helicobacter pylori eradication. Pediatrics 1999;104:E22. Oderda G, Rapa A, Marinello D, et al. Usefulness of Helicobacter pylori stool antigen test to monitor response to eradication treatment in children. Aliment Pharmacol Ther 2001;15:203–6. Kuster JG, Gerrits MM, van Strijp JAG, VandenbrouckeGrauls CMJE. Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 1997; 65:3672–9. MacDonald WC, Trier JS, Everett NB. Cell proliferation and migration in the stomach, duodenum, and rectum of man. Gastroenterology 1964;46:405–17.