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Evaluation of the Helicobacter pylori stool antigen test for monitoring eradication therapy
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2002 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 97, No. 3, 2002 ISSN 0002-9270/02/$22.00 PII S0002-9270(01)04097-7
Evaluation of the Helicobacter pylori Stool Antigen Test for Monitoring Eradication Therapy Takeo Odaka, M.D., Taketo Yamaguchi, Ph.D., Hidehiko Koyama, M.D., Hiromitsu Saisho, Ph.D., and Fumio Nomura, Ph.D. First Department of Medicine and Department of Laboratory and Clinical Medicine, School of Medicine, Chiba University, Chiba; and Department of Medicine, Social Insurance Funabashi Central Hospital, Funabashi, Japan
OBJECTIVE: We aimed to clarify the time course of the Helicobacter pylori stool antigen (HpSA) level during and after eradication therapy and to determine the appropriate time of measurement of HpSA to evaluate eradication. METHODS: The subjects were 47 patients who were positive for H. pylori. The patients underwent a 1-wk regimen of triple therapy. The outcome of the eradication therapy was judged by urea breath test, culture, and histology 6 wk after the end of treatment. The HpSA level was serially measured nine times from before therapy until 12 wk after the end of therapy. RESULTS: In the group with successful eradication, HpSA became negative immediately after the end of therapy and the negativity persisted. In contrast, in the noneradication group HpSA became negative immediately after therapy, but became positive again within 2 wk after the end of therapy. The mean absorbance value of the HpSA test on the 4th day after the initiation of eradication therapy was significantly higher in the noneradication group than in the group with successful eradication. The diagnostic accuracy of the HpSA test increased to ⭌90% 2 wk after therapy and thereafter. Comparison of the diagnostic accuracy at 4 wk after the end of therapy showed no significant differences with that at 2, 6, 8, and 12 wk after the end of therapy. CONCLUSIONS: The course of the HpSA levels during and after eradication therapy in patients with successful eradication was quite different from that in noneradication patients. Measurement of HpSA was useful to evaluate eradication, and the appropriate evaluation of the outcome of treatment could be made as early as 2 wk after the end of therapy. (Am J Gastroenterol 2002;97:594 –599. © 2002 by Am. Coll. of Gastroenterology)
INTRODUCTION Infection of the gastric mucosa with Helicobacter pylori causes chronic atrophic gastritis and peptic ulcer of the duodenum and stomach, and is also responsible for the development in the atrophic gastric mucosa (1–3). There are several methods of diagnosing H. pylori infec-
tion: invasive procedures using mucosa biopsied by endoscopy and noninvasive procedures without using endoscopy. Biopsy-based tests suffer from sampling errors, and only the local infection status in the stomach is observed. Two representative noninvasive tests are the detection of antibodies against H. pylori in serum or urine and the urea breath test (UBT). The serological tests are useful for diagnosing the presence of H. pylori before therapy. However, it is necessary to monitor the antibody titer for several months to accurately evaluate the outcome of eradication (4). The UBT allows a highly accurate diagnosis of H. pylori infection, and has been regarded as the golden standard to evaluate eradication (5–7). More recently, the H. pylori stool antigen (HpSA) test has been put on the market as another noninvasive technique. This new method is simple and does not require a technician or expensive equipment. Several reports indicate that diagnostic usefulness of the HpSA test is equivalent to that of the UBT in the pretreatment setting (8 –12). It has been shown that this test is useful not only for detecting H. pylori infection but also as a marker of the outcome of eradication treatment (13–16). The optimal time for testing HpSA to accurately evaluate the outcome of eradication treatment remains to be clarified, however. Therefore, we monitored the HpSA levels during and after eradication treatment in 47 patients in a prospective manner.
MATERIALS AND METHODS Patients A total of 47 patients with H. pylori infection participated in this study. There were 38 men and nine women, whose ages ranged from 18 to 67 yr (mean ⫽ 54.3). All patients were clinically diagnosed by upper GI endoscopy, and duodenal ulcer, gastric ulcer, gastroduodenal ulcer, and gastritis were observed in 17, 22, six, and two patients, respectively. None of the patients had been treated with proton pump inhibitors or antibiotics during the 2 months before endoscopic examination. Four of the 47 patients were lost to follow-up. Thus, the outcome of eradication was evaluated in 43 patients.
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Figure 1. Study design. RUT ⫽ rapid urease test; SA ⫽ HpSA.
Because the medical cost of H. pylori eradication therapy is covered by Japanese national health insurance only in patients with peptic ulcer, the majority of the patients enrolled had peptic ulcer. The study was explained to all participants before the start of eradication therapy, and the therapy and measurement of HpSA were performed after written informed consent was obtained. Diagnosis and Eradication Therapy In all patients, serum antibody tests (ImmunoCard, Meridian Diagnostics, Cincinnati, OH) and endoscopic examinations of the upper GI tract were performed before eradication therapy. During endoscopic examination, two specimens each were obtained from the antrum and the corpus of the stomach using a biopsy forceps, and H. pylori infection was examined by culture (selective blood agar) and histology (Giemsa stain). Furthermore, an additional specimen was biopsied from the corpus of the stomach, and the rapid urease test (PyloriTek, Serim Research, Elkhart, IN) was performed. Regardless of positivity or negativity on the culture test, when positivity was obtained in two of the three
tests (histology, rapid urease, and serum antibody) the patient was diagnosed as being H. pylori positive. As eradication therapy, the patients were administered a new regime consisting of triple therapy with 60 mg of lansoprazole, 1500 mg of amoxycillin, and 400 mg of clarithromycin, daily for 1 wk. Eradication was evaluated 6 wk after the end of the eradication therapy. The UBT, culture test, and histological examination were performed, and when H. pylori was negative in all tests, eradication was judged successful and test results were expressed as negative. When positivity was obtained on any one of these tests, eradication was judged unsuccessful and test results were indicated as positive. 13
C Urea Breath Test The 13C UBT was performed as previously reported (17). Briefly, the patients were asked to fast beginning the night before the test, and underwent the test in the morning before breakfast. In 100 ml of water, 100 mg of 13C urea was dissolved. The patients ingested the reagent and then thoroughly washed their mouths with water. The patients were in a sitting position during the test. Expired air was collected
Figure 2. Changes of the HpSA level in cases of successful eradication.
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Figure 3. Changes of the HpSA level in cases of successful eradication. Data presented in Figure 2 are shown with different scales, magnification of A values of 0.2 or lower.
before and 20 min after ingestion of the reagent. When ⌬13C value was 5‰ or higher, the test was judged positive. HpSA Assay To determine H. pylori antigen in feces, the HpSA assay kit (Meridian Diagnostics) was used. A fecal sample, 5 mm in diameter, was suspended in 200 l of diluting solution; 50 l of the diluted sample was dripped on microwells coated with rabbit anti–H. pylori antibody; and peroxidase-labeled anti–H. pylori antibody was added. The microwells were incubated for 1 h. After the microwells were washed well to remove nonreacted substances, the substrates (urea peroxide and tetramethylbenzidine) were added and the wells were reacted for 10 min. The absorbance (450/630 nm) of the color developed was measured using a microplate reader. All of these processes were performed at room temperature.
The results were judged according to the assessment criteria established by the manufacturer (A ⫽ 450/630 nm: positive ⭌ 0.12, 0.12 ⬎ equivocal ⭌ 0.10, negative ⬍ 0.10). HpSA was assayed at nine timepoints: before, during (on the 4th day after initiation of eradication therapy), immediately after the end of (on the next day after the end of eradication therapy), and 1, 2, 4, 6, 8, and 12 wk after the end of eradication therapy. For sampling, about 5 ml of feces excreted in the morning was collected in a highly closed container and immediately frozen at ⫺80°C. A schematic diagram of the whole study is shown in Figure 1. Statistical Analysis The A values of the HpSA test carried out on the 4th day after the initiation of eradication therapy for successfully
Figure 4. Changes of the HpSA level in cases of unsuccessful eradication.
94.1% (86.2–100%)
95.3% (89.0–100%)
90.9% (81.1–100%)
92.9% (85.1–100%) 93.0% (85.4–100%) 78.6% (66.2–91.0%) 76.2% (63.3–89.1%)
90.5% (81.4–99.4%)
94.1% (86.2–100%) 93.9% (85.7–100%) 97.0% (91.2–100%)
90.9% (81.1–100%)
95.3% (89.0–100%)
9 2 32 0 0 100.0% (66.4–100%)
97.1% (91.5–100%)
12 wk
9 3 30 0 1 100.0% (66.4–100%)
6 wk
8 1 33 1 0 88.9% (51.8–99.7%) 8 2 32 1 0 88.9% (51.8–99.7%)
4 wk 2 wk
8 3 30 1 1 88.9% (51.8–99.7%) 2 2 31 7 1 22.2% (2.8–60.0%)
1 wk 0 wk
0 1 32 9 1 0.0% (0–33.6%)
True positive False positive True negative False negative Equivocal Sensitivity (95% CI) Specificity (95% CI) Accuracy (95% CI)
Table 1. Diagnostic Usefulness of the HpSA Assay at Each Timepoint After Eradication Therapy
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eradicated and noneradicated cases were compared using Student’s t test. Sensitivity, specificity, and accuracy of the HpSA assay and 95% CIs of these items were calculated at each assay point by Ilstrup’s method (18). Furthermore, the receiver operating characteristic (ROC) curves were drawn at each assay point using Hanley’s method (19) to obtain the appropriate timing for the HpSA assay to evaluate eradication.
RESULTS Out of 47 patients defined as H. pylori positive, HpSA was positive in 45 in the pretreatment setting; the sensitivity was 95.7%. According to the above-mentioned criteria, eradication was successful in 34 and unsuccessful in nine; the success rate was 79.1%. In patients with gastric and/or duodenal ulcers who underwent successful eradication, the ulcer was in the active or healing stage before eradication therapy, and it had become a scar at the assessment of eradication in all patients. The time course of HpSA levels before, during, and after eradication treatment in 34 patients with successful eradication is illustrated in Figures 2 and 3 using two different scales. In the group with successful eradication, the high pretreatment values rapidly decreased upon initiation of therapy; HpSA tests became negative immediately after the end of therapy in 32 patients (94.1%). During the 12-wk follow-up period, HpSA tests transiently became positive in eight patients, but A values remained in the negative range in 26 other cases. Twelve weeks after the end of therapy, 33 were negative (97.1%) and one was false positive. In this positive patient, the A value 12 wk after the end of therapy was 0.138 (weakly positive), but both the UBT and the HpSA were negative in this case up to 24 wk after the end of therapy. The time course of HpSA levels in nine noneradication patients is shown in Figure 4. The A values decreased below the cutoff value at the end of therapy in all nine patients, as in the group with successful eradication. However, they rebounded and eventually exceeded the cutoff value in all nine patients. The rate of increase of the A values after the cessation of treatment and rebounded levels differed from case to case. The mean A values of the HpSA test on the 4th day after the initiation of eradication therapy were 0.363 ⫾ 0.509 and 0.911 ⫾ 0.863 for successfully eradicated and noneradicated cases, respectively. There was a significant difference between these two values (p ⫽ 0.018). The 95% CIs of sensitivity, specificity, and accuracy of the HpSA assay after eradication therapy are shown in Table 1. All patients in whom eradication was not successful presented false-negative results immediately after the end of therapy; the sensitivity was 0%. The sensitivity was 22.2% 1 wk after eradication therapy, and the overall accuracy was low (78.6%). After 2 wk, false negativity markedly decreased and the sensitivity increased; the overall accuracies
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Figure 5. Comparison of the ROC curve of the HpSA test and the AUC at each assay point. n.s. ⫽ no significance vs 4 wk. *p ⬍ 0.05 vs 4 wk; **p ⬍ 0.01 vs 4 wk.
2, 4, and 6 wk after the end of therapy were 90.5%, 93.0%, and 95.3%, respectively. Figure 5 shows the ROC curves obtained 0, 1, 2, 4, 6, 8, and 12 wk after the end of therapy. Sensitivity was based on nine noneradication patients and specificity was determined from the results of 34 successfully eradicated cases. The areas under the curves (AUCs) are also presented. With the AUC of the ROC curve 4 wk after the end of therapy regarded as the standard, the AUCs of the ROC curves immediately after and 1 wk after the end of therapy were significantly lower than that of the standard ROC curve, but there were no significant differences at 2, 6, 8, or 12 wk after the end of therapy. It is noteworthy that the AUC at 2 wk did not significantly differ from that at 4 wk.
DISCUSSION A novel enzyme immunoassay to detect H. pylori antigens in stools has recently been put on the market (8). This noninvasive test does not require expensive equipment and is relatively cheap. The diagnostic value of the HpSA test in the pretreatment setting is well recognized (8 –12). An increasing number of reports have indicated that HpSA would be useful not only to screen H. pylori infection, but also to monitor the outcome of eradication therapy (13–16). The optimal time to measure HpSA to judge whether eradication was successful or not has not been determined yet. The initial report by Vaira et al. (13) indicated that the HpSA test provided results equivalent to that of the UBT
when assessed 4 wk after the end of eradication treatment. This sample point, 4 wk after the end of therapy, appears to be the standard point to reassess H. pylori infection by the HpSA test in the posttreatment setting. Makristathis et al. (20), however, suggested that 1 month may be too short a period for follow-up evaluation of stool specimens by enzyme immunoassay. Moreover, Forne´ et al. (21) reported that the HpSA test lacks accuracy as compared to the UBT in evaluating the outcome of the therapy at 6 wk and 6 months after treatment. On the other hand, Oderda et al. (16) reported that HpSA was accurate to monitor treatment in children at both 2 and 6 wk after treatment. The first objective of this study was to know the appropriate time to carry out the HpSA test when one wants to judge whether eradication therapy was successful or not. We determined, in a prospective manner, serial changes of HpSA levels during an entire course of eradication therapy in patients with peptic ulcer up to 12 wk after the end of treatment. We found that the HpSA test could predict the outcome of eradication therapy as early as 2 wk after the end of treatment. It is worth noting that HpSA levels fluctuated even in patients with eventual eradication during the course, and in eight patients out of 34, HpSAs transiently became weakly positive. It is likely that the fluctuation in the test results was due to day-to-day variation of the HpSA assay, indicating that serial measurements to obtain a time course of HpSA levels rather than a single determination may be important in evaluating the outcome of eradication therapy. Alternatively, there is a possibility of a cross-reaction with
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Helicobacter bacteria other than H. pylori. This is, however, unlikely. The second objective of this study was to examine serial changes of HpSA levels during and after eradication therapy in a group of patients who failed to respond to eradication therapy. HpSA levels immediately after the end of therapy were below the cutoff value in all of these cases; the false negative rate was 100%. It is noteworthy, however, that the A values in these noneradication patients during therapy (on the 4th day) were significantly higher than those in patients with successful eradication. HpSA levels during eradication therapy might be a rough indicator of the outcome of treatment. Further studies are required to clarify this point. HpSA levels once below the cutoff levels in patients without successful eradication soon became positive after the end of therapy. The interval between the end of therapy and the point HpSA reappeared differed from case to case (1– 8 wk), but the A values were higher than the cutoff value at 2 wk after treatment in eight out of nine patients. ROC analysis confirmed that the overall performance of the HpSA test at 2 wk after treatment as an indicator of the outcome of eradication therapy was comparable to that at 4 and 6 wk. From these results, we conclude that the HpSA test has a diagnostic value comparable to that of the UBT in monitoring eradication therapy and that the judgment of treatment outcome can be done as early as 2 wk after treatment.
The HpSA Test and Monitoring Eradication Therapy
7.
8. 9. 10. 11.
12.
13.
14.
15. Reprint requests and correspondence: Takeo Odaka, M.D., First Department of Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Tyuo-ku, Chiba city, Japan 260-8677. Received Mar. 29, 2001; accepted Oct. 30, 2001.
REFERENCES 1. Kuipers EJ, Uyterlinde AM, Pena AS, et al. Long term sequelae of Helicobacter pylori gastritis. Lancet 1995;345: 1525– 8. 2. Marshall BJ. Helicobacter pylori. The etiologic agent for peptic ulcer. JAMA 1995;274:1064 – 6. 3. International Agency for Research on Cancer, World Health Organization. Schistosomes, liver flukes and Helicobacter pylori. Lyon: IARC, 1994:177–202. 4. Cutler AF, Prasad VM. Long term follow-up of Helicobacter pylori serology after successful eradication. Am J Gastroenterol 1996;91:85– 8. 5. Atherton JC, Spiller RC. The urea breath test for Helicobacter pylori. Gut 1994;35:723–5. 6. Cutler AF, Havstad S, Ma CK, et al. Accuracy of invasive and
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non-invasive tests to diagnose Helicobacter pylori infection. Gastroenterology 1995;109:136 – 41. Rollan A, Giancaspero R, Arrese M, et al. Accuracy of invasive and non-invasive tests to diagnose Helicobacter pylori infection after antibiotics treatment. Am J Gastroenterol 1997; 92:1268 –74. Vaira D, Malfertheiner P, Megraud F, et al. Diagnosis of Helicobacter pylori infection with new non-invasive antigen based assay. Lancet 1999;354:30 –3. 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. Ohkura R, Miwa H, Murai A, et al. Usefulness of a novel enzyme immunoassay for the detection of Helicobacter pylori in feces. Scand J Gastroenterol 2000;35:49 –53. Oderda G, Rapa A, Ronchi B, et al. Detection of Helicobacter pylori in stool specimens by non-invasive antigen enzyme immunoassay in children: Multicentre Italian study. BMJ 2000;320:347– 8. Husson MO, Rolland C, Gottrand F, et al. Evaluation of a Helicobacter pylori stool antigen test for the diagnosis and follow-up of infections in children. Eur J Clin Microbiol Infect Dis 2000;19:787–9. Vaira D, Malfertheiner P, Megraud F, et al. Noninvasive antigen-based assay for assessing Helicobacter pylori eradication: A European multicenter study. Am J Gastroenterol 2000;95:925–9. Ishihara I, Kaji T, Kawamura A, et al. Diagnostic accuracy of a new non-invasive enzyme immunoassay for detection of Helicobacter pylori in stool after eradication therapy. Aliment Pharmacol Ther 2000;14:611– 4. 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. BMJ 2000;320:148. 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. Miwa H, Murai T, Ohkura R, et al. Usefulness of the [13C]urea breath test for detection of Helicobacter pylori infection in fasting patients. J Gastroenterol Hepatol 1998;13:1039 – 43. Ilstrup DM. Statistical methods in microbiology. Clin Microb Rev 1990;3:219 –26. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 1982;143:29 –36. 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. Forne´ M, Domı´nguez J, Ferna´ ndez-Ban˜ ares 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.
Vol. 97, No. 3, 2002 ISSN 0002-9270/02/$22.00 PII S0002-9270(01)04097-7
Evaluation of the Helicobacter pylori Stool Antigen Test for Monitoring Eradication Therapy Takeo Odaka, M.D., Taketo Yamaguchi, Ph.D., Hidehiko Koyama, M.D., Hiromitsu Saisho, Ph.D., and Fumio Nomura, Ph.D. First Department of Medicine and Department of Laboratory and Clinical Medicine, School of Medicine, Chiba University, Chiba; and Department of Medicine, Social Insurance Funabashi Central Hospital, Funabashi, Japan
OBJECTIVE: We aimed to clarify the time course of the Helicobacter pylori stool antigen (HpSA) level during and after eradication therapy and to determine the appropriate time of measurement of HpSA to evaluate eradication. METHODS: The subjects were 47 patients who were positive for H. pylori. The patients underwent a 1-wk regimen of triple therapy. The outcome of the eradication therapy was judged by urea breath test, culture, and histology 6 wk after the end of treatment. The HpSA level was serially measured nine times from before therapy until 12 wk after the end of therapy. RESULTS: In the group with successful eradication, HpSA became negative immediately after the end of therapy and the negativity persisted. In contrast, in the noneradication group HpSA became negative immediately after therapy, but became positive again within 2 wk after the end of therapy. The mean absorbance value of the HpSA test on the 4th day after the initiation of eradication therapy was significantly higher in the noneradication group than in the group with successful eradication. The diagnostic accuracy of the HpSA test increased to ⭌90% 2 wk after therapy and thereafter. Comparison of the diagnostic accuracy at 4 wk after the end of therapy showed no significant differences with that at 2, 6, 8, and 12 wk after the end of therapy. CONCLUSIONS: The course of the HpSA levels during and after eradication therapy in patients with successful eradication was quite different from that in noneradication patients. Measurement of HpSA was useful to evaluate eradication, and the appropriate evaluation of the outcome of treatment could be made as early as 2 wk after the end of therapy. (Am J Gastroenterol 2002;97:594 –599. © 2002 by Am. Coll. of Gastroenterology)
INTRODUCTION Infection of the gastric mucosa with Helicobacter pylori causes chronic atrophic gastritis and peptic ulcer of the duodenum and stomach, and is also responsible for the development in the atrophic gastric mucosa (1–3). There are several methods of diagnosing H. pylori infec-
tion: invasive procedures using mucosa biopsied by endoscopy and noninvasive procedures without using endoscopy. Biopsy-based tests suffer from sampling errors, and only the local infection status in the stomach is observed. Two representative noninvasive tests are the detection of antibodies against H. pylori in serum or urine and the urea breath test (UBT). The serological tests are useful for diagnosing the presence of H. pylori before therapy. However, it is necessary to monitor the antibody titer for several months to accurately evaluate the outcome of eradication (4). The UBT allows a highly accurate diagnosis of H. pylori infection, and has been regarded as the golden standard to evaluate eradication (5–7). More recently, the H. pylori stool antigen (HpSA) test has been put on the market as another noninvasive technique. This new method is simple and does not require a technician or expensive equipment. Several reports indicate that diagnostic usefulness of the HpSA test is equivalent to that of the UBT in the pretreatment setting (8 –12). It has been shown that this test is useful not only for detecting H. pylori infection but also as a marker of the outcome of eradication treatment (13–16). The optimal time for testing HpSA to accurately evaluate the outcome of eradication treatment remains to be clarified, however. Therefore, we monitored the HpSA levels during and after eradication treatment in 47 patients in a prospective manner.
MATERIALS AND METHODS Patients A total of 47 patients with H. pylori infection participated in this study. There were 38 men and nine women, whose ages ranged from 18 to 67 yr (mean ⫽ 54.3). All patients were clinically diagnosed by upper GI endoscopy, and duodenal ulcer, gastric ulcer, gastroduodenal ulcer, and gastritis were observed in 17, 22, six, and two patients, respectively. None of the patients had been treated with proton pump inhibitors or antibiotics during the 2 months before endoscopic examination. Four of the 47 patients were lost to follow-up. Thus, the outcome of eradication was evaluated in 43 patients.
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Figure 1. Study design. RUT ⫽ rapid urease test; SA ⫽ HpSA.
Because the medical cost of H. pylori eradication therapy is covered by Japanese national health insurance only in patients with peptic ulcer, the majority of the patients enrolled had peptic ulcer. The study was explained to all participants before the start of eradication therapy, and the therapy and measurement of HpSA were performed after written informed consent was obtained. Diagnosis and Eradication Therapy In all patients, serum antibody tests (ImmunoCard, Meridian Diagnostics, Cincinnati, OH) and endoscopic examinations of the upper GI tract were performed before eradication therapy. During endoscopic examination, two specimens each were obtained from the antrum and the corpus of the stomach using a biopsy forceps, and H. pylori infection was examined by culture (selective blood agar) and histology (Giemsa stain). Furthermore, an additional specimen was biopsied from the corpus of the stomach, and the rapid urease test (PyloriTek, Serim Research, Elkhart, IN) was performed. Regardless of positivity or negativity on the culture test, when positivity was obtained in two of the three
tests (histology, rapid urease, and serum antibody) the patient was diagnosed as being H. pylori positive. As eradication therapy, the patients were administered a new regime consisting of triple therapy with 60 mg of lansoprazole, 1500 mg of amoxycillin, and 400 mg of clarithromycin, daily for 1 wk. Eradication was evaluated 6 wk after the end of the eradication therapy. The UBT, culture test, and histological examination were performed, and when H. pylori was negative in all tests, eradication was judged successful and test results were expressed as negative. When positivity was obtained on any one of these tests, eradication was judged unsuccessful and test results were indicated as positive. 13
C Urea Breath Test The 13C UBT was performed as previously reported (17). Briefly, the patients were asked to fast beginning the night before the test, and underwent the test in the morning before breakfast. In 100 ml of water, 100 mg of 13C urea was dissolved. The patients ingested the reagent and then thoroughly washed their mouths with water. The patients were in a sitting position during the test. Expired air was collected
Figure 2. Changes of the HpSA level in cases of successful eradication.
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Figure 3. Changes of the HpSA level in cases of successful eradication. Data presented in Figure 2 are shown with different scales, magnification of A values of 0.2 or lower.
before and 20 min after ingestion of the reagent. When ⌬13C value was 5‰ or higher, the test was judged positive. HpSA Assay To determine H. pylori antigen in feces, the HpSA assay kit (Meridian Diagnostics) was used. A fecal sample, 5 mm in diameter, was suspended in 200 l of diluting solution; 50 l of the diluted sample was dripped on microwells coated with rabbit anti–H. pylori antibody; and peroxidase-labeled anti–H. pylori antibody was added. The microwells were incubated for 1 h. After the microwells were washed well to remove nonreacted substances, the substrates (urea peroxide and tetramethylbenzidine) were added and the wells were reacted for 10 min. The absorbance (450/630 nm) of the color developed was measured using a microplate reader. All of these processes were performed at room temperature.
The results were judged according to the assessment criteria established by the manufacturer (A ⫽ 450/630 nm: positive ⭌ 0.12, 0.12 ⬎ equivocal ⭌ 0.10, negative ⬍ 0.10). HpSA was assayed at nine timepoints: before, during (on the 4th day after initiation of eradication therapy), immediately after the end of (on the next day after the end of eradication therapy), and 1, 2, 4, 6, 8, and 12 wk after the end of eradication therapy. For sampling, about 5 ml of feces excreted in the morning was collected in a highly closed container and immediately frozen at ⫺80°C. A schematic diagram of the whole study is shown in Figure 1. Statistical Analysis The A values of the HpSA test carried out on the 4th day after the initiation of eradication therapy for successfully
Figure 4. Changes of the HpSA level in cases of unsuccessful eradication.
94.1% (86.2–100%)
95.3% (89.0–100%)
90.9% (81.1–100%)
92.9% (85.1–100%) 93.0% (85.4–100%) 78.6% (66.2–91.0%) 76.2% (63.3–89.1%)
90.5% (81.4–99.4%)
94.1% (86.2–100%) 93.9% (85.7–100%) 97.0% (91.2–100%)
90.9% (81.1–100%)
95.3% (89.0–100%)
9 2 32 0 0 100.0% (66.4–100%)
97.1% (91.5–100%)
12 wk
9 3 30 0 1 100.0% (66.4–100%)
6 wk
8 1 33 1 0 88.9% (51.8–99.7%) 8 2 32 1 0 88.9% (51.8–99.7%)
4 wk 2 wk
8 3 30 1 1 88.9% (51.8–99.7%) 2 2 31 7 1 22.2% (2.8–60.0%)
1 wk 0 wk
0 1 32 9 1 0.0% (0–33.6%)
True positive False positive True negative False negative Equivocal Sensitivity (95% CI) Specificity (95% CI) Accuracy (95% CI)
Table 1. Diagnostic Usefulness of the HpSA Assay at Each Timepoint After Eradication Therapy
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eradicated and noneradicated cases were compared using Student’s t test. Sensitivity, specificity, and accuracy of the HpSA assay and 95% CIs of these items were calculated at each assay point by Ilstrup’s method (18). Furthermore, the receiver operating characteristic (ROC) curves were drawn at each assay point using Hanley’s method (19) to obtain the appropriate timing for the HpSA assay to evaluate eradication.
RESULTS Out of 47 patients defined as H. pylori positive, HpSA was positive in 45 in the pretreatment setting; the sensitivity was 95.7%. According to the above-mentioned criteria, eradication was successful in 34 and unsuccessful in nine; the success rate was 79.1%. In patients with gastric and/or duodenal ulcers who underwent successful eradication, the ulcer was in the active or healing stage before eradication therapy, and it had become a scar at the assessment of eradication in all patients. The time course of HpSA levels before, during, and after eradication treatment in 34 patients with successful eradication is illustrated in Figures 2 and 3 using two different scales. In the group with successful eradication, the high pretreatment values rapidly decreased upon initiation of therapy; HpSA tests became negative immediately after the end of therapy in 32 patients (94.1%). During the 12-wk follow-up period, HpSA tests transiently became positive in eight patients, but A values remained in the negative range in 26 other cases. Twelve weeks after the end of therapy, 33 were negative (97.1%) and one was false positive. In this positive patient, the A value 12 wk after the end of therapy was 0.138 (weakly positive), but both the UBT and the HpSA were negative in this case up to 24 wk after the end of therapy. The time course of HpSA levels in nine noneradication patients is shown in Figure 4. The A values decreased below the cutoff value at the end of therapy in all nine patients, as in the group with successful eradication. However, they rebounded and eventually exceeded the cutoff value in all nine patients. The rate of increase of the A values after the cessation of treatment and rebounded levels differed from case to case. The mean A values of the HpSA test on the 4th day after the initiation of eradication therapy were 0.363 ⫾ 0.509 and 0.911 ⫾ 0.863 for successfully eradicated and noneradicated cases, respectively. There was a significant difference between these two values (p ⫽ 0.018). The 95% CIs of sensitivity, specificity, and accuracy of the HpSA assay after eradication therapy are shown in Table 1. All patients in whom eradication was not successful presented false-negative results immediately after the end of therapy; the sensitivity was 0%. The sensitivity was 22.2% 1 wk after eradication therapy, and the overall accuracy was low (78.6%). After 2 wk, false negativity markedly decreased and the sensitivity increased; the overall accuracies
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Figure 5. Comparison of the ROC curve of the HpSA test and the AUC at each assay point. n.s. ⫽ no significance vs 4 wk. *p ⬍ 0.05 vs 4 wk; **p ⬍ 0.01 vs 4 wk.
2, 4, and 6 wk after the end of therapy were 90.5%, 93.0%, and 95.3%, respectively. Figure 5 shows the ROC curves obtained 0, 1, 2, 4, 6, 8, and 12 wk after the end of therapy. Sensitivity was based on nine noneradication patients and specificity was determined from the results of 34 successfully eradicated cases. The areas under the curves (AUCs) are also presented. With the AUC of the ROC curve 4 wk after the end of therapy regarded as the standard, the AUCs of the ROC curves immediately after and 1 wk after the end of therapy were significantly lower than that of the standard ROC curve, but there were no significant differences at 2, 6, 8, or 12 wk after the end of therapy. It is noteworthy that the AUC at 2 wk did not significantly differ from that at 4 wk.
DISCUSSION A novel enzyme immunoassay to detect H. pylori antigens in stools has recently been put on the market (8). This noninvasive test does not require expensive equipment and is relatively cheap. The diagnostic value of the HpSA test in the pretreatment setting is well recognized (8 –12). An increasing number of reports have indicated that HpSA would be useful not only to screen H. pylori infection, but also to monitor the outcome of eradication therapy (13–16). The optimal time to measure HpSA to judge whether eradication was successful or not has not been determined yet. The initial report by Vaira et al. (13) indicated that the HpSA test provided results equivalent to that of the UBT
when assessed 4 wk after the end of eradication treatment. This sample point, 4 wk after the end of therapy, appears to be the standard point to reassess H. pylori infection by the HpSA test in the posttreatment setting. Makristathis et al. (20), however, suggested that 1 month may be too short a period for follow-up evaluation of stool specimens by enzyme immunoassay. Moreover, Forne´ et al. (21) reported that the HpSA test lacks accuracy as compared to the UBT in evaluating the outcome of the therapy at 6 wk and 6 months after treatment. On the other hand, Oderda et al. (16) reported that HpSA was accurate to monitor treatment in children at both 2 and 6 wk after treatment. The first objective of this study was to know the appropriate time to carry out the HpSA test when one wants to judge whether eradication therapy was successful or not. We determined, in a prospective manner, serial changes of HpSA levels during an entire course of eradication therapy in patients with peptic ulcer up to 12 wk after the end of treatment. We found that the HpSA test could predict the outcome of eradication therapy as early as 2 wk after the end of treatment. It is worth noting that HpSA levels fluctuated even in patients with eventual eradication during the course, and in eight patients out of 34, HpSAs transiently became weakly positive. It is likely that the fluctuation in the test results was due to day-to-day variation of the HpSA assay, indicating that serial measurements to obtain a time course of HpSA levels rather than a single determination may be important in evaluating the outcome of eradication therapy. Alternatively, there is a possibility of a cross-reaction with
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Helicobacter bacteria other than H. pylori. This is, however, unlikely. The second objective of this study was to examine serial changes of HpSA levels during and after eradication therapy in a group of patients who failed to respond to eradication therapy. HpSA levels immediately after the end of therapy were below the cutoff value in all of these cases; the false negative rate was 100%. It is noteworthy, however, that the A values in these noneradication patients during therapy (on the 4th day) were significantly higher than those in patients with successful eradication. HpSA levels during eradication therapy might be a rough indicator of the outcome of treatment. Further studies are required to clarify this point. HpSA levels once below the cutoff levels in patients without successful eradication soon became positive after the end of therapy. The interval between the end of therapy and the point HpSA reappeared differed from case to case (1– 8 wk), but the A values were higher than the cutoff value at 2 wk after treatment in eight out of nine patients. ROC analysis confirmed that the overall performance of the HpSA test at 2 wk after treatment as an indicator of the outcome of eradication therapy was comparable to that at 4 and 6 wk. From these results, we conclude that the HpSA test has a diagnostic value comparable to that of the UBT in monitoring eradication therapy and that the judgment of treatment outcome can be done as early as 2 wk after treatment.
The HpSA Test and Monitoring Eradication Therapy
7.
8. 9. 10. 11.
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15. Reprint requests and correspondence: Takeo Odaka, M.D., First Department of Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Tyuo-ku, Chiba city, Japan 260-8677. Received Mar. 29, 2001; accepted Oct. 30, 2001.
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