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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References

Background:

Conventional 13C-urea breath testing (13C-UBT) includes a test meal to delay gastric emptying, which, theoretically, improves the accuracy of the test. Citric acid has been proposed as the best test meal. However, recent studies have suggested that a test meal may not be necessary.

Aim:

To investigate a new 13C-UBT protocol without a test meal in a Chinese population.

Methods:

Consecutive dyspeptic patients referred for upper endoscopy were recruited. 13C-UBT was performed on two separate days with or without a test meal (2.4 Gm citric acid) and compared with the ‘gold standard’ (CLO test and histology).

Results:

Two hundred and two patients were tested. Using receiver operating characteristics (ROC) analysis, the optimal delta-value and optimal measurement interval for UBT were 5% and 30 min, respectively, both with or without a test meal. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 13C-UBT with citric acid (96.5%, 97.7%, 98.2%, 95.6%, 97.0%) were similar to 13C-UBT without a test meal (94.7%, 97.7%, 98.2%, 93.5%, 96.0%).

Conclusion:

This simplified 13C-UBT protocol without a test meal produced highly accurate and reliable results in the Chinese population.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References

The 13C-urea breath test (13C-UBT) is a technically simple and noninvasive means of diagnosing Helicobacter pylori infection. It has been increasingly used in both hospital based and office based settings.1[2][3][4]–5 The reliability of the 13C-UBT in diagnosing H. pylori infection is very high, with a sensitivity of 90–98% and a specificity of 92–100%, respectively.6[7][8][9]–10 Since the first description of the 13C-UBT by Graham et al., several modifications have been published to simplify and optimize the test.5 The amount of substrate used, type of test meal, number of samples and the sampling time were the key variables being investigated.

The dose of 13C-urea varies among different investigators, from 75 mg to 350 mg. A dose of 125 mg 13C-urea was used in the validated US protocol11 and 100 mg 13C-urea was used in the standard European protocol.2 However, the 75 mg low dose protocol has been shown to be equally reliable.3, 7, 12[13][14][15][16]–17 The chief advantage is a lower cost of the test. The most commonly used cut-off value for the 75 mg 13C-UBT protocol between H. pylori infected subjects and H. pylori uninfected subjects was 4%.12 Although a lower cut-off value has been suggested by various studies, there is no universal agreement on the use of a lower cut-off value.2, 18 The commonly adopted sampling time is before and 30 min after the ingestion of 13C-urea, although the initial description of the test took breath samples every 10 min for 3 h.5 Conventional 13C-UBT uses a test meal to decrease gastric emptying and maximize the distribution of 13C-urea within the stomach. It allows a better and longer contact time between the bacteria and the substrate, and may improve the diagnostic accuracy of 13C-UBT by reflecting the urease activity of the whole stomach.5 Various liquid test meals have been proposed by several studies.1, 2, 8, 19 A citric acid drink has been recently proposed as the best liquid test meal. It delays gastric emptying and lowers the intragastric pH. It has better accuracy, palatability, and a lower cost when compared to other conventional test meals.12, 20, 21 However, recent studies suggested that 13C-UBT without a test meal may be equally reliable for the diagnosis of H. pylori infection.22, 23 Most of the published data about 13C-UBT have come from the Western population. Relatively few reports has been published assessing the use of 13C-UBT in a Chinese population. Thus the aims of this study were: (i) to investigate whether the low dose 75 mg 13C-urea protocol is useful in a Chinese population; (ii) to investigate the most reliable delta over baseline (DOB) cut-off value for Chinese population; (iii) to determine the optimal measurement interval for this low dose 13C-urea protocol; (iv) to investigate whether the same accuracy of 13C-urea breath can be achieved without a test meal.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References

Patient population

Two hundred and thirty patients referred to the endoscopy unit of the Department of Medicine, Queen Mary Hospital, for investigation of dyspepsia were recruited. Dyspepsia was defined as persistent or recurrent upper abdominal pain or discomfort over the preceding 3-month period. Informed written consent was obtained from all patients participating in the trial. Exclusion criteria included patients with previous gastric surgery or previous H. pylori eradication therapy and patients taking antibiotics, H2 receptor antagonists, bismuth compounds or proton pump inhibitors in the preceding 4 weeks before endoscopy.

Gastric biopsies

During upper endoscopy, three antral biopsies and two corpus biopsies were taken. One antral biopsy was used for rapid urease test (CLO test) and the rest were sent for histological examination of Helicobacter pylori status by haematoxylin and eosin stains and modified Giemsa staining if necessary. Specimens were examined by a pathologist specializing in gastroenterology who was blinded to all clinical information, including the CLO test results. The definition of H. pylori infection in this study required both CLO test and histology to be positive and was used as the ‘gold standard’ in this study. The absence of H. pylori infection required both tests to be negative. Equivocal cases (one test positive and the other test negative) were excluded from our analysis.

C13-urea breath test (13C-UBT) procedure

The 13C-UBTs, with or without a test meal, were performed in all patients after an overnight fast on two consecutive days. The test meal used was 200 mL 0.1 N (2.4 Gm) citric acid solution. Ten minutes after ingestion of the test meal, a baseline exhaled breath sample was collected in a vacutainer. Then 75 mg 13C-urea powder dissolved in 50 mL of water was given orally. Further breath samples were taken at 15, 30, 45 and 60 min (106 patients with baseline, 15 and 30 min only). All patients were kept in sitting position over the whole study period. Collected samples were analysed by the purpose built isotope ratio mass spectrometer in Simon KY Lee Digestive Disease Laboratory, Queen Mary Hospital, Hong Kong.

Results were expressed as the delta over baseline (DOB). The sensitivities, specificities, positive predictive values, negative predictive values and accuracies of the 13C-UBT with variation of the DOB cut-off levels were evaluated at different measurement intervals at 15, 30, 45 and 60 min. The appropriate cut-off points were obtained using receiver operating characteristics (ROC) curves for the 13C-UBT with test meal and 13C-UBT without test meal. The mean DOB with or without test meals at different time intervals was plotted to determine the peak DOB. The mean peak DOB and mean time to peak DOB with or without test meal were also determined. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the 13C-UBT using the best cut-off values with different sampling time were calculated.

Statistical analysis

The statistics used included a χ2-test and Fisher’s exact test when appropriate. A P-value of 0.05 or less was considered statistically significant. The sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy were calculated.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References

Two hundred and thirty patients were recruited into the study. Twenty-eight cases were excluded from the analysis. Five cases lacked histology data. Thirteen cases could not be classified according to our gold standard for the diagnosis of H. pylori infection, i.e. only one positive test out of the two. Ten cases had incomplete data during the breath test analysis. Thus, a total of 202 patients were available for analysis. The mean age of the 202 patients was 49 years (range 18–80) years. There were 90 males and 112 females. All the patients were ethnic Chinese. One hundred and fourteen patients (56%) were diagnosed as H. pylori positive by the gold standard. Among these 114 H. pylori positive patients, seven (6.1%) had gastric ulcer, 16 (14.0%) had duodenal ulcer, 10 had gastric and duodenal ulcers (8.8%), one had duodenitis (0.9%) and two had gastric polyps (1.8%).

Eighty-eight patients were H. pylori negative by our gold standard. Among these 88 patients, six had duodenal ulcer (6.8%), two had gastric and duodenal ulcers (2.3%), one had duodenitis (1.1%), one had gastric carcinoma (1.1%), one had oesophageal carcinoma (1.1%) and one had gastric polyps (1.1%). H. pylori status correlated strongly with the presence of peptic ulcer diseases (P < 0.001).

Citric acid as a test meal for 13C-UBT was well tolerated by all the patients. The accuracy of the 13C-UBT with variations in the DOB cut-off levels at 15, 30, 45 and 60 min are shown in Table 1. The cut-off values with the highest accuracy, including those both with and without test meals at 15, 30, 45 and 60 min were 6%, 5%, 3.5% and 3%, respectively. The best cut-off values were confirmed by ROC analysis (data not shown). The cut-off values with the highest accuracy were then used to calculate the sensitivity, specificity, positive predictive value and negative predictive value of the 13C-UBT at 15, 30, 45 and 60 min (Table 2). The 30 min measurement interval with the test meal gave the highest specificity, positive predictive value and accuracy for 13C-UBT at the cut-off value of 5%, and highest sensitivity and negative predictive value at a cut-off value of 4.5%. The 30 min measurement without a test meal gave the highest specificity and positive predictive value at both cut-off values of 4.5 and 5% (Table 2). The highest accuracy at 30 min measurement interval without test meal was 96% at cut-off values of 4%, 4.5% and 5%.

Table 1.  . Accuracies of the 13C-urea breath tests (with/without test meal) with different cut-off values at 15, 30, 45 and 60 min Thumbnail image of
Table 2.  . Sensitivities, specificities, positive and negative predictive values, and accuracies of 13C-urea breath test with or without test meals at different sampling time with the cut-off values determined by ROC analysis and the highest accuracy obtained from Table 1Thumbnail image of

The mean DOB with or without a test meal in H. pylori positive patients at different sampling times is plotted in Figure 1. There was no difference in the mean DOB at different sampling times with respect to the use of test meal or not. Furthermore, there was no difference in the mean peak DOB either with or without a test meal in H. pylori positive patients as well as in H. pylori negative patients. The mean time to peak DOB with or without test meal is shown in Figure 2. The mean time to peak DOB was significantly longer in H. pylori positive patients using citric acid as the test meal for 13C-UBT (25.3 min vs. 19.4 min, P=0.018). The use of a citric acid drink shifted the mean peak DOB towards the 30 min measurement interval.

image

Figure 1. .  Mean delta over baseline (DOB) with or without test meals in Helicobacter pylori positive subjects over 60 min. Solid line represents 13C-UBT with test meal and dotted line represents 13C-UBT without test meal.

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image

Figure 2. .  Mean time to peak delta over baseline (DOB) of 13C-UBT with or without test meals in Helicobacter pylori positive and negative subjects. (Column 1, 13C-UBTs with test meal in H. pylori positive subjects; 2, 13C-UBTs without test meal in H. pylori positive subjects; 3, 13C-UBTs with test meal in H. pylori negative subjects; and 4, 13C-UBTs without test meal in H. pylori negative subjects.) *P=0.018 for the comparison of time to peak DOB for 13C-UBT with test meal vs. without test meal in H. pylori positive subjects.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References

The 13C-UBT is one of the most important noninvasive methods for the detection of H. pylori infection. Since it was first described by Graham et al., the test has been extensively modified, including variations in the dose of 13C-urea, sampling time, test meal and cut-off values. The 13C-urea breath test has been studied extensively in Western populations. However, relatively few reports had been published which have studied the optimal 13C-UBT protocol in a Chinese population. Since H. pylori infection has a strong association with peptic ulcer diseases, gastric lymphoma and gastric cancer, which has a high prevalence in Asian populations, the establishment of an optimal 13C-UBT protocol becomes an important issue in this regard.24

Decreasing the dose of 13C-urea permits a reduction in the cost of the test. This is particularly important for developing countries like China which has a high prevalence of H. pylori infection. This potentially permits a wider application of this noninvasive test for the diagnosis of H. pylori infection in such a high-risk region.

The sampling time of the 13C-UBT has been progressively simplified since the first report by Graham et al. At present, there is universal agreement that two samples should be taken: one collected before, and another 30 min after the ingestion of 13C-urea. We have taken a breath sample every 15 min after the intake of 13C-urea for 97 patients. The best measurement period appears to be at 30 min and was well correlated with the published data (Table 2). The use of a citric acid test meal shifted the peak DOB towards the 30 min interval (Figure 2) but there was no statistical difference between the mean DOB with or without test meal at 15, 30, 45 and 60 min (Figure 1).

A commonly used cut off value between H. pylori infected subjects and H. pylori uninfected subjects for this low dose 75 mg 13C-urea protocol is 4%.12 In the present study, the highest accuracy at 30 min measurement interval was 97% (cut-off values of 5% and 6%) for 13C-UBT with the test meal and 96% (cut-off values of 4%, 4.5% and 5%) for 13C-UBT without the test meal. The highest accuracy for UBT without the test meal was 96.9% at the 45 min interval, using a cut off value of 3.5%. This has to be balanced against practicality, operator time and patient convenience. We therefore conclude that in our Chinese population, the best cut off value for a low dose 75 mg 13C-urea protocol with a test meal at 30 min is 5%, while the best cut off value for that without a test meal at 30 min is the same, from 4 to 5%. For simplicity, we advocate that the cut off value for breath tests with or without a test meal should be 5%.

The use of a test meal in the 13C-UBT was employed to increase the residence time of the 13C-urea in the stomach and to improve the contact between urease produced by H. pylori and the substrate. A number of different test meals have been suggested. Recently, citric acid has been suggested as the best liquid test meal.12, 25 Graham et al. evaluated the effect of citric acid as a test meal for the 13C-UBT and found a dose–response relationship between the mean DOB and the amount of citrate used.21 The optimal dose of citric acid suggested by their study was around 1–2 Gm. A further modification of the 13C-UBT, such as the omission of a test meal has also been suggested. 13C-UBT in the fasting state produced comparable sensitivity and specificity with other 13C-UBT protocols using a test meal.22, 23 The dose of 13C-urea used in the two published 13C-UBT studies which did not use a test meal were 100 mg and 125 mg. Our study is the first to evaluate and compare the low dose 75 mg 13C-urea breath test protocol without a test meal vs. citric acid (2.4 Gm), which has been proposed as the best test meal. Our findings suggested that the 13C-UBT in a fasting state has equally reliable and excellent results in the diagnosis of H. pylori infection in a Chinese population. Atherton et al. evaluated the effect of test meals on intragastric distribution of urea and suggested that 13C-UBT without a test meal may mainly reflect antral urease activity.26 Although the use of a test meal allowed a better delivery of urea solution to the gastric body and fundus, the contribution of urease in the body and fundus appeared to be small in their study.

Furthermore, simplifying the 14C-urea breath test studies by others have already shown that omitting the test meal was a feasible option and gave a satisfactory sensitivity and specificity.27, 28 It has been shown that 13C-UBTs at 10 min were unaffected by the use of test meal while values were significantly greater at 30 min and later in their study.26 However, the mean DOB was similar between 13C-UBTs with or without a test meal at all time intervals in our study. It appears that the distribution, delivery and emptying of 13C-urea may be different in Chinese populations. An analogous situation was observed in the acid secretory pattern in an earlier study reported by us, in which acid output was significantly greater in the Scottish population compared to Chinese, both in normal controls and duodenal ulcer patients.29 It will be interesting to perform an isotope-labelled urea study in the Chinese population to study the distribution, delivery and emptying of 13C-urea as described by Atherton et al.26

In conclusion, we have shown that for a low dose 75 mg 13C-urea breath test protocol with or without a test meal, the optimal measurement interval was 30 min and the optimal DOB cut-off value was 5%. This low dose protocol without a test meal was highly accurate in the diagnosis of H. pylori infection in a Chinese population. This simplified protocol is more practical and economical in Asia and may become the standard protocol in Chinese populations and other parts of Asia.

ACKNOWLEDGEMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References

This study was supported by the Peptic Ulcer Research Fund and the Simon KY Lee Gastroenterology Research Fund, University of Hong Kong, Queen Mary Hospital, Hong Kong. We would like to thank Nurse Specialist M. Chong, and Endoscopy Nurses Y. C. Fan, V. S. Y. Tang, M. Y. Lee, K. K. Chang and H. S. Lee for their nursing assistance and providing care to the patients and senior technician Ms E. Kwok for technical assistance.

Footnotes
  1. These authors contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. References
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