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Evaluation of a 20 minute 14C urea breath test for the diagnosis of Helicobacter pylori infection   Back Bookmark and Share
McCarthy CF

The use of 14C-urea breath testing for diagnosis of Helicobacter pylori infection in gastric mucosa has gained widespread acceptance and utilisation. We evaluated a 14C urea breath test (UBT) in 116 patients undergoing endoscopy. Seventy four patients were administered 185 kBq (5 mCi - conventional dose), and 42 patients reduced dose (92.5 IBq, 2.5 mCi) of l4C-urea. All were tested for H. pylori using culture, direct microscopy of gastric biopsies and histological evaluation of paraffin stained sections. Using the mean + three standard deviations as the cut-offvalue, a sensitivity of 96% and specificity of 100% was found for the conventional dose test. At reduced dose, sensitivity was 100% and specificity 96%. Positive and negative predictive values were 100% and 93% for the conventional dose test, and 96% and 100% for testing at reduced dose. We conclude that the UBT is a simple, non-invasive and useful diagnostic alternative for detection of H. pylori in infected patients. We advocate its use in patients less than 45 years of age without alarm symptoms, and also in cases where the need for endoscopic evaluation is not vital, such as after eradication therapy.

Author : McCarthy CF, Abukhadir BA , Heneghan MA, Kearns M, Little CL

Abstract

The use of 14C-urea breath testing for diagnosis of Helicobacter pylori infection in gastric mucosa has gained widespread acceptance and utilisation. We evaluated a 14C urea breath test (UBT) in 116 patients undergoing endoscopy. Seventy four patients were administered 185 kBq (5 mCi - conventional dose), and 42 patients reduced dose (92.5 IBq, 2.5 mCi) of l4C-urea. All were tested for H. pylori using culture, direct microscopy of gastric biopsies and histological evaluation of paraffin stained sections. Using the mean + three standard deviations as the cut-offvalue, a sensitivity of 96% and specificity of 100% was found for the conventional dose test. At reduced dose, sensitivity was 100% and specificity 96%. Positive and negative predictive values were 100% and 93% for the conventional dose test, and 96% and 100% for testing at reduced dose. We conclude that the UBT is a simple, non-invasive and useful diagnostic alternative for detection of H. pylori in infected patients. We advocate its use in patients less than 45 years of age without alarm symptoms, and also in cases where the need for endoscopic evaluation is not vital, such as after eradication therapy.

Introduction

With the recognition that Helicobacter pylori (H. pylori) plays an etiological role in the pathogenesis of peptic ulcer disease, gastric malignancies and non ulcer dyspepsia, the accurate diagnosis of H. pylori gastritis has taken on a greater importance.1-4 Epidemiological surveys give an indication of the extent of dyspepsia, with the one year community prevalence of dyspepsia and reflux symptoms being of the order of 40%.5,6 It has been suggested also that screening of patients under the age of 45 years for H. pylori infection may significantly reduce the number of endoscopies required.7,8 In adults less than 45 years of age the risk of missed diagnosis of serious disease due to endoscopy has been calculated to be minimal. No single test is accepted as the "gold standard" for diagnosis of active H. pylori infection owing to the patchy distribution of H. pylori in the gastric mucosa. Combinations of tests such as histology, culture and urease enzyme activity of gastric biopsy specimens are considered the "gold standard" in diagnosis of active H. pylori infection.9,10

There is therefore a great need for a simple accurate diagnostic test for H. pylori. At present, urea breath tests (UBTs) are increasingly used in the diagnosis of H. pylori infection. The literature indicates that the 14C UBT is reliable.11,17 This report is an evaluation of the first 116 tests carried out at University College Hospital Galway. Our aims therefore were to validate the 14C UBT, to examine the feasibility of a simplified protocol using lower radiation doses and the omission of 12C "cold urea" and a test meal. 

Patients and Methods

One hundred and sixteen patients undergoing endoscopy were evaluated. The first 74 patients (group A), 60 men and 14 women (mean age 54 years, range 20-84 years) were administered 185 kBq (5 mCi) l4C-urea. Group B (the next 42 patients), 33 men and 9 women, mean age 54 years (Range 28-72 years), had 92.5 kBq (2.5 mCi) 14C-urea. All patients underwent routine endoscopy, with one antral biopsy taken for standard histological evaluation using Haematoxylin and Eosin staining of paraffin embedded tissue. Two additional biopsy specimens were evaluated bacteriologically, with culture of one biopsy and direct microscopy of the second. Exclusion criteria included women who might be pregnant, or those planning to become pregnant within the ensuing 3 months, patients with a past history of gastric surgery, intake of non steroidal anti-inflammatory agents, acid suppressant drugs, or antibiotics within 1 month of the test. Patients were defined as being H. pylori positive if histopathology or bacteriology confirmed the presence of the organism. 

Preparation of isotope

Firstly, 9250 kBq (250 mCi) l4C-urea (Amersham, Bucks, UK) were dissolved in 25 ml of sterile water. 500 l doses of the urea solution was added to 5 ml of sterile water giving a dose of 185 kBq per vial while 250 l doses of urea solution were added to 5.0 ml of sterile water for a dose of 92.5 kBq. The l4C-urea doses were frozen at -20 C until use. 

Urea breath test 

To define the pattern of l4CO2 excretion curves in H. pylori positive and H. pylori negative subjects, breath samples were collected before the test and at 5, 10, 15, 20, 30, 45 and 60 minutes after ingestion of 185 kBq l4C-urea from 2 patients who were H. pylori positive and 2 who were H. pylori negative. Patients presented in the morning after an overnight fast. False teeth were removed and mouth rinsed with tap water. 14C-urea was dissolved in 20 ml of water and swallowed in a single swallow by the patients. Breath samples were collected at 20 minutes following the administration of l4C-urea. The subjects were asked to blow through a disposable drinking straw directly into a 20 ml vial containing trapping solution (1.5 ml ethanol, 1 mmol hyamine hydroxide, 1 drop of thymophthalein (a pH indicator giving a dark blue colour)) until the blue colour of the liquid trap became colourless. 10 ml of scintillation liquid (Beckman, Ireland) was then added to each vial and the 14C activity of each sample analysed by a scintillation counter (Beckman LS 7000). Results are expressed as counts per minute (CPM), with the background activity subtracted.

Statistical analysis 

The Mann-Whitney U-test was used for comparisons between groups. A p value less than 0.05 was considered significant. 

Results

Definition of the l4C excretion curve 

In those who were positive for H. pylori, a sharp rise in l4C-excretion occurred within the first 5 minutes, peaked at 15 minutes and declined thereafter. In patients who were H. pylori negative, this decline began at 10 minutes (Figure 1). Lack of overlap between the curves of patients who were H. pylori positive and negative confirmed that urease producing organisms within the mouth did not have a significant effect on the hydrolysis of orally administered urea in H. pylori positive subjects.

185 kBq 14C UBT

Results are shown in figure 2. Forty seven patients were found to be H. pylori positive by either histological or bacteriological evaluation, while 27 were H. pylori negative. Mean CPM in H. pylori positive subjects, was 33852130 compared to 343237.7 for those who were H. pylori negative, p < 0.001.

92.5 kBq 14C UBT

In patients who received reduced dose 14C UBTs, 18 patients were H. pylori positive and 24 were H. pylori negative by the previous criteria. Mean CPM in H. pylori positive patients was 19181088 versus 113.965.4 for those who were H. pylori negative. p < 0.001 (Figure 3). The cut off value of the test is the CPM below which the test is considered negative, and above which the test is considered positive for H. pylori infection. Negativity for the test was the CPM value below the mean +3 standard deviation obtained for H. pylori negative group. These calculated cut off points were 1056 CPM and 310 CPM for the conventional and reduced dose 14C UBTs respectively. In patients who were administered 185 kBq of 14C-urea, all H. pylori negative patients had CPM below the cut off value. 45/47 patients who were H. pylori positive were found to have CPM greater than the cut off value. Two patients were therefore falsely negative based on this test. In patients who received the reduced dose test, 24 were H. pylori negative, by conventional analysis. One of these had a CPM slightly higher than the cut-off-value (357 CPM). Although H. pylori was neither cultured, nor found in paraffin stained sections, a chronic active gastritis with lymphoid aggregates was found, findings consistent with chronic H. pylori infection by histology. Sensitivity and specificity of the conventional and reduced dose UBTs are listed in Table 1. 

Table 1: Test characteristics of 14C UBTs.
 

14C UBT Sensitivity(%) Specificity(%) PPV(%) NPV(%)
185 kBq 96 100 100 93
92.5 kBq 100 96 96 100

PPV = positive predictive value.
NPV = negative predictive value.

Discussion

We evaluated a UBT for the detection of H. pylori following its introduction into routine use at University College Hospital Galway, both at conventional, and at a reduced dose compared to our current "gold standard," combining the results of histology and bacteriology. The prevalence of H. pylori infection was 64% in group A and 42% in group B. This prevalence is consistent with other studies of patients presenting for endoscopy in whom prevalences from 42-68% are reported.11-15 Presence of H. pylori by either histological or bacteriological evaluation was accepted as being consistent with infection even though it is recommended that at least two diagnostic tests concur. The taking of three biopsy specimens from within 3 cm of the pylorus is in keeping with current practice, and although it has been suggested that additional diagnostic biopsies from the high lesser curve or body of stomach may increase the likelihood of H. pylori positivity, the added benefit appears to be small.16UBT has been called "the most important non-invasive test for Helicobacter pylori."17 Two similar methods, using either radioactive 14C,13-16 or the stable isotope 13C,11-18 are in use. The principle of the test is simple. Urea, isotopically labelled with either 14C or 13C is administered orally and if H. pylori is present in the stomach, the urease enzyme hydrolyses the urea producing isotopically labelled CO2. This diffuses into the circulation and is excreted by the lungs. Although H. pylori was described in 1983 by Warren and Marshall,19 it was not until one year later that the presence of urease was noted.20 McNulty in 1985,21 described the first biopsy urease test and in 1987 the first UBT designed specifically to detect H. pylori using 13C labelled urea was described.22 This was quickly followed by reports of a UBT using 14C.23UBTs differ depending on whether 14C or 13C is used. For 13C UBT the main problem is that the isotope occurs naturally and at variable concentrations, being present in 1.11% of expired CO2. Thus, a small incremental rise in expired 13C may give a positive test result. As 13C is a stable isotope, detection is by isotope ratio mass spectrometry, and although this detection process is extremely accurate, most hospitals do not have access to a mass spectrometer and commercial analysis is expensive.Controversy exists also in relation to the optimal protocol for 14C UBTs. Some authors have used essentially the same protocol as for 1 13C.24 However, because 14C is a -emitter, analysis is performed by liquid scintillation counting. Because 14C does not occur naturally, an intrinsic advantage exists over 13C as an isotopic label. We adapted sampling at 20 minutes according to the protocol of Marshall,13 and although sampling at 10 minutes has also given successful results,16 it may increase the risk of false positive results resulting from urease activity of oral bacteria. This problem can be avoided by mouth washing prior to administering the test. In terms of radiation exposure, that derived from a 14C UBT is negligible. A typical test of 185 kBq gives radiation exposure to bone marrow of 3 x 10-6 Sv, similar approximately to one day's background radiation, a transatlantic plane journey and 10 times less than the marrow exposure from a chest x-ray.13 Our reduced dose UBT, which appears almost equally discriminative, gives half this exposure. In cases where doubt exists, repeat UBT is possible. Alternatively, these patients could be referred for endoscopic evaluation. A problem with radioisotopes like 14C are the regulations relating to licensing, security, storage, recording and moving of stock. It has been suggested that 14C UBTs are only manageable in the context of a medical physics department or a fully equipped research department.17 However, we would contend that in most other respects 14C UBTs are preferable, due to the relatively low cost of 14C-urea, trapping solution, analysis, the presence of simplified protocols for usage and the ability to give results almost immediately. Because of the long half life of 14C, a delay in the counting of a sample is not important. The importance of this, is such that the test may be carried out in centres without a scintillation counter, breath samples stored and sent in batch to an institution with suitable detection equipment. Our results are similar to the published literature in terms of sensitivity and specificity. Sensitivities of 90-100% and specificities from 78-100% are reported when compared to biopsy based tests.11,13,17 We report sensitivities of 96 and 100% for normal and reduced dose tests respectively, with specificities falling into the same range. The two false negative tests in patients who received the normal dose test are unexplained. In practice however, the commonest reason for false negative tests is breath testing too soon after a course of bismuth, antibiotics or proton pump inhibitors. False positive tests, on the other hand occur if other urease producing bacteria are present in the stomach, as may occur in patients with achlorhydria from gastric atrophy.17 Because little significant difference was found between normal dose and the reduced dose tests, we have since introduced the reduced dose test into routine clinical practice.Many institutions have hesitated in setting up a UBT service, on the basis that serological methods are constantly improving. However, when direct comparisons are made between UBTs and serology, it is apparent that serology is less accurate.11,15-25 Antibody response may not be mounted in a small number of patients with H. pylori infection,26 and the test is considered unreliable in the elderly.22 Furthermore, in terms of following up patients who have received eradication therapy, serological evaluation is unreliable for up to six months post treatment.11,28,29 Because one of the main clinical indications for a non-invasive test is to evaluate the result of attempted eradication therapy, the fact that UBT can be used one month after the end of treatment gives it significant advantages to both the patient and clinician alike. In conclusion, the 14C UBT we describe is a simple, safe, rapid, non-invasive and relatively inexpensive test when compared to endoscopic techniques. Sensitivity and specificity is high, making it a valuable tool in clinical practice. In an era of increased awareness of both health care economics and H. pylori as a general health care issue, UBTs will have a pivotal role, especially in the management of patients less than 45 years without "alarm symptoms." In the long term, the establishment of open-access UBT services may further improve the interaction between gastroenterologists and primary care practitioners, reduce the number of unnecessary endoscopies such as after attempted curative therapy and offer an alternative, when other invasive and non- invasive tests fail to provide an adequate diagnosis. 

Acknowledgments

The authors wish to express their thanks to the nursing staff of the endoscopy unit at UCHG,

  • Dr Andrew Shearer, Dept. of Physics, UCG,
  • Dr Eilish Hynes, Department of Physiology, UCG and to
  • Una O'Connor for her secretarial assistance. 

Correspondence:

Prof CF McCarthy,
 
Dept of Medicine,
Clinical Science Institute,
University College Hospital,
Galway.

References

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