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The urea breath test (UBT) is one of the most important non-invasive methods for detecting Helicobacter pylori infection. The test exploits the hydrolysis of orally administered urea by the enzyme urease, which H pylori produces in large quantities. Urea is hydrolysed to ammonia and carbon dioxide, which diffuses into the blood and is excreted by the lungs. Isotopically labelled CO2 can be detected in breath using various methods.
Labelling urea with 13C is becoming increasingly popular because this non-radioactive isotope is innocuous and can be safely used in children and women of childbearing age. Breath samples can also be sent by post or courier to remote analysis centres. The test is easy to perform and can be repeated as often as required in the same patient. A meal must be given to increase the contact time between the tracer and the H pylori urease inside the stomach. The test has been simplified to the point that two breath samples collected before and 30 minutes after the ingestion of urea in a liquid form suffice to provide reliable diagnostic information. The cost of producing 13C-urea is high, but it may be possible to reduce the dosage further by administering it in capsule form.
An isotope ratio mass spectrometer (IRMS) is generally used to measure13C enrichment in breath samples, but this machine is expensive. In order to reduce this cost, new and cheaper equipment based on non-dispersive, isotope selective, infrared spectroscopy (NDIRS) and laser assisted ratio analysis (LARA) have recently been developed. These are valid alternatives to IRMS although they cannot process the same large number of breath samples simultaneously.
These promising advances will certainly promote the wider use of the13C-UBT, which is especially useful for epidemiological studies in children and adults, for screening patients before endoscopy, and for assessing the efficacy of eradication regimens.
Many diagnostic methods have been developed over the past 15 years to detect Helicobacter pyloriinfection—some invasive (rapid urease test, histology, culture, and polymerase chain reaction) because they cannot be performed without endoscopy, and others non-invasive (serology, urea breath test (UBT) and, more recently, H pylori antigen determination on faeces). Of the latter, the UBT is being increasingly used both in pretreatment and post-treatment phases.
Since it was first described by Graham et al,1 the test has been modified extensively to simplify and optimise it, including changes to the dose of urea used, sample timing, test meal, and cut off values to distinguish infected from uninfected subjects. Despite these modifications, the accuracy of the test has remained high and this is the best confirmation of its robustness.
The test exploits the large amount of urease produced byH pylori, as this enzyme hydrolyses the orally administered, labelled urea into ammonia and labelled CO2, which is absorbed through the mucus layer of the stomach and then transported to the lungs via the bloodstream for excretion. Isotope enrichment can be measured by various methods in breath samples collected at appropriate times.
Urea can be labelled with two different carbon isotopes:14C and 13C. The main difference between them is that the former is radioactive, whereas the latter is stable. The advantages of using 14C-urea are that it is cheap, so rapid that administering 14C-urea in a gelatin capsule allows an accurate response to be obtained from a single 10 minute breath sample,2 ,3 and does not require any test meal. However, although the dose of 14C has become progressively smaller and the test can now be performed with 1 μCi,3 which is equal to the natural background radiation received in one day,3 ,4 the main problems are still the availability of a nuclear medicine department or centres licensed for storage and disposal of radioactive substrates, shipping difficulties, and the copious amounts of labelled tracer needed to perform large scale epidemiological studies. The use of this unstable tracer can be recommended when the number of UBTs per annum in a given gastroenterological centre is less than 2500 and 14C facilities are available on site.5
In contrast, 13C is a non-radioactive isotope that can be used safely for repeated testing, which is frequently required in clinical practice, and for detecting H pylori infection in children and women of childbearing age. Furthermore, 13C-urea has been the most widely used substrate in methodological studies performed to validate this kind of diagnostic test. Another relevant advantage of using the stable isotope is that breath samples can be sent by post or courier to remote analysis centres, thus promoting the distribution of the test, which can even be performed at home if the patients are adequately selected and instructed.6 The major drawbacks of13C-urea are the higher cost compared with14C-urea, and the need for expensive mass spectrometry, which is the most preferable device for measuring 13C enrichment in breath samples of subjects infected withH pylori.
This review focuses on the most recent advances in the machines used to measure the 13C isotope and on the most important aspects regarding the main UBT variables.
13C is measured as the13CO2:12CO2 isotope ratio and is expressed as delta over baseline (DOB) per mil (‰) with respect to the international reference standard represented by the Pee Dee Belemnite limestone. The difference in isotope masses (45:44) is detected with extreme accuracy by the sector magnet contained in the mass spectrometer (IRMS). This equipment also requires a gas chromatograph because CO2 has to be carefully purified from the whole breath prior to introduction into the mass spectrometer. The precision of measurements made by conventional IRMS can be as high as 0.01‰7 and this allows very low isotopic enrichments to be detected. As 13C is measured as the ratio of13CO2 to 12CO2, there is no need for a large volume of expired air and a 10 ml sample is enough to obtain reliable diagnostic information.5Furthermore, the examination results do not depend on the body mass, so it is not necessary to take this variable into account.8
If a mass spectrometer is the most preferable machine for detecting13C excess in breath samples, its high price has limited the spread of the test and has prompted the biomedical industry to develop new instruments that are capable of measuring the stable isotope at a lower cost. Over the past few years, analytical devices besides IRMS have been developed to perform13CO2 and 12CO2analysis, such as infrared spectrometry9 ,10 and laser optogalvanic spectroscopy.11 Several comparative studies12-15 have shown the reliability of these new, cheaper machines in measuring 13C enrichment, thus allowing them to be considered as valid alternatives to IRMS.
Table 1 presents the main characteristics of IRMS, non-dispersive, isotope selective, infrared spectroscopy (NDIRS), and laser assisted ratio analyser (LARA) equipment. The advantage of IRMS is that it processes the highest number of breath samples and works in an automated manner. However, it is the most expensive and requires the longest analysis time. NDIRS does not require helium as the carrier gas and has the lowest weight and price, but it can analyse only a small number of samples and so does not need multitasking software. The LARA system has the quickest analysis time; the other characteristics are average compared with the other two machines, especially with regard to the number of samples it can process and the cost. Integrated bar code readers and the use of multitasking programs have made running IRMS instruments much easier; however, they do require more maintenance than NDIRS and LARA systems. IRMS and LARA are more suitable for gastroenterological centres requiring large quantity, automated analysis, whereas NDIRS is more suitable for small laboratories in which the daily number of assays is not high. In this light, a small, new, cheap, infrared device for examination of only two breath samples has been developed to be used exclusively in the doctor’s office.16 In the endless attempt to render the test cheaper and to increase the number of laboratories where it can be performed, other investigators have shown that a gas chromatograph coupled with a mass selective detector, which is available in many analytical and biomedical settings, can also be reliably used for13C-UBT.17 ,18
The 13C-UBT is a simple and innocuous assay which requires only few precautions in order to obtain accurate results. Besides having to avoid several dietary constituents with a natural abundance of 13C,19 such as maize, cane, and cornflour, there is still some debate concerning both the type of antisecretory drugs that may influence the test and the optimal timing of breath testing after their discontinuation in order to exclude false negative results. This is a relevant point because many patients requesting UBT are dyspeptic and thus reluctant to discontinue their antisecretory drugs. Although there is general consensus20-25 regarding the adverse effect of proton pump inhibitors (PPIs) on the UBT (false negative results range from 17 to 61%), the timing of their cessation prior to testing is not so clear. In many clinical trials aimed at assessing the efficacy of eradication regimens, for instance, it is generally reported that patients must not have taken PPIs for at least one month before the UBT, but it has been shown that five to seven days is sufficient to reverse their adverse effect.21-23 Laineet al,24 however, suggest that patients should remain off PPIs for 14 days in order to be sure of avoiding false negative results. Although H2 blockers are regarded as antisecretory drugs with no effect on UBT,26some recent investigations have shown that even standard and high dose ranitidine can cause up to 20% of false negative breath tests.22 ,25 These studies have shown that the reversal of this negative effect occurs within five to seven days of drug cessation and therefore withdrawal for one week suffices to rule out any adverse influence the drugs may have on the UBT.
Antibiotics, particularly those with anti-helicobacter action, and bismuth preparations should be withdrawn at least one month prior to UBT, as is usually suggested.
Amount and formulation of 13C-urea
The urea dose has been progressively reduced from the 350 mg (5 mg/kg) dose initially used in the study by Grahamet al,1 thus decreasing the cost of the substrate needed to perform the test. At present three different dosages are utilised. A dose of 125 mg has been validated in the United States by Klein et al,27 who showed that a sensitivity and specificity as high as 100% can be reached with this dosage. Loganet al used 100 mg in their proposal of a standard diagnostic protocol28 and this dosage has received the greatest amount of attention in Europe. Finally, 75 mg (approximately 1 mg/kg) has been shown to be as reliable as the higher doses29 and is used satisfactorily in an increasing number of research studies.13 ,15 ,17 ,30-34 This low dosage is also included in many commercial kits for13C-UBT,4 as it limits the cost of the test. However, it may be possible to reduce the dosage of13C-urea further by administering the tracer in capsules. It has been shown that a dose of either 38 mg35 or 45 mg,36 given in rapidly dissolving gelatin capsules, maintains the high sensitivity and specificity of the test and also reduces its duration to 15–20 minutes. In fact, this peculiar formulation bypasses the problem of contact of the substrate with the urease producing bacteria present in the oropharynx, thus avoiding false positive results if breath samples are taken too soon. Obviously, there is no need to delay gastric emptying by an appropriate meal, which is fundamental in tests performed with the liquid form of13C-urea.
One of the most important advantages of the UBT is that it samples the whole stomach and is not prone to sampling error as are biopsy based tests, which can be influenced by the patchy distribution ofH pylori infection within the gastric mucosa. This global test can be improved if prolonged contact of the substrate with the urease enzyme is promoted. The use of test meals capable of increasing the residence time of 13C-urea in the stomach is recommended in most protocols. The usefulness of turning the patient instead of leaving him/her seated, in order to favour the contact between labelled substrate and urease activity, has recently been disproved.37
Test meals containing fat are usually chosen due to the well known ability of this nutrient to delay gastric emptying. Recently, citric acid, which acts by lowering duodenal pH, which in turn reduces antral motility and relaxes the gastric fundus,38 has been shown to be an optimal test meal.34 In this study citric acid, which was compared with two other frequently used semiliquid meals, Meritene and Ensure/Calogen, provided the highest and earliest δ peaks; however, data from uninfected patients were not shown in the published paper and the extent of the excess δ13CO2 excreted because of the citric acid was only reported as the ratio above the baseline excess δ13CO2 values, making it difficult to judge.4 One advantage of using citric acid is that the liklihood of contamination by urease produced by bacterial flora in the mouth is reduced greatly.29
Is pretest fasting necessary?
Many protocols recommend that patients fast for at least four hours before testing in order to avoid any interference from food. Some recent studies, however, have addressed the question of whether fasting is really necessary in patients undergoing UBT. Perri and colleagues31 and Moayyedi and colleagues39found that there is no statistical difference between tests performed in fasting and non-fasting conditions and therefore do not recommend fasting before UBT. On the contrary, Epple and colleagues33 and Savarino and colleagues13observed that feeding causes a significant decrease in the 30 minute δ values compared with the fasting ones and an increase in false negative results. They suggest that fasting before testing should be mandatory. These contradictory findings probably result from the different meals used in the various studies, but at present there are no clear guidelines in this area.
Time of breath collection
The test has been progressively simplified since the first report by Graham et al,1 who took breath samples every 10 minutes for three hours. At present, there is universal consensus on the use of a two point examination—that is, a basal sample collected before and another sample collected 30 minutes after ingestion of the tracer, using whatever dosage of13C-urea is chosen.5 ,20 ,29-34 ,40 As already mentioned, the use of citric acid as test meal has been shown to be better than other semiliquid fatty meals in the maximal recovery of 13CO2 at this sampling time.34
Reducing the number of breath samples to only one by omitting the baseline collection in order to decrease cost and duration of the test further has also been proposed.30 ,40 Lastly, shortening the sampling time to 20 minutes and omitting the meal has been shown to maintain the excellent sensitivity and specificity of the test41; however, this study was performed using 125 mg of13C-urea, which is the usual dose in the USA but not in Europe.
Cut off values
Discrimination between infected and uninfected subjects is based on a strict cut off value which has been validated in many studies. The most widely used value is 5‰, which has been proposed by Logan and colleagues28 in their European standard protocol. Later, by using receiver operator characteristic (ROC) curves, Johnston and colleagues42 showed that this cut off value can be lowered to 3.5‰ without compromising the sensitivity and specificity of the test. Cluster analysis was used by Mion et al,43 who also suggested adopting 3.0‰ as the optimal cut off point with the need, however, for a grey zone in which the results of UBT are inconclusive; there was no reference to a gold standard in their study. However, there is no universal agreement on the use of a lower than usual cut off value. Zagari and colleagues44 also used ROC analysis on a large sample of patients and were not able to decrease the cut off level below the value of 4.5‰ with either 75 mg or 100 mg doses of13C-urea. It must be stressed, however, that the 5‰ value remains a strong index for distinguishing infected subjects from uninfected ones and therefore this cut off value is still widely used.
Diagnostic accuracy and clinical applications
The 13C-UBT can be used in many clinical settings because of its non-invasiveness, simplicity, and safety. Furthermore, the sensitivity and specificity in untreated subjects are very high and range from 90 to 98% and from 92 to 100%, respectively.5 ,13 ,28 ,39 ,40 ,45 ,46 Accuracy in the assessment of eradicating efficacy is more controversial because some investigators47-50 found excellent reliability even in the post-treatment phase, whereas others51 did not confirm these good results. Accordingly, it has been recommended recently that two UBTs, performed at separate post-treatment time points (for example, two months apart), can equate to two different tests52 when assessing the success of anti-helicobacter treatments. Obviously, this applies to research trials on eradication treatments rather than to routine clinical practice.
It must be stressed, however, that the UBT has sometimes proved to be poorly diagnostic, because its accuracy has frequently been assessed against gastric biopsies as gold standard4 and it is well known that this latter test is susceptible to sampling error. As confirmation of this fact, Epple and colleagues33 have shown that several apparently false positive UBT results were in fact correct findings, as clearly shown by the analysis of additional multiple biopsy specimens taken in the same patients who underwent a second gastroscopy. Furthermore, in a recent multicentre trial performed in Belgium, Glupzcynski and colleagues53 showed that the UBT was the most reliable test for assessing the efficacy of eradication treatment, whereas the sensitivity of histology was low because of the frequently insufficient size and/or number of biopsy samples.
Considering the above mentioned points, the UBT is the test of choice for screening patients before endoscopy, in epidemiological studies on the prevalence of H pylori infection in various populations, and in the non-invasive evaluation of the efficacy of eradication regimens. The other non-invasive test, serology, has relevant limits in assessing the success of anti-helicobacter regimens due to the considerable amount of time (at least six months) needed to show a consistent decrease in serum antibody levels. Lastly,13C-UBT is the ideal test for children, using a lower dose of urea (50 mg); provided that a mask is used, breath samples can be collected from those under three years of age.4
Currently, the UBT is considered to be one of the most important and reliable non-invasive methods for the diagnosis ofH pylori infection. The examination is simple, innocuous when 13C is used, easy to repeat, highly accurate, and requires a low number of precautions in order to obtain reliable results. It is particularly suitable for epidemiological studies in children and adults, in all clinical conditions where endoscopy is not strictly necessary, and to check the success of eradication regimens. The new and cheaper measuring equipment, the possibility of using a lower urea dose in capsules, thus omitting the meal, as well as shorter than usual sampling times, will certainly promote an increasingly broader application of this test.
Abbreviations used in this paper
- urea breath test
- isotope ratio mass spectrometer
- non-dispersive, isotope selective, infrared spectroscopy
- laser assisted ratio analysis
- proton pump inhibitor
- receiver operator characteristic
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