13C-urea breath test

What do we expect from a visit to a gastroenterologist? Competent treatment that can eliminate the problem in the shortest possible time. The basis for this is the timely formulation of the correct diagnosis.

Currently, there are various methods that can detect the disease with high accuracy even in the early stages. However, not every diagnostic method is as useful for making a diagnosis as it is expensive for the patient’s wallet. In their quest not to make a mistake, doctors prescribe various examinations. Considering that some of them cost decent money, before shelling out a round sum, it would be nice to know what’s what.

Take the case when a gastroenterologist suspects you have an infection. Helicobacter pylori. Most often, the carriage of this infection in the body is asymptomatic. But in some cases the bacterium Helicobacter pylori can cause a number of serious diseases, for example: gastritis, peptic ulcer of the stomach or duodenum, duodenitis, stomach cancer and iron deficiency. Unfortunately, at the moment it remains unclear why in some people the presence of Helicobacter in the stomach does not cause any problems, while in others it leads to serious illness.

And so the doctor prescribes a breath test –¹³C, explaining that it is allegedly the “gold standard” in diagnosing an infection Helicobacter pylori. At the same time, the doctor mentions that the test is non-invasive and will allow you to avoid pain from EGD, which is a diagnostically useful procedure, but so unloved, probably, by every patient of gastroenterologists. What is this mysterious panacea for swallowing the hated “pipe”? And is the result of this test really enough to make a correct diagnosis?

Method 13C-UDT

It’s time to figure out what is ¹³C-urea breath test.

For the first time about breath tests to diagnose infection Helicobacter pylori started talking in the late 80s of the last century, when Barry Marshall described the principles for implementing this study.

Barry Marshall is a Nobel laureate. They proved that gastric ulcers are most often caused by a bacterium Helicobacter pylori.

According to these principles, urease breath tests consist of administering a safe amount of urea to the patient and then analyzing the air exhaled by him for the presence of hydrolysis products of this urea. These products are carbon dioxide and ammonia. The presence of these gases in the exhaled air is a positive indicator of the presence of Helicobacter pylori in a patient. In the case of diagnostics based on the study of carbon dioxide, it is assumed that urea contains isotope labels – carbon atoms ¹³With or ¹⁴C. The subject’s breath is then analyzed for the presence of isotopically labeled carbon dioxide. ^[1].

Let’s turn to Wikipedia for more detailed explanations.

“The patient drinks a load solution – urea labeled with a carbon isotope ¹³C. To improve the reliability of the test results, the investigators recommend using a citric acid solution or citrus juice.”

“For the study, two samples of exhaled air are sufficient, taken before and after (after 30 minutes) taking a solution of urea. Breath samples are taken in special bags, which are then transported to the measuring equipment in analytical centers,” – says the Internet Encyclopedia ^[2].

So, ¹³C-UDT makes it possible to detect the presence Helicobacter pylori. But when studying the materials attached to the instructions of domestic and foreign manufacturers of test kits, we come across a curious contradiction. Russian manufacturers delicately bypass some of the information that European ones do provide. According to the latest, ¹³The C-urease breath test should not be used to diagnose patients with gastric ulcers, gastric infection, and atrophic gastritis (which, by the way, can be caused by the bacterium of interest), as these pathologies can affect the test result. ^[3].

Keep this in mind when referring to domestic specialists: doctors may not have an idea about these contraindications, and such diagnoses are not uncommon.

Nevertheless, some curious points still await attentive people in section 11 of the instructions for the test kit of a Russian manufacturer: “Factors affecting the reliability of the test” ^[4].

Impressive.

A number of questions immediately arise, which, unfortunately, remain rhetorical. Is it realistic to observe the necessary methodology in polyclinics and private clinics? To what extent do the physical and chemical processes of the oral cavity affect the reliability of the result? Regarding the last point: what exactly is included in the concept of “physical activity”? Ten pushups is obvious. Can running to the minibus and climbing the stairs to the fifth floor of the clinic also affect the result of the miracle test?

The concept of “gold standard”

As mentioned above, you may hear from your doctor that the breath test ¹³C is a kind of “gold standard” in diagnosis Helicobacter pylori. Of course, for obvious reasons, domestic test kit manufacturers claim the same thing.

Indeed, European scientists studying H. pylori infection organized a series of conferences with the participation of leading experts. Based on the standards of evidence-based medicine and a large number of controlled clinical trials, approaches to diagnosis and treatment have been developed. Helicobacter pylori. The first conference was held in Maastricht (Netherlands) in 1996.

At the next conference, namely at “Maastricht-II”, held in 2000, for a breath test –¹³Very optimistic recommendations were made. The test has been presented as the best of the best for the primary diagnosis of helicobacteriosis, the so-called “gold standard”, with which others simply cannot compare. ^[5].

Almost 20 years have passed since then, and the concept of the “gold standard” in relation to the test is¹³C was never used again. So, at subsequent conferences “Maastricht”, the same scientists and doctors no longer put this method in first place among the rest. ^{[6] [7] [8]}. Firstly, this test is intended for primary diagnosis when there is no possibility of endoscopy, and this is extremely rare. Secondly, if it is the “gold standard”, it is exclusively for the control of eradication – the treatment of helicobacteriosis, but not for primary diagnosis. ^[9]. However, in domestic sources, the opinion of twenty years ago still prevails.

test kit

Basically, a test kit includes a breath sample container and a load. The load is carbamide (urea) in powder form, and this powder is dissolved in water, with or without the addition of citric acid. More on this later.

Some researchers recommend using a citric acid solution or citrus juice to dissolve the load to increase the accuracy of the test results. But even here there are doubts: the researchers did not agree on whether it is worth dissolving the load in these fluids or whether it is better for the patient to drink these fluids before the procedure. ^[10]. Since there is no single methodology, the moment is extremely controversial. Apparently, people conducting this testing are forced to rely only on their intuition.

How do Russian manufacturers of test kits answer this question? To date, three manufacturers of this type of product are registered in Russia. The HELICARB set produced by the ISOCARB company has a certain popularity in the market.

The manufacturer of the load “HELICARB” recommends using orange or grapefruit juice in the study, but does not indicate in the instructions that, according to scientists, these drinks should be used with caution when testing patients with diabetes. In addition, quite a few people are allergic to citrus fruits, and even a small amount of such juice can cause a reaction. Recall also that citrus juice aggressively affects the gastric mucosa due to its high acidity. And since people with diseases of this organ pass the test, besides on an empty stomach, taking an acidic liquid can become unpleasant (and even painful) for the patient. It is sad that the experts conducting the test are forced to choose between the convenience of the patient and the reliability of the result. Another manufacturer, creator of the set “¹³S-CARBAMIDE-TEST”, and completely suggests using citric acid in the study, the effect of which on the stomach is also aggressive ^[11].

Let’s figure out what else the manufacturer of the HELICARB test kit offers us.

Доза ¹³C-urea: 50 mg.

What does the manufacturer’s instructions for the test kit say:

“The generally accepted single dose for testing in adults is 1 mg per kg of body weight, which is, on average, 75-80 mg of the drug. The dose for children 5 years of age and older is 40 mg per test.

It turns out that an adult will take a child’s dose of the drug? The composition, of course, is expensive, and, apparently, this was the reason for calculating the dosage by the Russian manufacturer, who distinguished himself with his 50 mg. In the USA and Europe, the minimum dose of urea for this test has been approved, namely 75 mg. ^[13].

Even more suspicious is the manufacturer’s attempt to explain its own choice of dosage. Again, let’s look at his instructions. Highlight:

“In order to optimize the dose ¹³C-urea when conducting ¹³In 2015, we performed a study with C-UDT … 23 volunteers took part … were studied on the mass spectrometer “Heli View” (South Korea)” ^[11].

When conducting the study, the manufacturer assumed that the optimal dosage of the labeled urea preparation, obtained as a result, would be used to test thousands, if not millions of people. But how optimal was the number of subjects? I remind you: 23 people.

When conducting such studies, as a rule, the minimum number of patients is 200 people. This can be seen in a number of foreign publications of this kind. ^{[14] [15] [16]}. Usually, the authors of publications indicate diagnostic methods, with the results of which the results are compared. ¹³S-UDT. Load manufacturer “HELICARB” does not do this in his manual.

By the way, samples of these volunteers were examined on the Heli View apparatus (South Korea), which is not in the state registry of Roszdravnadzor, which deprives this study of the manufacturer of the last trust.

The manufacturer also refers to the Swedish company Kibion, which produces test kits with the same (50 mg) dosage. However, these tests are available in the form of tablets, and this eliminates the loss of load on the oral cavity and esophagus, that is, urea hydrolysis occurs exclusively in the stomach. According to the Swedish manufacturer, this helps to minimize false positive results. ^[12]. Nevertheless, among foreign manufacturers, such a dosage and form of loading is rather an exception. Accordingly, the comparison is unacceptable.

Measuring instruments

To reveal the ratio in selected samples ¹³CO₂/¹²CO₂, the following devices are used:

• mass spectrometers;
• IR spectrometers;
• laser spectrometers.

Mass spectrometers are considered among the most highly sensitive and accurate instruments for ratio measurement. ¹³CO₂/¹²CO₂, however, due to complex maintenance and high price, they are not used for the task of mass diagnostics.

In this regard, fundamentally cheaper IR spectrometers are offered, for example, an IR spectrometer for respiratory tests with stable isotopes approved for use in practical healthcare: “IRIS.Doc” (Kibion/Wagner, Sweden/Germany, Registration certificate No. ФСЗ 2008/ 03312 of December 22, 2008).

This device has been produced in the EU for a long time ^[17], in the Russian Federation it was first registered in 2008. No changes have been made to the instrument since that time, as this would have required changes to the marketing authorization. And from the changes in the registration certificate from Roszdravnadzor, you can see only a change in the name of the manufacturing organization and the address of the applicant organization of the medical device in 2015, as well as the OKP code to the OKPD2 code in 2017 (however, this is due to changes in the classification system in the Russian Federation) ^[18]. The question is, to what extent does the retro-device of 2008 meet modern requirements?

There is also a device from the Chinese manufacturer “HEADWAY”, on the example of which you can learn about the calibration of any IR spectrometer.

The instrument manufacturer recommends that you calibrate once a month. Apparently, it does not matter at all how many samples will be examined during this period of time. But this is not the worst.

The instructions say: “The operator blows into the sampling bag.” Surprisingly, because usually special bags and cylinders with a gas mixture are used to calibrate such devices. Is it assumed that the operator, who in a medical institution is usually a nurse or laboratory assistant, has the ratio of gases in the exhalation as constant as in the mentioned cylinders and bags? So, while German instrument manufacturers supply special calibration bags filled with a gas mixture, in our country a nurse’s exhalation will be used as a reference. Interesting, isn’t it? And the same person will “breathe as a reference”? What if that particular nurse or technician gets sick and doesn’t show up for work on the day of the calibration? What now, will another person calibrate (if, of course, such a term can be used in relation to this at all)?

Transporting sample air bags to the instrument

However, in order to make measurements on amazing instruments, samples must first be taken. In general, the sampling procedure is about the same, regardless of which test kit is used. The first sample is taken before the load is received in a special bag, and the second one is taken some time after.

By the way, “some time” is a loose concept. Various manufacturers disagree on when exactly it is better to take a second sample: someone recommends doing it 10 minutes after taking the load, someone – after half an hour. This is a fairly significant spread, since it concerns the diagnosis of a serious illness.

The sampling procedure has its own complexity. Carbon dioxide, formed as a result of the hydrolysis of ingested urea and containing labeled carbon atoms, is collected in the lower parts of the lungs – the alveoli. In fact, it is this air that is the target analyte for the study, since it contains the highest concentration of labeled atoms. The difficulty is that the patient needs to exhale so deeply that the alveolar air, and not just the air from the external environment that fills the larynx, trachea and oral cavity, can be collected in a special bag.

The foreign manufacturer BreathTek supplies a straw in its test kit so that it is possible to dose exhaled air ^[19]. It is assumed that difficulties arise here: it is not so easy for the patient to hold his breath, and the straw is poorly fixed in the bag, so losses occur during sampling. Nevertheless, responsible foreign manufacturers are still trying to offer at least some solutions to increase the accuracy of the test. This is the difference between the approaches: the supposedly “unreasonable complications” of foreign manufacturers are really aimed at improving accuracy, while everything is “easy and simple” for domestic manufacturers.

Further, the selected samples must be delivered to the measuring equipment. Typically, measuring devices are not installed in a health care facility: they are located in a centralized laboratory, possibly in another city. The test samples are delivered to the device from various clinics.

Neither in the instructions nor on its website does the Russian manufacturer of the test kit specify the timeframe during which the sample must be delivered and tested. It is strange that such important information is missing for a medical device. And the sample taken is a gas mixture, which can be very sensitive to the conditions of transportation: air temperature, pressure, method of packing bags. Add to this the Russian realities: harsh winters and delivery difficulties.

Interpretation of results

“During the breath test-¹³With measure the indicator δ – the relative difference between the ratio ¹³С/¹²C, determined in the test sample of exhaled air, R_std = (¹³С/¹²FROM)_x and the standard isotope ratio R_std = (¹³С/¹²FROM)_stdmeasured in parts per thousand. δ = (R_x/R_std – 1)x1000, where R_std = 0,011237 for carbon.

As a rule, the test is considered positive at ≥ 4,0‰; negative — at ≤ 3,0‰; intermediate (threshold) – at δ from 3,0‰ to 4,0‰ “, is a summary from Wikipedia.

By the way, it also says that there are no single and generally accepted values ​​for the parameter δ, and its value as a positive, negative or threshold result is completely determined by the attending physician. Subjective and very vague, despite the fact that the criteria for interpreting the result are an important thing in diagnosis. You can read about the difficulties in establishing reference values ​​here. ^[20].

By the way, the Russian manufacturer of the test kit claims: “In any case, in order to make a clinical diagnosis and determine the tactics of managing a patient with helicobacteriosis, it is necessary to compare the results ¹³C-UDT with the clinical picture of the disease, data from endoscopy, biopsy, laboratory and other studies.

Plus to this: “With threshold values ​​of δ from 3‰ to 4‰, asymptomatic H. pylori bacteriocarrier is possible […] which may require repeated testing ¹³C-UDT or other studies on helicobacteriosis, taking into account the totality of clinical factors “ ^[21]. Given the problem with interpreting the results, be prepared to spend money on additional research.

Not all doctors think ¹³C-UDT is ideal. There are publications, including those of foreign authors, where much has been called into question. So, for example, we can recall the abstracts of the scientific work “Respiratory examination for HP in an open access system has a high level of potentially false negative results due to a violation of the test procedure” ^[22].

It would seem that the non-invasiveness of the test could serve as a significant plus when examining young patients. However, its sensitivity significantly depends on the age of the child. For children under 5 years of age, its use is not recommended, because at an early age, due to the small volume of exhaled air, the accuracy of the results of the research method is significantly reduced. In children under two years of age, the sensitivity and specificity of the test are 10% and 80%, respectively, from two to five years old – 40% and 90%, over five years old – 70% and 90% ^[23].

As with adults, test results can also be affected by a child’s physical activity. ^[24].

Gastric urease-producing agents can cause false-positive breath test results. ¹³C. Such microorganisms are found in the oral cavity, in the gastrointestinal tract, in the organs of the genitourinary system and on the skin. Excessive colonization of the body by urease-producing bacteria other than Helicobacter easily causes the test to fail. ^[25].

Finally, the review article states in plain text that at the moment there is no agreed and standardized methodology for conducting a breath test.¹³С ^[26].

There are different views on the dosage of urea, divergence of opinions regarding the use of citrus juice in the study, as well as obvious problems with sampling and transportation of samples, outdated equipment for their analysis. Doctors and laboratories have different approaches to conducting this test and further deciphering the data obtained. What will the patient end up with?

From all of the above, we can conclude that the breath test is¹³C, like any other diagnostic method Helicobacter pylori, has its drawbacks and limitations. Therefore, it is incorrect to single out one method as the “gold standard”.

Sources of