The detection and monitoring of H. pylori colonization of the gastric mucosa has become a usual analysis by the 13C-labeled urea breath test (TRU) because it is non-invasive and now available in almost total medical analysis laboratories. The objective of this work is the validation of the TRU to verify and confirm the analytical results frequent in our laboratory. Samples were taken using the Taukit Isomed Pharma kit and analyzes were performed by infrared isotope ratio spectrometry. The results obtained show that the TRU is efficient, faithful, accurate and robust and we can apply it daily on patients with great confidence.

The H. pylori is mostly acquired during childhood and is usually asymptomatic [1]. This infection can lead to various disorders such as inflammation of the stomach (gastritis), peptic ulcer (10% to 20% of cases), adenocarcinoma of the distal stomach (1% to 2% of cases) and lymphoma of the lymphoid tissue associated with the mucous membranes of the stomach [2]. The prevalence of H. pylori infection in Canada is estimated at 7.1% in children aged 5 to 18 years and at 30% in adults according to the World Gastroenterology Organization [3], in Morocco this prevalence is of the order of 36

II.1- Instrumentation

 The analyzes of H. Pylori by the 13C urea breath test were carried out by an Isotope Ratio Infrared Spectrometer (SIRI), IR-force (IR 3000) on samples of air exhaled by the patient in air samples 10 ml dry tubes [7]. 

II.2- Sampling 

According to the information provided by the applicant, TRU 13C is performed using the Taukit kit from Isomed Pharma. First, the patient on an empty stomach (for at least 3 hours) should drink a citric acid drink and then blow, through a straw, into two tubes. Then he should drink another citric acid drink in which the 13C urea has been dissolved, then wait 30 minutes. Finally, he must blow again, with a straw, in two other tubes. In addition to fasting, the patient should prepare by following guidelines for the consumption of certain medications. The collection should be done with the tubes of the kit, which, afterwards, must be hermetically sealed and then stored and transported at room temperature. Thus, the samples will be analyzed by SIRI (IR-force). These samples are indeed stable for long periods (5 weeks) [8]. 

II.3- Experimental protocol 

The IR-force analyzer designed for the determination of 13C labeled CO2 in exhaled air. The analysis focuses on the measurement and determination of the 12CO2 / 13CO2 quotient in the air exhaled by the patient. 

II.3.1- Instrument calibration 

We blow into the exhaled air sampling bag, which we place in the device's adapter before launching the automatic calibration of the device which is necessary to exclude any impact of the 12CO2 concentration on the value. delta during measurement. 

II.3.2- Analysis of samples 

The analysis is carried out on the 2 sampling tubes (T0 and T30) which are placed on the adapter of the device. Before starting the analysis, the patient identification must be entered on the list of samples to be analyzed, launch the test, after a few minutes the device displays the result of the analysis of each sample which is the delta (∆), expressed in part per thousand (‰), which represents the relative difference between the isotope ratio of the sample and that of a reference substance. The difference between the deltas of the samples (T = 30 and T = 0) from the same patient represents the delta over base-line (DOB). The final test result is generally considered positive when the BOD is greater than 5 ‰ [9]. 

II.4- Validation parameters 

The 13C urea breath test is considered semi-quantitative therefore the validation parameters that we have verified are; repeatability, intermediate precision, inter-operator variability, uncertainties and comparison of methods.

The analysis of H. pylori by infrared spectrometry with isotopic relation gives results in delta of difference of isotopic rations between T30 and T0 expressed in part per thousand of the same sample. 

T30 -T0 in delta / 1000:                <3>

                                          3 ≤ R ≤5: Undetermined result.

                                                 > 5: Positive result [10]. 

III.4.1- Repeatability 

The repeatability of this test was determined on 2 levels of controls (6 Negative and 6 Positive). The coefficients of variation (CVr) calculated are lower than the limits retained by the Laboratory [11], see table 1.

Table 1: Result of the repeatability study 

II.4.2- Intermediate reliability 

Reproducibility was evaluated at 2 control levels (6 Negative and 6 Positive). The calculated coefficients of variation (CVR) are lower than the limits retained by the Laboratory [11], see table 2.

Table 2: Result of the reproducibility

II.4.3- Inter-operator variability 

Each control was run 8 times by all operators with the same lot of reagent and the same instrument. The results of this inter-operator correlation are consistent (Table 3). 

Table 3: Results of the inter-operator correlation II.4.4- Uncertainty       

2 levels of controls (7 Negative and 7 Positive) were analyzed internally at our Laboratory (CIQ) and externally in a foreign laboratory (CEQ) (Table 4 and 5). 

Table 4 : Results of the uncertainty calculation (Control Negatif) 

Table 5: Results of the uncertainty calculation (Positive Control) 

II.4.5- Method comparison 

A volume of 13 control samples were analyzed by the IR-force machine and by The HeliFANplus machine (table 6). The results found are consistent with the comparison interval (CI). 

Table 6: Result of the method comparison 

The non-invasive analysis of H. pylori by the 13C urea breath test is efficient compared to other serological or even classical bacteriological techniques for diagnosing infection, faithful, accurate and robust. It is applied directly to samples of patient exhaled air without any pre-treatment and is also used to confirm post-treatment eradication of H. pylori.