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On-line version ISSN 1678-9946
Rev. Inst. Med. trop. S. Paulo vol.41 n.1 São Paulo Jan./Feb. 1999
VALIDATION OF 14C-UREA BREATH TEST FOR DIAGNOSIS OF Helicobacter pylori
Rejane MATTAR, Fernando Marcuz SILVA, Ana Maria ALEXANDRINO & Antonio Atílio LAUDANNA
The aim of this study was to validate the 14C-urea breath test for use in diagnosis of Helicobacter pylori infection. Thirty H. pylori positive patients, based on histologic test and thirty H. pylori negative patients by histology and anti-H. pylori IgG entered the study. Fasting patients drank 5 uCi of 14C-urea in 20 ml of water. Breath samples were collected at 0, 5, 10, 15, 20 and 30 min. The difference of cpm values between the two groups was significant at all the time intervals, besides time 0 (p<0.0001). At 20 min, the test gave 100% sensitivity and specificity with a cut-off value of 562 cpm. Females were higher expirers than males (p=0.005). 14C-urea breath test is highly accurate for Helicobacter pylori diagnosis. It is fast, simple and should be the non-invasive test used after treating Helicobacter pylori infection.
KEYWORDS: 14C urea breath test; Helicobacter pylori diagnosis
After the Australian Easter holiday weekend in 1982 an incubator was opened in the Microbiology Department of the Royal Perth Hospital revealing the first ever culture of a spiral-shaped bacterium from gastric biopsies of patients with gastritis. The discovery of this organism, known as Helicobacter pylori, by Drs. Robin Warren and Barry Marshall has revolutionized gastroenterology7.
Helicobacter pylori is now recognized as the main risk factor in the multifactorial pathogenetic cascade of peptic ulcer disease. Clinical studies continue to provide evidence that ulcer disease can be cured by eradication of this bacterium9.
The initial diagnostic tests for H. pylori relied on gastric mucosal biopsy for urease testing22, histological examination16, or culture4. There has been great interest in H. pylori diagnostic tests based on non-invasive techniques that are inexpensive and sensitive17.
14C- urea breath test has been considered a very sensitive (97%) and specific (95%) non-invasive test15. As H. pylori produces large amounts of urease, the enzyme that hydrolyses 14C-urea to form HCO3- and NH4+, 14CO2 is driven towards the mucosa into the bloodstream and expired through the lungs10.
Most experts agree that the urea breath test will be the test of choice to establish H. pylori eradication after a course of appropriate therapy1. In the Hospital of Clinics of São Paulo, this was the first time that the 14C-urea breath test (UBT) was performed, so the purpose of this study was to validate UBT and to determine the diagnostic ranges for H. pylori-positive and H. pylori-negative patients.
MATERIALS AND METHODS
The study was approved by the Scientific Ethics Committee and the Council of the Department. Symptomatic patients who had been determined to have H. pylori, based on histologic findings, rapid urease CLO test and anti-H. pylori IgG were studied. These consisted of thirty patients with duodenal ulcer (twelve males and eighteen females) with a mean age of 46 + 2 yr. In addition, symptomatic patients were studied who had undergone upper endoscopy but were negative for H. pylori, based on histology, rapid urease CLO test and anti-H. pylori IgG, or patients who had previously been infected by H. pylori but had a successful eradication. There were twenty four patients with duodenal ulcer, two with gastric ulcer, three with dyspepsia and one with esophagitis (fifteen males and fifteen females) with a mean age of 46 + 3 yr.
Antral biopsies were taken for histological techniques and CLO tests. Histology samples were prepared and stained with hematoxylin and eosin (H&E) and with Giemsa stain. H. pylori was identified after examination of H&E and Giemsa stained biopsy sections by a histopathologist blinded to the UBT and H. pylori serology. The discordant CLO test, histology, or breath test patients were submitted to a second blinded histological examination.
H. pylori serology
Sera were assayed for H. pylori IgG antibodies using an enzyme immunoassay (EIA- Cobas Core Roche, 2nd generation, France). Incorporation of urease into a well-characterized and highly immunogenic purified H. pylori- specific multicomponent antigen preparation, free of cross-reading flagella proteins, forms the basis of this test. In the first step, patient samples and controls were diluted with sample diluent and incubated with beads coated with highly purified H. pylori antigens. Specific antibodies were bound to the beads. After removal of the unbound material by a washing step, the antigen-antibody complex on each bead was detected with peroxidase (POD)-conjugated goat anti-human IgG antibody. After removal of unbound conjugate, the beads were incubated with a substrate solution containing tetramethylbenzidine (TMB) and hydrogen peroxide. A blue color developed, the intensity of which was proportional to the amount of H. pylori-specific IgG bound to the beads. The enzymatic reaction was stopped by the addition of acid, and absorbance values were determined at 450 nm. For quantitative results a standard curve was obtained by dilution series of the positive control and plotting each absorbance value versus the corresponding standard value. The concentrations of IgG antibody to H. pylori in patient samples were determined by interpolation from this standard curve18.
Antral mucosal biopsy specimen was inserted into a homemade urease test tube4. Urease reagent was made up to 100 ml with bacto-yeast extract 0.010g, KH2PO4 0.0091g, Na2HPO4 0.0095g, urea 2g and Phenol red 0.5% 15 drops, the pH of the solution was adjusted to 6.9. It was sterilized by filtration, dispensed into 0.5 ml aliquots and stored -20º C. If the urease enzyme of H. pylori was present in the gastric biopsy being tested, the resulting breakdown of urea caused the pH to rise and the color of the solution turned from yellow to bright magenta. The urease test tube was examined over the next 24 h.
14C-urea breath test (UBT)
UBT was performed according to MARSHALL et al.10 14C- urea (250 uCi, Amersham) was reconstituted with 25 ml of sterile distilled water. Aliquots of 0.5 ml were frozen at -20o C until ready to use. Subjects were fasted for at least six hours prior to the test. They had to remove false teeth (if present), and cleanse their mouth with antiseptic solution (thymol, salol, menthol, saccharin, fuchsin, water and ethanol) provided by the Pharmacy Division of the Hospital; a baseline breath sample was collected and identified as Time 0. Then, they swallowed 5 uCi of 14C- urea dissolved in 20 ml of water. According to MARSHALL et al.10, when 14C-urea solution was given as a mouthwash, caused an immediate peak of 14CO2 excretion (urease from commensal mouth organisms) in a two min breath sample, reaching baseline levels after 12 min. However, when was given directly into the esophagus in Hp- subjects, isotope excretion did not increase more than 50 cpm above the baseline value. Therefore, patients rinsed their mouth again two min after they had swallowed 14C-urea, spitting the water out and making sure that none was ingested. Breath samples were collected at 5, 10, 15, 20 and 30 min. Patients were instructed to blow through tubing attached to a safety trap into a scintillation vial containing 2.5 ml of 400 mM Hyamine (Sigma) in methanol with 15 mg/l thymolphthalein (blue alkaline color). They had to blow until the solution became colorless indicating the collection of 1 mmol of CO2. Once the breath samples had been collected, scintillation fluid (10 ml- 5.5 g PPO/ 0.2g POPOP of 2:1 v/v Toluene/Triton-X) was added to the vial; counting proceeded for 5 min per vial, and the results were expressed as cpm/mmol CO2. Counting efficiency of the Beckman LS 100C was 93%. Patients were not allowed to take antibiotics or bismuth-containing compounds within 30 days before the test and proton pump inhibitors, sucralfate or H2-RAs within a minimum of 7 days before the test.
Determination of H. pylori status
Patients were considered H. pylori negative (Hp -), if histology and anti-H. pylori IgG were negative. Patients H. pylori positive (Hp +) were those with positive histology. The CLO test was used to provide corroborative evidence of H. pylori status.
The results were analyzed by GraphPad prism software version 2.0.
Breath samples were collected at 0, 5, 10, 15, 20 and 30 min after 5 uCi of 14C-urea had been swallowed by twenty seven Hp - patients and twenty two Hp + patients. With the exception of time 0, at all other times of breath collection there was a striking difference of 14CO2 excretion between the two groups (p< 0.0001, Fig. 1).
Fig. 1- 14CO2 excretion for patients who were Hp + and Hp- . Graph shows means and bars represent SD of breath samples collected at 0, 5, 10, 15, 20 and 30 min after 5 uCi of 14C-urea intake. Values are presented as cpm.
Accuracy analysis of UBT was shown in Table 1. Cut-off value (Mean + 3 SD) was established at all the time points, except at time 0. The specificity of the test was the percent of Hp - patients presenting 14CO2 excretion below the cut-off value and the sensitivity of the test was the percent of Hp + patients with 14CO2 excretion above the normal range. The specificity of the test was 96% at all the times, except at 10 and 20 min when it was 100%; the sensitivity of the test was 59% at 5 min and 100% at all other times of breath collection.
As the specificity of the test was 100% at 10 and 20 min, sixty patients (thirty Hp - and thirty Hp +) entered the study to establish the best time for breath collection. The differences between Hp - and Hp + were larger at 20 min (Fig.2), consequently this was the time point found as the best for breath collection with a cut-off of 562 cpm.
Fig. 2- 14C-urea breath tests from 30 Hp+ patients and 30 Hp - patients after 10 min (A) and 20 min (B) of 5uCi of 14C-urea intake. Each dot represents a single value in cpm.
Females Hp + were higher expirers than males at 20 min, this difference was significant by t test (p=0.0051, Fig.3). Hp + patients had higher concentrations of antibodies IgG anti-H. pylori than Hp - patients (p=0.0019 by t test, Fig. 4).
Fig. 3- 14C-urea breath test from 30 Hp + patients, 18 females and 12 males, after 20 min of 14C-urea intake. Each dot represents a single value in cpm.
Fig. 4- Concentration of IgG anti-H. pylori (U/ml) in Hp + and Hp - patients. Each dot represents a single value.
Thirty Hp + patients and thirty Hp - patients entered the study to validate 14C-urea breath test. Breath was collected at 0, 5, 10, 15, 20 and 30 min, and unexpectedly the difference between the two groups was significant (p<0.0001) at all these time points, except time 0. The sensitivity and specificity of the assay at 20 min was 100%; therefore, as was described previously1, 10, this was the time of choice for breath collection after 5 uCi of 14C-urea intake. When a capsule containing 14C-urea (1 uCi) was given, a single time point of 10 min was chosen for accuracy analysis15; nonetheless, in other authors' 1 experiments the 20-min sample gave the most consistent and reproducible data.
According to MARSHALL et al.10, counting until 10 min corresponded to bacterial urease of the oropharynx, therefore it is important to perform a mouthwash before taking 14C-urea, cleansing the mouth again 2 min later and spitting the water out. Even though at 5 and 10 min, most probably there was an addition of the mouth bacteria and Helicobacter pylori urease activities, the difference between the Hp - and Hp + groups was too significant (p<0.0001) to consider the possibility of false positive results. In Hp- patients at the 10 min the level of 14CO2 started to fall reaching almost baseline values at 30 min, however in Hp+ patients, the 14CO2 count was still high at this time point.
We obtained a cut-off value of 562 cpm at 20 min, different from Marshall et al.10 which was 928 cpm. Sensitivity and specificity depend on the chosen cut-off point11; ours gave 100% of sensitivity and specificity.
The study of metabolic fate of the radioactive carbon in the 14C-urea breath test showed that the mean combined urinary and breath recovery for high expirers in 72 h was 86% (SD=7%) and for low expirers it was 97% (SD=3%), thus the long-term retention of 14C was low12, consequently the radiation exposure was trivial10,15.
Females Hp + were higher expirers than males at 20 min, this difference was significant (p=0.005), authors10 that had the same result accounted for the lower endogenous 12CO2 production by females. Furthermore, there was a significant correlation between 14C-urea breath test result and the number of H. pylori colonies in culture2,17 . In addition, a direct correlation between the 13C-urea breath test values and the density of Helicobacter pylori in antrum biopsies was seen20, 21.
We hypothesize that there is a direct correlation between cpm values of 14C-urea breath test and the density of Helicobacter pylori on the gastric mucosa, and females have a higher number of bacteria, perhaps explaining their worse response to treatment. However, we are planning to prove whether this hypothesis is correct or incorrect in the near future. If this was true, 14C-urea breath test could predict treatment success. Otherwise, the percentage of women in therapeutic failures was greater than expected as they harbored metronidazole-resistant strains more than twice as often as men. The explanation for this was the previous use of antibiotics for gynecologic complaints3,19.
Serology is of limited use, in that it is only good for the diagnosis of pre-treatment patients, as antibodies do not fall immediately after H. pylori has been eradicated. In the present study, even though many patients of Hp - group were previously infected, the concentration of antibodies in this group was lower than of Hp+ group (p<0.0001). Therefore, sequential serological samples could be used to determine if H. pylori has been eradicated.
The original version of the urea breath test was reported by GRAHAM et al.5 with the stable isotope 13C that can be used in children and pregnant women. Otherwise, 13C-urea breath test requires patients to drink a test meal (50 ml) to delay gastric emptying, holding the isotopic urea in the stomach, also a relatively large dose of 13C-urea substrate is necessary to measurably increase 13CO2 excretion8. The 14C isotope is cheaper and more sensitive6, as it does not require a test meal, allowing 14C-urea to be fully exposed to gastric mucosal urease10.
In nature, 14C is produced by the collision of cosmic radiation and nitrogen, therefore, a small fraction is present in the air, being 1: 1012 the proportion of 14C to 12C. Plants, human tissues and bones, normally have 14C in such a way to allow archeologists to identify the age of fossils by measuring this long living isotope in bones13.
In conclusion, the 14C-urea breath test should be the non-invasive test for choice for H. pylori infection diagnosis. It is highly sensitive and specific at 20 min of breath collection, and is also fast, simple and inexpensive.
Validação do teste expiratório com 14C-uréia para o diagnóstico do Helicobacter pylori
O teste expiratório com 14C-uréia diagnostica infecção pelo Helicobacter pylori no estômago. A urease, produzida por esta bactéria, quebra a 14C-uréia, resultando em HCO3- e NH4+, sendo expirado 14CO2 pelos pulmões e quantificado, então, por contador Beta. O objetivo do nosso estudo foi validar o teste expiratório com 14C-uréia, determinando o tempo ideal de coleta da amostra e o valor de corte. Foi definido o tempo de 20 min para coleta do exame após a ingestão de 5 uCi de 14C-uréia com valor de corte de 562 cpm. A sensibilidade e a especificidade do teste foram de 100% aos 20 min. As diferenças de contagens entre os grupos Helicobacter pylori negativos e Helicobacter pylori positivos foram estatisticamente significativas (p<0,0001). As pacientes do sexo feminino, aos 20 min apresentaram contagens superiores às dos pacientes do sexo masculino (p=0,005). É preciso verificar se as mulheres têm maior densidade bacteriana, ou se é pelo fato dos homens expirarem mais CO2, diluindo a amostra. O teste expiratório com 14C-uréia é método diagnóstico não invasivo, muito sensível, específico e barato.
All the materials were obtained from the Hospital of Clinics of the University of São Paulo School of Medicine. The authors thank Arlete F. das Dores and Clodoaldo F. Leite.
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Laboratory of Functional Tests of the Digestive System, Department of Gastroenterology, Hospital of Clinics, University of São Paulo School of Medicine, São Paulo, Brazil.
Correspondence to: Rejane Mattar, Laboratório de Provas Funcionais do Aparelho Digestivo, Hospital das Clínicas, Av. Dr. Eneas de Carvalho Aguiar 255, 2º andar bloco 6, 05403-000 São Paulo, SP, Brazil, (fax 005511-2827599) E-mail: firstname.lastname@example.org
Received: 31 July 1998
Accepted: 09 November 1998