Limitations of urease test in diagnosis of pediatric Helicobacter pylori infection

Abstract

The diagnosis of Helicobacter pylori (H. pylori) infection is usually based on the results of urease test and histology. The urease test known as a simple and cheap method does not need special skills to perform or to read the result. The time needed for the test to turn positive depends on the concentration of bacteria, and the accuracy is up to the density of H. pylori density in the biopsy sample, which is generally lower in children than adolescents and adults. Therefore, there are debates about the sensitivity of the urease test in children. The reason for lower sensitivity of the urease test in children was not identified, but might be related to the low density and patchy distribution of bacteria. In this review, we discuss the limitations of the urease test in children according to age, histology, number of biopsy samples, and biopsy site. In children under 5 years old, the differences in positivity rate when the urease test used one or three biopsy samples, and samples from the antrum or the gastric body, were larger than those in children aged 5-15 years. Thus, three or more biopsy samples from both the antrum and body would improve the sensitivity of H. pylori infection diagnosis in children under 5 years old.

Key Words: Helicobacter pylori infection; Urease test; Children

Core tip: The diagnosis of Helicobacter pylori (H. pylori) infection is usually based on the findings of histology and the results of the urease test. However, the sensitivity of urease test in children was lower compared to adults. The lower sensitivity of urease test of children might be related to the low density and patchy distribution of bacteria. In urease test, three or more biopsy samples from both the antrum and body would improve the sensitivity of H. pylori infection diagnosis in children under 5 years old.

INTRODUCTION

Helicobacter pylori (H. pylori) is the main pathogen of acute and chronic gastritis, peptic ulcer disease, and gastric cancer[1,2]. Although primary H. pylori infection occurs during early childhood, most adults eventually become H. pylori carriers in developing countries[3,4]. In children, abdominal pain is the most common symptom in H. pylori infection[5]. Whether or not H. pylori-infected children with recurrent abdominal pain should be treated did not reach consensus[6]. However, a positive family history of gastric cancer is one of the risk factors of adulthood gastric cancer in children with H. pylori infection[7]. Thus, a reliable diagnostic test to detect H. pylori infection in children is required. The most reliable diagnostic method of H. pylori infection in children is endoscopy with gastric biopsy[8].

The diagnosis of H. pylori infection is usually based on the findings of histology and the results of the urease test[9]. Histology plays an important role in detecting H. pylori and also indicates the degree of chronic and active inflammation in the stomach[10]. The urease test known as a simple and cheap method does not need special skills to perform or to read the result. The urease test uses phenol red, which changes from yellow to pink or red as the pH increases. H. pylori can stay alive in the human stomach and activate its own cytoplasmic urease, converting urea into carbon dioxide and ammonia and increasing the pH of the surrounding environment[11]. Buffered urease tests need at least 10⁵ organisms so as to produce a positive result[12]. Therefore, the urease test may be negative, if biopsy samples in which the histology reveals only one or two bacteria in the entire section[13]. The time needed for the test to show a positive result depends on the concentration of bacteria[14], and the accuracy of the urease test primarily is up to the density of H. pylori in the gastric sample. Generally the H. pylori density is found to be lower in children than adolescents and adults[15]. The time of the urease test turns positive is up to the concentration of bacteria and the temperature. Most commercially available urease tests will turn positive within 2-3 h but it is best to hold those that appear negative for 24 h regardless of age[14]. The urease enzyme activity of H. pylori at the diagnosis also affects the urease test. Acid suppression by proton pump inhibitor is a well-known cause of the false negative result of urease test[14].

However, there is currently a debate about the sensitivity of the urease test in children without consideration of the urease enzyme activities. A review of the literature about H. pylori diagnostic tests in children from 1999-2009 suggested that prompt urease tests have better sensitivity than histology to detect the occurrence of H. pylori[16]. In 1590 pediatric patients from 1989 to 2009, urease tests had lower sensitivity (83.4%) and comparable specificity (99.0%) with histology[17]. The reason for this lower sensitivity in children was not identified, but it might be related to the low density and patchy distribution of bacteria[15,18]. In this review, we discuss the limitations of the urease test in children based on the literature reporting on pediatric patients.

AGE OF CHILDREN

Age is another factor influencing the sensitivity of the urease test[17,19,20]. The age ranges of the children in different studies were wide, and varied from ≤ 15 to ≤ 18 years of age[5-8]. In children, a low density of H. pylori has been reported[15,21], which can be attributable to sampling errors such as very low numbers of H. pylori in the tissue samples or patchy distribution of the organism in the stomach’s mucosa[22]. Among 530 children infected with H. pylori, the urease test was positive in 442, and the rate of urease tests was positively associated with the children aged 5 years old and above compared with those below 5 years old[17]. The false negative rate for the urease test was 16.6% in these children, mostly found in those below 5 years old[17]. In our previous studies, we divided pediatric patients into three age groups: 0-4 years, 5-9 years, and 10-15 years[19,20]. The positivity rate of the urease test increased with each increasing age group, with the lowest rate in the 0-4 years group and highest in the 10-15 years group (Figure 1)[20]. We then compared the time points at which the positive reaction produced, dividing these into 0-1 h, 1-6 h, 6-24 h, and 24-48 h. This was considered within each of 4 age groups: 0-4 years, 5-9 years, 10-14 years, and 20-29 years[20]. Positive results occurred within 1 h in the older age groups, 10-15 years and 20-29 years. Conversely, most positive results occurred at 6-24 h in the 0-4 years group using body biopsy specimens (Figure 1)[20]. This result suggested that a low degree of H. pylori colonization in gastric biopsy samples might be more frequent in children under 5 years old.

Figure 1 The positivity rate and positive timing of the urease test both in the antrum and body according to age. The positivity rate of the urease test in the antrum was higher in 20-29 years group than that in other three age groups, and the positivity rate of the urease test in the body decreased with increasing age (P < 0.0001). The highest positivity timing was within 1 h in the 20-29 years group, and within 6-24 h in children (P < 0.0001)[20].

HISTOPATHOLOGICAL GRADES AND THE POSITIVITY RATE OF THE UREASE TEST

H. pylori infection causes chronic inflammation in stomach mucosa. Histology has been regarded as the golden compass for H. pylori detection[9], as it can detect the bacteria as well as the severity of inflammation. However, just as a positive urease test requires approximately 10⁵H. pylori in the biopsy sample[12], histopathological results are limited by the distribution and density of the bacteria in the sample. Therefore, the patchy distribution and low density of H. pylori in gastric mucosa in children might be related to the absence of bacteria in some histopathological findings. Nevertheless, the positive urease test is highly correlated with the density of bacteria, the severity of chronic gastritis, and the presence of active gastritis, as determined by histopathology[15,17].

When histopathological evaluations found severe degrees of chronic and active gastritis, or H. pylori infiltration, the urease tests revealed a quick change of color, indicating positive results in samples from both the antrum and gastric body, regardless of age, compared with those from milder histopathological grades[20]. In 38 children, aged 2-18 years, the accuracy of the rapid urease test was 95% for samples determined to be positive by histology[23]. The accuracy of the urease test is up to the density of H. pylori in the gastric sample. However the H. pylori density is overall lower in children than in adolescents and adults[15]. Therefore, both histology and a urease test should be performed in children for diagnosis of H. pylori infection.

EFFECT OF THE NUMBER OF GASTRIC BIOPSY SPECIMENS

Two or more numbers of gastric biopsies improve the sensitivity and saves the time for the positive result for diagnosis of urease test[19,24]. There were several studies comparing positivity rate of urease test according to the biopsy numbers diagnosis (Table 1): three were adults studies and one was children study. In children, the positivity rate of the urease test was higher in three biopsy samples than single biopsy sample, in the same child[19]. The discrepancy between positivity rates of the urease test utilizing one vs three biopsy samples was elevated in children below 5 years old[21]. Thus, when the urease test is performed on three or more biopsy samples, it is possibly a more accurate diagnostic modality.

Table 1 The studies about the positivity rate of urease test by comparing to biopsy sites and biopsy numbers.

Ref.	Published year	No. subjects	Mean age (yr)	Comparison	Positivity rate of urease test (%)
Siddique et al[24]	2008	100 adults	36.1	One biopsy	52
				Two biopsies	68
				Three biopsies	76
				Four biopsies	96
Seo et al[19]	2014	255 children	NA	One biopsy	32.2
				Three biopsies	40.1
Moon et al[25]	2012	214 adults	53.6	Antrum	58.9
				body	62.1
				Antrum + body	69.2
Lan et al[26]	2012	164 adults	NA	Antrum	83.3
				Antrum + corpus	100

NA: Not available.

In adults, the positivity rate of urease test also increased with using two to four biopsy specimens than a single biopsy specimen[24]. The discrepancy rates of one and two or more biopsy specimens were higher in adults (10.3%, 16.7% and 16%) than in children (7.9%) (Table 1)[19,24-26]. In adults, the time for positive urease test was faster in united test (1.69 h for antrum and body) than in separate test (3.58 h for body or antrum) on average[25]. In children, it is reported to be a low degree of H. pylori colonization and more patchy distribution of H. pylori[21]. Thus, when the urease test is performed on three or more biopsy samples, it may be a more accurate diagnostic modality.

EFFECT OF BIOPSY SITE ON UREASE TEST RESULTS

Most patients were taken of biopsy specimens from the antrum, as one biopsy from the gastric angle for urease test had the maximum probability for detecting H. pylori infection[27]. The use of an additional biopsy from the gastric body can improve the detection rate of H. pylori (Table 1)[20,25,26]. In our previous study[20], the discrepancy of urease test results was bigger between the antrum and body in children compared with that in adults, and the most difference was found in the samples that showed a positive color change late in testing, from 6-24 h (Figure 1). In our study, no difference was observed between the speed of a positive reaction in samples from the antrum and from the body in adults[20]. But the time of positive test was 3.58 h for the separate test and 1.69 h for the united test on average[25]. The colonization degree was remarkably lower in the body compared with the antrum[21]. In our previous study, the histopathologic findings of the antrum and body revealed similar degrees of chronic and active gastritis and infiltration of bacteria in children, the positivity rate of the urease test was higher in samples from the body than from the antrum[20]. This result was similar to the study of Korean adults, where the positivity rate of urease test in body was higher than in antrum (Table 1)[25]. In adults, the sensitivity of urease test decreased along with the degree of gastritis with atrophy increasing from 100% in normal, 97% in mild, 91% in moderate to 66% in severe[26]. However additional corpus biopsy resulted in increased sensitivity to 16.67% compared to single antrum biopsy (Table 1)[26]. This might also be related to the patchy distribution of H. pylori and low density of the organism in body. There has been a continuing debate about the optimum site and number of gastric biopsies for the diagnosis of H. pylori.

CONCLUSION

Early diagnosis of H. pylori infection is crucial for symptomatic children with a family history of gastric cancer. In children, endoscopic examination and biopsy for histology and urease test is still the most reliable method for diagnosis of H. pylori infection. The difference in the positivity rate of the urease test between using one or three biopsy samples, or using biopsy samples from the antrum or body, were larger in children aged 0-5 years than 5-15 years. Use of three or more biopsy samples from both the antrum and body would improve the sensitivity of H. pylori detection in children.