Oral urease rapid test serve as an effective complement to the detection of Helicobacter pylori infection through urea breath test

Abstract

The urea breath test (UBT) is the clinic’s most popular and accurate non-invasive diagnostic method for Helicobacter pylori (HP) infection. Yet, it is time-consuming and unavailable to some individuals. The oral rapid urease test (RUT) is an emerging non-invasive convenience method but lacks clinical data support. A double-center, double-blind, methodological comparative design experiment is invited to evaluate the consistency of the oral RUT with UBT. 247 participants were enrolled. Their Dental plaque and tongue coating samples were collected, blind and tested with RUT. The significant association between the RUT and UBT was analyzed. The oral RUT showed a high level of agreement with the UBT. The sensitivity of the test reagent was 98.63% (95% CI: 92.64%, 99.76%), and the specificity was 96.34% (95% CI: 92.25%, 98.31%). The overall conformity rate was 97.05% (95% CI: 94.03%, 98.56%). The positive predictive value was 92.31%, and the negative predictive value was 99.37%. The positive likelihood ratio was 26.96, and the negative likelihood ratio was 0.01. The Kappa value was 0.97. The efficacy of oral RUT for HP infection diagnosis is extremely consistent with the UBT.

Trial registration

Clinical trial of rapid test paper (chemical reaction method) for Helicobacter pylori (NO.ChiCTR2500097395), Registration Date2025.02.19.Retrospectively registered.

Introduction

Helicobacter pylori (HP) is a Gram-negative bacterium characterized by its spiral-shaped, flagellated, microaerophilic structure, primarily inhabiting the human gastric antrum [1, 2]. Most importantly, HP is currently the only bacterium classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) [3, 4]. If left HP infection untreated, chronic inflammation in the stomach can develop over time, leading to ulceration, metaplasia, and ultimately, gastric cancer (GC). Epidemiological studies [5, 6] have shown that HP infection is highly prevalent in the general population, with a global infection rate exceeding 50%. Most HP -infected individuals are asymptomatic and occult; some may exhibit nonspecific symptoms, including upper abdominal discomfort, pain, bloating, loss of appetite, nausea, vomiting, and dark or tarry stools. Factors influencing HP infection rates include economic status, living conditions, educational level, occupation, and drinking habits, with higher rates generally observed in developing countries [7].

Diagnostic methods for HP infection can be broadly categorized into invasive and non-invasive. Invasive procedures rely on sample collection through gastroscopy [8, 9], including histological tests (such as toluidine blue staining, acridine orange staining, immunohistochemistry), bacterial culture, rapid urease test (RUT), and HP nucleic acid detection. Non-invasive methods include serology (antibody) testing [10, 11], stool-based detection of HP antigens (HPSA) [12, 13], genes [14], which are also accurate for establishing HP status. However, HP-diagnostic methods currently available have certain limitations, such as invasion, costly, complex, and sometimes involving the use of radiation. Therefore, it is worth developing a convenient, non-invasive, and reliable method for HP detection that is clinically significant.

As mentioned above, traditional RUT was applied for HP diagnosis during endoscopy. Although it was accurate and time-saving, it was invasive. The oral RUT was a noninvasive method. However, to our knowledge, there are few clinical trials to evaluate the diagnosis effect of oral RUT. In this study, we compared the diagnosis effect of oral RUT with UBT. The oral RUT exhibits a comparable accuracy with UBT.

Patient and methods

Clinical trial design and participation

A multi-center (two centers), synchronous double-blind, methodological comparative design clinical trial was performed to determine the sensitivity, specificity, and accuracy of the novel urease HP test, compared with the golden standard method UBT test (registering Number: XXLB20220014). People who meet the following criteria will be eligible for inclusion: Individuals who need to undergo UBT testing based on their clinical diagnosis and voluntarily sign an informed consent form, including factors such as different ages and genders. Sample collection and handling should comply with the reagent instructions or relevant regulations. Those who met any of the following criteria were excluded: (1) Poor compliance (failure to cooperate with sample collection). (2) Failure to meet the inclusion criteria. (3) With signs of intestinal tuberculosis. (4) Use of proton pump inhibitors or H2 receptor antagonists within the past two weeks. (5) The researcher determines that the sample does not meet the testing requirements (sample contamination or sample preservation process does not comply with the reagent instructions). The trial was obeyed by the experiment flow (Fig. 1). In brief, an adequate size of sample (approximately 1 mm diameter of tongue coating and/or dental plaque) from participants was collected by special personnel. After blinding, the samples were stored and tested following the RUT kit instructions (Rapid Urease Test Strip for HP, Pandian Medical Equipment Co., Ltd., Hunan, China), while the positive and negative were estimated (Fig. 2). Then, the UBT was performed and documented, and the C14 or C13 urea breath tests were permitted. All the results were unblinded and analyzed in the final. The trial was performed with approval from the Ethics Committees of Jiangmen Central Hospital (No.202301 A) and Panyu District Central Hospital(No.PYZXYYEC2022-034-01). All procedures were carried out following relevant guidelines and regulations. All participants signed the informed consent form for the clinical trial.

Fig. 1.

The trial flow: this is a synchronous double-blind, methodological comparative design trial. There are total 240 participants recruited from Jiangmen Central Hospital and Panyu District Central Hospital, including 3 pre-experimental cases, resulting in an analysis dataset of 237 participants. Samples, including dental plaque and tongue coating, were collected and coded blinded. All participants underwent the UBT test, while the coded blinded sample were test by the novel rapid test strip. The data was unblinded and analyzed conclusively. No participants quitted, and no samples were dropped out

Fig. 2.

The positive (left) and negative (right) results of the novel reagent

Sample size estimation and sample allocation

In this clinical trial, assuming a two-sided α error probability of 0.05 and a β error probability of no more than 0.2, for parameter estimation, it is only necessary to ensure that the confidence interval of the evaluation index meets the requirements without a target acceptance value. The following formula can be used for sample size estimation:

In the formula, n represents the sample size, Z1-α/2 represents the quantile of the standard normal distribution, P represents the positive or negative compliance rate, and Δ represents the allowable error size of P. Δ is usually taken as half of the width of the 95% confidence interval of P, commonly ranging from 0.05 to 0.1.

Based on preclinical small sample experiments, the positive compliance rates of tongue coating, dental plaque, and UBT were set at 95%, while the negative compliance rates were set at 90%. Fixing the allowable error Δ as 0.05, the required sample sizes were calculated as follows:

Number of tongue coating and tartar samples (positive) :

Number of tongue coating and tartar samples (negative)

.

Considering a 5% dropout rate, the minimum total sample size for tongue coating in this clinical trial is 223 cases, and the minimum sample size for dental plaque is 223 cases.

Statistical indicators and clinical evaluation indicators

In terms of descriptive statistics, normally distributed continuous data are presented as mean ± sem, and categorical data are presented as counts and proportions (%).The chi-square test in a crosstable is used to identify the significant association between two detecting method, and the positive agreement rate, negative agreement rate, overall agreement rate, and their 95% confidence intervals were calculated using formulas or the SPSS statistical software. while p < 0.05 was considered statistically significant.

Results

Participants characteristic

From February 22, 2023, to April 4, 2023, 240 participants were recruited from Jiangmen Central Hospital and Panyu District Central Hospital, including 3 pre-experimental cases, resulting in an analysis dataset of 237 participants, and all were included (as shown in Table 1). Among these participants, 98 (41.35%) were male, and 139 (58.65%) were female. The average age was 48.18, with the youngest 20 years old and the oldest 85 years old. 35 participants (14.77%) were below 30 years old, 134 participants (56.54%) were between 30 and 59 years old, and 68 participants (28.69%) were 60 years old or above. 73 (30.80%) were diagnosed with HP infection through the UBT test (Table 2).

Table 1.

Participant summary

Center	Participant ID	Cases Number	First Testing Date	Last Testing Date
01	1001–1124	124	2023/03/13	2023/04/04
02	2001–2116	116	2023/02/23	2023/03/10
Total	-	240	2023/02/23	2023/04/04

Center 01 refers to Jiangmen Central Hospital, while Center 02 refers to Panyu Central Hospital in Guangzhou

Table 2.

Participant information

Characteristics	Value
Participant Number (n)	237
Age, years (mean ± SD)	48.18 ± 17.40
Gender, n (%)
Male		98 (41.4)
Male		139 (58.6)
UBT Test Results, n (%)
Positive		73 (30.8)
Negative		164 (69.2)

The novel urease test strip exhibits respectable performance in HP detecting

In this trial, both the dental plaque and tongue coating samples underwent the novel urease test. The paired samples produced identical results. Among the 237 participants in the analysis dataset, the positive rate of the novel urease test was 32.91% (78/237). In comparison to the UBT, the sensitivity was 98.63% (95% CI: 92.64%, 99.76%), specificity was 96.34% (95% CI: 92.25%, 98.31%), accuracy was 97.05% (95% CI: 94.03%, 98.56%), positive predictive value was 92.31%, negative predictive value was 99.37%, positive likelihood ratio was 26.96, negative likelihood ratio was 0.01, and the Kappa value was 0.97 (Tables 3 and 4). The area under the ROC curve (AUC) was 0.958 (Fig. 3), indicating excellent diagnostic discrimination according to conventional benchmarks (where AUC > 0.9 is considered excellent).

Table 3.

Paired count data table for novel urease test and UBT

Novel Urease Test	UBT		Total
	Positive	Negative
Positive	72	6	78
Negative	1	158	159
Total	73	164	237

Table 4.

Analysis results of novel urease test

Indicators	value(%)	95%CI(%)
Sensitivity	98.63%	92.64%,99.76%
Specificity	96.34%	92.25%,98.31%
Accuracy	97.05%	94.03%,98.56%
PPV	92.31%	/
NPV	99.37%	/
LR(+)	26.96	/
LR(-)	0.01	/
Kappa	0.97	/

Fig. 3.

ROC curve of oral RUT, area under the ROC curve was 0.958

Discussion

HP infection is highly prevalent in the general population and is considered a risk factor for Gastric cancer. UBT is one of the gold standards for HP detection, with nearly 95% sensitivity and specificity [5, 15, 16]. However, UBT requires standardized procedures for sample collection and ingestion of radioactive material, which pregnant women and children may not accept. It also shows poor sensitivity in patients undergoing gastric surgery and pediatric patients [17]. In this study, we found an oral urease test performed a consistent result with UBT with the advantage of an easy process, lower cost, and non-radioactive. The novel test may become a potential tool for UBT complement.

HP produces a large amount of urea. Therefore, a rapid urease test was widely applied to diagnose HP infection [18, 19]. Nevertheless, the traditional urease test requires gastric biopsy specimens through a gastroscope, and it is an invasive examination. In addition, accurate diagnosis with traditional rapid urease tests like Pyloritek takes one hour to obtain [20]. In this study, we collected dental plaque and tongue coating samples instead of endoscope biopsy specimens. The new test strip takes less than 3 min. Notably, the novel urease test strip showed high favorable agreement rates (98.63% for dental plaque and tongue coating) and negative agreement rates (96.34% for dental plaque and tongue coating). With rapid and non-invasive sampling, the oral urease test has a broader application than the endoscopic urease test.

As the entrance and first component of the gastrointestinal system, the oral cavity is a reservoir for extra-gastric HP storage [21, 22]. Recent studies indicate that HP can be detected using molecular biology and immunology methods in dental plaque, saliva, and tongue coating [23–25]. Meanwhile, genotyping results of HP in the oral cavity are consistent with those in the stomach, suggesting that HP’s oral and gastric strains are likely homologous [22]. The prevalence of HP in dental plaque is different in various studies. Chaudhry et al. reported that HP infection was detected in 51.6% of the patients’ dental plaque using PCR [26], while Agarwal and Jithendra reported that the prevalence is 18% employing microbial culture [23]. Our result showed that 78 of the 237 participants (32.9%) tested positive using the oral RUT. Various conditions cause wide variations, such as Different inclusion criteria, sampling procedures, and HP detection methods. The prevalence was generally low in studies where culture was employed [27]. This can be explained by the coccoid form of HP, which is metabolically active but unculturable in dental plaque [28].

Gürbüz et al. reported that the RUT for detecting HP in dental plaque exhibited a sensitivity of 89.7% but a low specificity of 42.9% when compared to endoscopy RUT [29]. However, our study included a larger number of participants from two centers and observed an HP infection rate of 30.8%, which is lower than the rate of 81% from the former study. In our research, the novel oral RUT exhibited higher sensitivity (96.34%) and specificity (96.34%) compared to the urea breath test (UBT). Consequently, both dental plaque and tongue coating can be utilized as sample sources for detecting HP infection.

Nevertheless, there are several limitations to this study. Firstly, some urease-positive bacteria, such as Streptococcus, Haemophilus, and Actinomyces, may be detected in the oral cavity and present a false positive result. Secondly, the participants enrolled in the digestive department in the hospital, most of them arguing varying degrees of abdominal discomfort. and it is necessary to conduct further investigation to determine whether the outcome can be popularized in society. Thirdly, some persons, such as children and adolescents, were not included in this study, and they were the most suitable community for oral RUT HP detection.

Conclusion

In conclusion, we have found a timesaving, more economical, non-invasive, and user-friendly oral RUT for HP detection. Its diagnostic accuracy is comparable to the traditional UBT. This novel RUT can be applied to the first routine examination and provides a quick and accurate diagnosis for HP.

Authors’ contributions

Yaoming He, Chengxing Wang, Yu Wan, Jinglin Zhao were involved in the study design. Chengxing Wang, Wenfeng Luo, Weixing Lin, Shangren Li, Fangmei Xie, Chaorong Zhou and Jian Shen collected the patients’ data and biological samples. Data analysis and interpretation were performed by Yaoming He, Weiqiang Zou and Jietao Zhong. All authors contributed to the article and approved the submitted version.

Funding

None.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

The clinical trial was performed with approval from the Ethics Committees of Jiangmen Central Hospital (No.202301 A) and Panyu District Central Hospital(No.PYZXYYEC2022-034-01). All procedures were carried out following relevant guidelines, regulations and adheres to CONSORT guidelines. The clinical trial complies with the Declaration of Helsinki, and all participants signed the informed consent form for the clinical trial.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.