Artículos
The effect of warming specimens of rapid urease test on its diagnostic accuracy
El efecto del calentamiento de las muestras de la prueba de ureasa rápida en su precisión diagnóstica
Jafar Nasiri jhnasiri@yahoo.com
Arshya Allahdin
Reza Imani
Soleiman Kheiri
Abolfazl Khoshdel
The effect of warming specimens of rapid urease test on its diagnostic accuracy
Revista Latinoamericana de Hipertensión, vol. 14, no. 1, pp. 15-20, 2019
Sociedad Latinoamericana de Hipertensión
This work is licensed under Creative Commons Attribution-NoDerivs 4.0 International.
Abstract:
Introduction: Helicobacter pylori (H. pylori) is a prevalent cause of dyspepsia, peptic ulcer and gastric cancer in developing countries. Among various diagnostic methods, rapid urease test (RUT) is the ideal test for its diagnosis in patients undergoing endoscopy. Studies have identified the factors causing false negative and positive results. One of these factors is proposed to be the temperature of keeping specimens, but the results have been controversial. We aimed to compare the diagnostic accuracy of RUT specimens warmed at 37.C or kept at room temperature (22-25.C) in different time intervals until 24 hours. Methods: 100 patients with dyspepsia who were indicated for endoscopic examination were selected based on convenience sampling method from patients referring to Endoscopy center of Hajar Hospital during August-September 2006. After recording the demographic and medical history of patients, three biopsy specimens were taken from one portion of the antrum; the first two samples were placed in home-made RUT solution and the third biopsy was placed in formalin solution; one tube was placed in incubator with 37.C and the other at room temperature (22-25.C). Positivity of RUT test at different times was compared to histopathological examination. Results: Infection with Helicobacter pylori was confirmed by histological examination in 66% of patients. After 24 hours, sensitivity of incubated RUT was 71% at 37.C, and 68% at room temperature (P=0.85). Specificity at 37.C was 59% and at room temperature was 76% (P=0.19). Median time to positive tests was 2 hours at 37.C and 3 hours at room temperature, while RUT became positive faster at 37.C (P=0.553). Conclusion: The non-significant difference between groups revealed that warming could not improve the diagnostic accuracy of RUT test, thus, the standard RUT method is recommended.
Keywords: Helicobacter pylori, Diagnostic Tests, Routine, Endoscopy, Gastrointestinal; Urease.
Resumen:
Introducción: Helicobacter pylori (H. Pylori) es una causa frecuente de dispepsia, úlcera péptica y cáncer gástrico en países en desarrollo. Entre los diversos métodos de diagnóstico, la prueba rápida de ureasa (RUT) es la prueba ideal para su diagnóstico en pacientes sometidos a endoscopia. Los estudios han identificado los factores que causan resultados falsos negativos y positivos. Se propone que uno de estos factores es la temperatura para conservar las muestras, pero los resultados han sido controvertidos. El objetivo fue comparar la precisión diagnóstica de las muestras RUT calentadas a 37°C o mantenidas a temperatura ambiente (22-25°C) en diferentes intervalos de tiempo hasta 24 horas. Métodos: se seleccionaron 100 pacientes con dispepsia que estaban indicados para un examen endoscópico según el método de muestreo de conveniencia de los pacientes que se referían al centro de Endoscopia del Hospital Hajar entre agosto y septiembre de 2006. Después de registrar la historia demográfica y médica de los pacientes, se tomaron tres muestras de biopsia. Una porción del antro; las dos primeras muestras se colocaron en una solución de RUT hecha en casa y la tercera biopsia se colocó en una solución de formalina; un tubo se colocó en una incubadora con 37°C y el otro a temperatura ambiente (22-25°C). La positividad de la prueba de RUT en diferentes momentos se comparó con el examen histopatológico. Resultados: la infección con Helicobacter pylori se confirmó mediante examen histológico en el 66% de los pacientes. Después de 24 horas, la sensibilidad del RUT incubado fue del 71% a 37°C y del 68% a temperatura ambiente (P=0,85). La especificidad a 37°C fue del 59% y la temperatura ambiente fue del 76% (P=0.19). El tiempo medio para las pruebas positivas fue de 2 horas a 37°C y 3 horas a temperatura ambiente, mientras que el RUT se convirtió en positivo más rápido a 37°C (P=0.553). Conclusión: la diferencia no significativa entre los grupos reveló que el calentamiento no podría mejorar la precisión diagnóstica de la prueba RUT, por lo que se recomienda el método RUT estándar.
Palabras clave: Helicobacter pylori, Pruebas de diagnóstico, de rutina, Endoscopia gastrointestinal, Urease.
Introduction:
Helicobacter pylori (H. pylori) is a gram-negative bacterium that affects a large number of patients worldwide, with a significantly high prevalence in developing countries and low socio-economic communities, although its prevalence differs by age, sex, race, and other factors1. H. pylori infection is reported to have a high prevalence in Iran with a prevalence of more than 80% reported in many cities2.
H. pylori infection is of great importance, as it leads to chronic gastritis, peptic ulcer disease, gastric cancer and mucosa-associated lymphoid-tissue (MALT) lymphoma3. The age-standardized risk of gastric cancer varies from 10.2 to 50 per 100,000 person-year in different cities2. Therefore, H. pylori infection requires early and accurate diagnosis4.
Although international guidelines suggest endoscopy after empirical acid suppression, it is suggested that this approach, named as “test and treat”, is not beneficial in countries with high prevalence of H. pylori infection5, like Iran, and endoscopic-based methods are recommended for eradication of H. pylori infection4.
Although none of the various methods suggested have been proven as the gold standard, studies have suggested various invasive and noninvasive methods for diagnosis of H. pylori infection: non-invasive methods include serology, immunoblot, stool antigen test, and urea breath test (UBT), and invasive tests include, histologic examination polymerase chain reaction (PCR) for antibiotic-resistant types of H. pylori, and rapid urease test (RUT)6.
RUT and histopathologic tests are suggested as excellent accurate diagnostic tests7. RUT is a rapid, cheap, and simple test that can indirectly assess the presence of bacteria through urease existence8. It requires about 105 H. pylori bacteria to change color in an agar-based like CLO test (campylobacter–like organism) and has high sensitivity varying from 80-100% and specificity of 97-100%, based on the methods and techniques used8. Obtaining two samples from antrum can result in a sensitivity of 95% and specificity of 100%9. In addition, although most specimens turn positive in the first 120-180 minutes, investigation of specimens for 24 hours and no longer results in most accurate detection of H. Pylori bacteria8.
Studies have investigated other factors that play a role in diagnostic accuracy of RUT; some have suggested that using proton pump inhibitors (PPI) by the patient reduces its sensitivity and increases false negative results10. Moreover, other bacterial species may rarely cause false positive11. Some studies have also advised that the temperature of reaction might influence the diagnostic value of RUT; some researchers suggested that using a warmer with 38oC had a 20% higher diagnostic ability in the first 30 minutes, while overall results did not differ significantly12, while other researchers proposed that warming the specimens at 37oC increased the sensitivity of RUT until 2 hours and had an earlier mean time to positive test13.
Due to the significance of RUT in diagnosis of H. pylori, beside the controversial results regarding the effect of temperature on diagnostic accuracy of RUT, the present study aimed to evaluate the diagnostic accuracy of RUT, in specimens kept at 37oC, compare with specimens kept at room temperature in different intervals until 24 hours.
Materials and methods:
Study design: In this prospective study, 100 patients (including 50 males and 50 females) with dyspepsia who were indicated for endoscopic evaluation were selected based on convenience sampling method among patients referring to Endoscopy center of Hajar Hospital from July to September 2006. Sample size was calculated to estimate the sensitivity and specificity of RUT with the 10% precision and 95% confidence, using the overall sensitivity/specificity equal to 50%.
Patients with dyspepsia aged over 14 years who were referred to Endoscopy center of Hajar Hospital from July to September 2006 were included into the study. Any patient with recent gastrointestinal bleeding, and urgent cases undergoing endoscopy was excluded from the study.
Demographic characteristics, past medical and therapeutic history of patients were recorded. Then, three biopsy specimens were taken from the one section of antrum. To prevent formalin contamination, the first two samples were placed in similar RUT and the third biopsy was placed in formalin solution.
Home-made RUT solution was prepared each day by mixing 0.02 g red phenol, 1 g potassium dihydrogen orthophosphate, and 10 g urea (Merk Company, Germany) with de-ionized distilled water; the solution was kept out of fridge 15-20 minutes before each test.
The exact time of sampling, and patients’ name were documented on each tube and the tubes were immediately sent to laboratory of Hajar Hospital; one tube was kept in incubator with 37oC and the other at room temperature (22-25oC). The results of the RUT test were evaluated by one laboratory expert each 15 minutes for the first hour, then each hour for the next four hours, and then at 8th, 12th, and 24th hour after sampling; the temperature conditions were maintained during the evaluation period. The results were reported positive, when the color changed to purple and was considered negative when no color change occurred; the time when the test became positive was recorded by the researcher.
Samples were sent to laboratory for pathologic examination at the end of each day. Specimens were stained by Giemsa, and hematoxylin-eosin and were evaluated by a pathologist. The specimens were fixed in formalin 10%; after processing two slides (stained with hematoxylin-eosin and Giemsa) were prepared and the presence of bacteria in any slide was considered positive pathology result and lack of bacteria in both stains were considered as negative pathology result.
Ethical considerations:The protocol of the study was approved by the Ethic Committee of Shahrekord University of Medical Sciences. The design and objectives of the study were explained to all participants and written informed consent was obtained from those who were willing to participate in the study and they were ensured that their data will be kept confidential and analyzed anonymously.
Statistical analysis; Results were presented as mean ± standard deviation (SD) for quantitative variables and were summarized by frequency (percentage) for categorical variables. Continuous variables were compared using T test or Mann-Whitney U test, whenever the data did not appear to have normal distribution or when the assumption of equal variances was violated across the study groups. Categorical variables were, on the other hand, compared using chi-square test and Fisher’s exact test. For the statistical analysis, the statistical software SPSS version 21.0 for windows (SPSS Inc., Chicago, IL) was used. P values of 0.05 or less were considered statistically significant.
Results:
Mean and SD of age of participants was 47.6±18.9 (range: 14-83) years. Half of patients were male and half were female; 82% of males and 78% of females were married, but patients’ sex and marital status had no association with the pathologic results (P=0.832, and 0.774, respectively). Mean years of history of gastrointestinal disorder was 3.8±5.3 (range 1-30) years with 80% giving a history of using medication for their gastrointestinal disorder. The most used medication was omeprazole (48 patients), H2-blocker (40 patients); 8 patients used both medications, and 8 used bismuth with omeprazole or ranitidine. There was no association between anti-acid therapy and pathologic result (P=0.102).
Infection with Helicobacter pylori was confirmed by histologic examination in 66% patients, among whom 18 patients had negative RUT results (false negative RUT) and 34 patients had positive RUT at both temperatures tested, while 13 cases had positive results only at 37oC, and one case at room temperature (P<0.001) (Tables 1 and 2). Negative histologic and RUT results were reported in 34 patients, among whom 20 had negative RUT results at both temperatures, and 4 patients had positive RUT (false negative RUT). In 10 patients, RUT was only positive at 37oC (P=0.022) (Tables 1 and 2).
| Table 1: Comparison of RUT results with pathologic results in both groups | ||||
| Positive pathology | Negative athology | Total | p-value | |
| Positive at 37oC | 47 | 14 | 61 | 0.004 |
| Negative at 37oC | 19 | 20 | 39 | |
| Total | 66 | 34 | 100 | |
| Positive at room temperature | 45 | 8 | 53 | <0.001 |
| Negative at room temperature | 21 | 26 | 47 | |
| Total | 66 | 34 | 100 | |
| Table 2: Comparison of RUT results between the two temperatures tested | ||||||||
| Sensitivity (%) | Specificity (%) | Positivity (%) | ||||||
| At 37oC | At room temperature | P-value | At 37oC | At room temperature | P-value | At 37oC | At room temperature | |
| After 15 minutes | 23 | 20 | 0.83 | 100 | 100 | 1.00 | 22.7 | 19.69 |
| After 30 minutes | 26 | 21 | 0.68 | 100 | 100 | 1.00 | 3.03 | 1.5 |
| After 45 minutes | 32 | 21 | 0.85 | 100 | 100 | 1.00 | 7.5 | 0 |
| 1st hour | 42 | 30 | 0.205 | 100 | 100 | 1.00 | 9.09 | 9.09 |
| 2nd hour | 50 | 38 | 0.22 | 94 | 100 | 0.49 | 7.5 | 7.5 |
| 3rd hour | 52 | 41 | 0.29 | 94 | 97 | 1.00 | 1.5 | 3.03 |
| 4th hour | 53 | 45 | 0.49 | 91 | 97 | 0.61 | 1.5 | 4.5 |
| 8th hour | 59 | 52 | 0.48 | 91 | 94 | 1.00 | 6.06 | 6.06 |
| 12th hour | 62 | 53 | 0.38 | 79 | 88 | 0.51 | 3.03 | 1.5 |
| 24th hour | 71 | 68 | 0.85 | 59 | 76 | 0.19 | 9.09 | 15.05 |
Among all positive RUT results, 26 cases were positive at both temperatures and 23 cases got positive faster at room temperature; the difference was more than one hour in 19 cases and less than one hour in 4 cases; also, 3 cases became positive later at 37oC.
Mean response time at 37.C was 7.69±9.41 hours (median: 2 hours), which was 8.55±9.95 (median: 3 hours) at room temperature (P=0.553).
The endoscopic results revealed gastric ulcer in 8 patients (7 positive patients), duodenal ulcer in 7 patients (5 positive patients), antral nodularity in 50 cases (30 positive patients), and other pathologies in 22 patients (14 positive patients); 12 patients had antral nodularity with duodenal ulcer (10 positive patients), and one patient had simultaneous gastric and duodenal ulcer. The endoscopic presentation was not associated with pathologic results (P=0.352).
The sensitivity of RUT increased gradually and was higher at all intervals at 37.C than room temperature, but was not statistically significant; the sensitivity at 24th hour was 71% at 37.C and at room temperature was 68% (P=0.85) (Table 3). The RUT specificity was greater than 90% until 8th hour in both temperatures, but then decreased and was higher at room temperature after the first hour (Table 3). At the 24th hour, specificity of the test was 59% at 37.C and 76% at room temperature, but was not statistically different (P=0.19).
| Table 3: Comparison of sensitivity and specificity between two temperatures | |||||
| Positive at room temperature | Negative at room temperature | Total | P-value | ||
| Positive at 37oC | 52 | 1 | 53 | <0.001 | |
| Negative at 37oC | 9 | 38 | 47 | ||
| Total | 61 | 39 | 100 | ||
Conclusion:
The results of the present study revealed a prevalence of 66% for H. Pylori infection in patients with dyspepsia. But the higher sensitivity of RUT at 37oC was not statistically different from room temperature and the specificity did not differ statistically, as well25,26.
Although some endoscopists keep the RUT specimens in their pocket to keep them warm, few studies have evaluated the effect of temperature on diagnostic accuracy of RUT8. Yousfi and colleagues suggested that using a warmer with 38oC for RUT specimens resulted in a 20% higher diagnostic accuracy in the first 30 minutes, while overall results did not differ significantly12. Also, in the present study, the diagnostic accuracy of warmed specimens were higher in first hours, although the difference was not statistically significant, which could be a result of sampling error or other confounding factors, such as high prevalence of using PPIs by patients in the present study, as studies have indicated that anti-acid medications reduce the sensitivity of RUT10,14. Laine and coworkers have obtained four antral specimens from 200 patients; two for histologic examination, and two for CLO test, one of which were incubated at 37oC and the other at room temperature13. The prevalence of H. pylori infection in their study was 61%, which is close to the prevalence reported in the present study. Laine and colleagues reported that the specimens kept at 37°C had a significantly earlier median time to a positive test and greater sensitivity until 2 hours, while specifities were similar13. The differences between the results of the studies might be due to the different RUT techniques, as we used home-made solution, while Laine and colleagues have used CLO solution for RUT. Moreover, there are many factors affecting sensitivity of the RUT, including using anti-acid medications10,14, presence of blood in samples15, the duration of the gastric ulcer, and the underlying gastric disease that may reduce the bacterial load8,16, which might have resulted in negative results in the present study.
In the present study, the rate of sensitivity and specificity of both groups were higher than some previous studies17,18, while lower than some others7,19, although some studies have adjusted the cut-off reported by the manufacturer.. The differences in diagnostic accuracy rates can be justified by the differences in the methods and techniques used.
The prevalence of positive H. pylori infection was similar to some studies13, whereas most studies in developed countries have reported a prevalence of less than 40% and developing countries have reported a prevalence of 80-90%1,20. Also, Iranian studies have determined various prevalence rates in different cities of Iran2. In Ardebil, Shiraz and Babol, a prevalence of 80% has been reported21,22, while it was reported about 60-70% in Tehran, Nahavand, and Rafsanjan23,24. As indicated by studies, the prevalence depend of various factors such as age, gender, race, and other factors1.
Conclusion:
One of the strengths of the present study included reporting sensitivity and specificity in several intervals that can give researchers a wider spectrum through analysis of RUT. In addition, all the results of RUT and histologies were reviewed by one expert, thus increasing the reliability of the results. On the other hand, the present study had also some limitations, including the confounding factors in the results, such as duration of the disease, the underlying gastric disease, high prevalence of using PPIs by patients, and other factors. Thus, it is suggested that future studies evaluate the effect of warming samples in different RUT methods, considering the confounders, especially duration of the disease and anti-acid therapy, in order to be able to evaluate the pure effect of temperature on diagnostic accuracy of RUT. In conclusion, the results of the present study indicated no significant difference in overall diagnostic accuracy of RUT by warming the specimens.
Conflict of interest: all authors declare that they have no conflict of interest.
Funding: None
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