Abstract

AIM To develop a “semi-quantitative RUN test” method for diagnosing Hp infection in human gastric mucosa.
METHODS After quantitating the relative number of Hp organisms in biopsy specimens and determining the OD value of parallel specimens, the result of the RUN test was designated into three grades. Three hundred and fifty patients undergoing gastroscopy were diagnosed by the semi-quantitative RUN test.
RESULTS ① The added urea could amplify the li-near range of “RUN test”, but not affect the absorption peeks of tested solution. ② The correspondence rate between Hp infection degrees and OD values was 88.9%. ③ Slight Hp infection was found in over half of the 350 patients tested, suggesting that the development of chronic gastritis might be associated with different Hp infection degrees.
CONCLUSION The semi-quantitative RUN test method could be used for both qualitative and semi-quantitative diagnosis of Hp infection.

INTRODUCTION

Today Helicobacter pylori (Hp) is regarded as an essential factor in the pathogenesis of chronic gastritis and peptic ulcer^[1] . The relationship between Hp infection and gastric cancer has also been confirmed^[2] . It has been recommended that all patients with Hp infection and gastric mucosal lesions should receive antimicrobial therapy^[1,3] . This implies that the diagnosis of Hp infection and the confirmation of its eradication are necessary for guiding the clinical therapy of the patients^[4] , and a quantitative method for determining the degree of Hp infection may be a useful guideline.
In our previous article, we reported a rapid and sensitive method for diagnosing Hp infection in human gastric mucosa based on Nessler′s reaction, the “Rapid Urease-Nessler′s test” (RUN test)^[5] . In this study, a semi-quantitative method for determining the degree of Hp infection with a definite judging criterion was developed, and results of the clinical use were analyzed.

MATERIALS AND METHODS
Specimens

Gastric mucosal biopsy specimens were collected from patients undergoing gastroscopy and routine Hp examination, and at the same time blood samples were taken for an Hp test as described^[5] .

Scan of absorption spectra

The samples of 0.6 mmol/ L (NH₄ )₂ SO₄ solution (containing 0.133 mol/L urea or without urea) and Hp positive biopsy specimens, were reacted with Nessler′s reagent and scanned with spectrophotometer (BECKMEN DU 65). The results of the scanning were analyzed.

Determination of linear range of RUN test system

To each of the wells coated with 0.133 mol/ L urea or without urea, added 100 μl of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 or 1.4 mmol/ L standard (NH₄ )₂ SO₄ solutions separately and then 50 μl 1∶4 diluted Nessler′s reagent^[5] . After 10 minute reaction, the wells were read with microtiter reader (MINIREADER Ⅱ, Dynate Inc.) at a wavelength of 410 nm. The microtiter reader was calibrated in advance with a control well (100 μl distilled water +50 μl diluted Nessler′s reagent).Designation of Hp infection degrees
For designating the judging criterion of “semi-quantitative RUN test”, 2 specimens were taken from the same mucosal site of each Hp positive patient diagnosed with RUN test. A total of 36 specimens from 18 patients were collected. One specimen of each patient was fixed in 10% formalin and its stained sections were examined by a pathologist. The mean relative number of Hp organisms of each specimen was calculated from 3 fields under oil-immersion objective. The Hp infection degrees were divided as follows: a Hp number of 1-15 field was designated as light Hp infection (+); 15-30, mo-derate Hp infection (+ +); and over 30, severe Hp infection (+ + +). The other specimen of each patient was reserved for the semi-quantitative RUN test. The coincidence rate was 88.9% between Hp infection degrees and OD values.

Semi-quantitative RUN test

In the semi-quantitative RUN test, the reagents and procedures were the same as in the RUN test^[5] . The only difference was in the way of result judgment. When a specimen was judged as Hp positive with RUN test within 5 minutes by naked-eye observation^[5] , the test well had to be placed under room temperature for another 5 minutes and then was read with a microtiter reader at 410 nm. The microtiter reader was calibrated in advance with 3 control wells [100 μl 0.25 mmol/ L (NH₄ )₂ SO₄ solution + one drop diluted Nessler′s reagent]. According to the above designation of Hp infection, 01.00, severe Hp infection (+ + +).

RESULTS
Absorption spectra of reacted solution

Figure 1 shows the absorption spectra of the reacted samples: 0.6 mmol/ L (NH₄ )₂ SO₄ solution (curve a), 0.6mmol/ L (NH₄ )₂ SO₄ containing 0.133 mol/ L urea (curve b) and Hp positive specimen (curve c), reacting with Nessler′s reagent respectively. The results showed that all the 3 kinds of test solutions had the same major absorption peaks at 415 nm and a shoulder peak at 410 nm, which might permit the test solution to be read by a microtiter reader with a filter of 410 nm or 415 nm.

Effect of urea on linear range of “RUN test” system

Figure 2 shows that the serial concentration of (NH₄ )₂ SO₄ solution (containing 0.133 mol/ L urea or without urea) reacted with diluted Nessler′s reagent in microtiter wells for 10 minutes and the reacted wells were read by microtiter reader at 410 nm while the microtiter reader was calibrated with distilled water. The results showed that the reacted solution containing urea (curve b) had wider linear range than that without urea (curve a). When the microtiter reader was re-calibrated with control wells [100 μl 0.25 mmol/ L (NH₄ )₂ SO₄ + 50 μl dilu-ted Nessler′s reagent], no linear range change occurred in curve c. These results not only proved that urea could inhibit the Nessler′s reaction, but also showed that urea could amplify the linear range of RUN test system.

Results of semi-quantitative RUN test

Three hundred and fifty Hp positive patients were studied with the semi-quantitative RUN test. Among them, 54.6% patients had light Hp infection (+) and 34.0% moderate Hp infection (+ +), but only 11.4% had severe infection (+ + +). These results revealed that most of the patients (about 88.6%) had light and moderate Hp infection. The association between Hp infection degrees and histological changes of gastric mucosa was also analyzed (Table 1). The results revealed that there was a significant correlationship between Hp infection degrees and chronic superficial gastritis.

Figure 1 The absorption spectra of reacted solution with Nessler′s reagent
a. 0.6 mmol/ L (NH₄ )₂ SO₄ solution
b. 0.6 mmol/ L (NH₄ )₂ SO₄ +0.133 mol/ L urea
c. Hp positive biopsy specimen

Figure 2 Effect of urea on the linear range of “RUN test”
a. (NH₄ )₂ SO₄ sultion
b. (NH₄ )₂ SO₄ +0.133 mol/ L urea
c. rereading curve b after re-calibrating microtiter reader
Table 1 Association between Hp infection degrees and gastric diseases

DISCUSSION

In our previous paper^[5] , we described that the principle of RUN test was different from the traditional urease test. The RUN test method was based on detecting the NH₄ ⁺ in gastric mucosa produced by the high Hp urease hydrolytic activity on urea. The difference of ammonia contents between Hp infected biopsy specimens and non-infected specimens could be quantitatively determined with the Nessler′s reaction^[5] . This result gave us an idea of developing a quantitative method for diagnosing Hp infection degrees in gastric mucosa. In practice, however, we found that the sizes of biopsy specimens were not uniform and that the absolute quantitative detection was impossible and unnecessary. Consequently we divided the Hp infection into three grades: light (+), moderate (+ +) and severe (+ + +). This designation accomplished a good correspondence rate of 88.6% between Hp infection degrees and instrument determining results of the semi-quantitative RUN test, which might mean a good repetition. The procedure of the RUN test was also changed a little, so that it could be used for both qualitative and semi-quantitative diagnosis of Hp infection.
The results of the semi-quantitative RUN test showed for the first time that the severe Hp infection patients account for only 11.4%. The clinical significance of this result needs further studies. An analysis of the association between the degree of Hp infection and histological changes of gastric mucosa suggested that the gastric diseases were not only associated with Hp infection^[1,2,5] , but also correlated with the degree of Hp infection.
After all, our studies showed and proved that the newly established “RUN test” method is a rapid and sensitive method for both qualitative and semi-quantitative determination of Hp infection.

¹ Cancer Research Center of Xiamen University, Xiamen 361005, China
² Department of Gastroenterology, Xiamen Zhongshan Hospital, Xiamen 361005, China
Dr. CHEN Rui-Chuan, male, born on 1963-05-02, in Xiamen, Director of Biochemical Laboratory, having 15 papers published.
Correspondence to: Dr. CHEN Rui-Chuan, Cancer Research Center of Xiamen University, Xiamen 361005, China
Received 1997-08-21

REFERENCES

1 Sipponen P, Hyvarinen H. Role of Helicobacter pylori in the pathogenesis of gastritis, peptic ulcer and gastric cancer. Scand J Gastroenterol, 1993;28(suppl. 196):3-6
2 Bechi P, Balzi M, Becciolini A, Maugeri A, Reggi CC, Amorosi A et al. Helicobacter pylori and cell proliferation of the gastric mucosa possible implications for gastric carcinogenesis. Am J Gastroenterol, 1996;91(2):271-276
3 Craanen ME, Blok P, Dekker W, Tytgat GNJ. Helcobacter pylori and early gastric cancer. Gut, 1994;35(10):1372-1374
4 National Institutes of Health. Consensus Conference. Helicobacter pylori infection in peptic ulcer disease. JAMA, 1994;272(1):65-69
5 Chen RC, Zhong CG, Wu YH, Yang SM. Diagnosis of Helicobacter pylori infection Ⅰ: a qualitative method and its application. WCJD, 1998;6(3)192-194