DETECTION OF HELICOBACTER PYLORI GENETIC MATERIAL IN GALL SAMPLES BY PCR METHOD

Alimentary tract and pancreas

Alimentarni trakt i pankreas

ARCH GASTROENTEROHEPATOL 2001; 20 ( No 3 – 4 ):

Detection of Helicobacter pylori genetic material in bile samples by PCR method

Detekcija genetskog materijala Helicobacter pylori u uzorcima žuči pomoću PCR metoda

( accepted December 15th, 2001 )

¹Milutin Bulajić

²Bojan Štimec

³Miroslav Milićević

⁴Matthias Loehr

⁵Ivan Boričić

³Nada Kovačević

³Mirko Bulajić

¹Clinical Center “Dr Dragiša Mišović”, Belgrade,

² Institute for Anatomy, School of Medicine, University of Belgrade,

³Institute for Digestive Diseases, University Clinical Center of Serbia, Belgrade,

⁴ Department of Gastroenterology, University of Rostock, Germany,

⁵ Institute for Pathology, School of Medicine, University of Belgrade.

Address correspondence to: Dr Milutin Bulajić

Clinic for Internal Medicine

Clinical Center ”Dr Dragiša Mišović”

Heroja Milana Tepića 1 St. YU-11000 Belgrade, Serbia, Yugoslavia

Email: [email protected]

……………………. ……………………………

H.pylori, bile, PCR. Gastroenteroloska sekcija SLD-

01719, 2001

Abstract

The scientists’ interest in the relation of H. pylori infection to various extradigestive diseases has increased recently. The aim of our study is to check and evaluate the specificity and reliability of the polymerase chain reaction (PCR) method, define its significance in research on the H. pylori bile infection and analyze the prevalence of H. pylori infection in some diseases of the biliary tree. The tested group of 87 patients included 41 men and 46 women (47.2% and 52.8%, respectively). Samples of bile were obtained by aspiration during endoscopic retrograde cholangio-pancreatography in 83 patients, and after cholecystectomy in 4 patients. The presence of the genetic material in bile was preliminary triaged through PCR detection of pyruvate dehydrogenase (PDH) gene, and the presence of H. pylori was determined by the nested PCR detection of ure A gene. The results of this reaction were given by electrophoresis on 1% agarose gel by the 256 base pair line. The majority of the patients included in our study suffered from biliary lithiasis (55.2%), while in the rest the inflammation (19.5%) or the tumor of the biliary tree (17.2%) were diagnosed. Seven patients (8.15%) had normal medical findings, being eligible for the control group. The tested patients with normal ERCP findings had positive PCR on H. pylori in 14% cases, while the tested patients with common bile duct and gallbladder tumors had positive PCR on H. pylori in 80% cases. All of the tested patients with the H. pylori positive findings in bile also had positive PCR findings of PDH gene. On the basis of these clinical and laboratory data it is possible to conclude that the PCR method is highly specific for the determination of H. pylori genetic sequence, particularly in cases where its genetic material is found in extremely low concentrations, such as in bile. The prevalence of H. pylori infection was highest in the tested patients suffering from the malignant diseases of the biliary tract.

Key words:

H. pylori infection, PCR, PDH, ureA gene, bile, biliary lithiasis, biliary tumour.

Sažetak

Od skora postoji povećano zanimanje naučnika za vezu infekcije H. pylori i vandigestivnih oboljenja. Cilj našeg istraživanja je bio da se proveri i vrednuje specifičnost i pouzdanost metoda lančane reakcije polimeraze (PCR), odredi njegov značaj u istraživanjima infekcije H. pylori i ispita prevalencija ove infekcije u nekim oboljenjima bilijarnog stabla. Ispitivanu grupu od 87 pacijenata činili su 41 muškarac i 46 žena (47.2% i 52.8%). Uzorci žuči dobijeni su aspiracijom tokom endoskopske retrogradne holangiopankreatografije u 83 pacijenata, i nakon holecistektomije u 4 pacijenta. Prisustvo genetskom materijala preliminarno je trijažirano pomoću PCR detekcije gena za piruvat dehidrogenazu (PDH), a prisustvo H. pylori određeno je “ugneždenom” PCR detekcijom ureA gena. Rezultati ove reakcije prikazani su elektroforezom na 1% agaroza gelu u nivou 256-tog para baza. Većina pacijenata iz studije imala je bilijarnu litijazu (55.2%), dok su kod ostalih postavljene dijagnoze inflamacije (19.5%) ili tumora bilijarnog stabla (17.2%). Sedam pacijenata (8.15%) imalo je normalan nalaz, što ih je uvrstilo u kontrolnu grupu. Ispitivani pacijenti sa normalnim ERCP nalazom imali su pozitivan PCR na H. pylori u 14% slučajeva, dok su ispitanici sa tumorima žučne kesice ili žučovoda imali pozitivan PCR na H. pylori u 80% cases. Svi pacijenti sa pozitivnim PCR na H. pylori imali su takođe i pozitivan PCR na PDH gen. Na osnovu naših kliničkih i laboratorijskih istraživanja moguće je zaključiti da je PCR metod visoko specifičan za određivanje genske sekvence H. pylori, pogotovu u slučajevima kada se genetski materijal nalazi u izrazito niskim koncentracijama, kao što je slučaj u žuči. Prevalenca infekcije H. pylori bila je najveća u pacijenata koji su imali maligna oboljenja bilijarnog trakta.

Ključne reči:

H. pyloriinfekcija, PCR, PDH, ureA gen, žuč, bilijarna litijaza, tumor žučnih puteva

INTRODUCTION

Helicobacter pylori (H. pylori) is a microaerophile gram-negative microorganism which is the main causal agent in the antral gastritis and duodenal ulcer development (1). In the last few years, the interest in the relation of various extradigestive diseases to the H. pylori infection has increased (2). Because of the complexity of the pathophysiological infection mechanisms produced by this microorganism, the diagnostic procedures for the detection of H. pylori infection failed to satisfy the high standards of finding H. pylori in extragastric specimens. A fast urease test for detection of H. pylori infection in stomach oftenly showed false negative or false positive results (3). The microscopic analysis of tissue specimens in various kinds of diseases also proved a non-specific test with low sensitivity for the detection of this organism. The H. pylori cultures require a period of even 3 up to 7 days of incubation. Based on such results, it became necessary to clone the gene for H. pylori urease, a gene specific for this kind. In this manner, a diagnostic procedure with high sensitivity and specificity was achieved (4). Urease genes were sequenced (EMBL acc. N₀ X17079) and the specific H. pylori oligonucleotide primers synthetised (5). By using a pair of these oligonucleotide primers for the detection and the identification of H. pylori, the PCR (polymerase chain reaction) method has now been developed, which, by multiplying the aimed genetic sequence of the specific ure A gene, allows recognition of H. pylori even if present only in a trace, theoretically in only one nucleus. The aim of our study is to check and evaluate the specificity and the reliability of the PCR method, define its significance in the research on H. pylori bile infection and the prevalence of H. pylori infection in some diseases of the biliary tree.

METHODS AND PATIENTS

The study was carried between September 1998 October 1999 at the Institute for the Digestive Diseases (Clinic for Gastroenterology and Hepatology and The First Surgical Clinic) of the University Clinical Center of Serbia in Belgrade. It included a group of 87 patients, 41 men and 46 women (47.2% and 52.8%, respectively). The age range was 11-90 years (average age 57.69±16.45). Eighty-three patients underwent endoscopic retrograde cholangiopancreatography (ERCP) with subsequent aspiration of bile. The samples of bile were obtained during cholecystectomy in 4 patients.

The first step in our examination was a routine check of the entire genetic material in the contents of the examined bile. The reaction by which the presence of the genetic material was proved was pyruvate dehydrogenase (PDH) PCR, i.e. it proved that the gene was responsible for the synthesis of piruvate dehydrogenase, which is the enzyme necessarily present in all cells both eucariotes and lower species, so as in H. pylori. If the existence of PDH genes were proved in some samples of bile, then those PDH positive samples would be included in further examination of the presence of H. pylori in bile. The nucleotide sequence characteristic for the PDH gene is: 5’-GGTATGGATGAGCTGGA-3’ and is used as primer-1, and its pair, primer-2, denoting the opposite direction of the DNA elongation has the following sequence: 5’-CTGCCACAGCCCTCGACTAA-3’.

In order to maintain the sensitivity of the reaction as high as possible, the so-called nested PCR reaction is performed (6). The mechanism of this reaction is that the desired sequences are amplified twice and in that way made highly selective. That is why two PCR reactions are conducted, in which the purpose of the first one is to amplify the examined genome segment, and the other one is to amplify even more the desired segments of the newly made products of the first reaction. The second segments are shorter than the first ones, but more precise in the sense of the identity to the original gene. In that way it is sure that it is the H. pylori, i.e. one of its genes, which is characteristic of that bacteria. These two pairs of primers are showed in Table 1.

The results of PCR were visually presented by electrophoresis on 1% agarose gel by the 256 base pair line (Figure 1).

Table 1. Two pairs of primers designed according to the published H. pylori ureA gene
Outer sense	5’-GCCAATGGTAAATTAGTTCC-3’
Outer antisense	5’-TTACTCCTTAATTGTTTTTAC-3’
Inner sense	5’-TTCTTTGAAGTGAATAGATGC-3’
Inner antisense	5’-ATAGTTGTCATCGCTTTTAGC-3’

RESULTS

On the basis of the clinical testing, the majority of the patients were diagnosed as having biliary lithiasis (55.2%), while in the rest the inflammation of the biliary tree (19.5%) or the tumour of the biliary tree (17.2%) dominated. Seven (8.1%) of the patients had normal medical findings, being eligible to form the control group.

The presence of the genetic material in the samples of bile was verified by the presence of the PDH gene by the PCR method. The genetic material was detected in 75 (87.3%) samples. The specific ure A gene of H. pylori in the samples of bile was positive in 48 (55.2%) of the tested patients, according to the PCR method.

By determining the presence of H. pylori infection in the bile samples by the PCR method, we achieved the following results in patients with normal and abnormal ERCP findings. The patients with normal ERCP findings had a positive PCR in 14% cases, while the patients with common bile duct and gallbladder tumour had a positive PCR in 80% cases. This represents a statistically highly significant difference (Fischer test p=0.006). The patients with non-malignant pathology, compared to the control group, presented no statistical difference in the prevalence of H. pylori infection in bile (Fischer test, p=0.0533). Table 2.

Table 2. H. pylori ureA PCR in bile

	non-malignant biliary diseases	malignant biliary diseases	controls
PCR positive	35	13	1
PCR negative	30	2	6

Apart from that, the examination results indicate the absolute correlation of PDH and ure A nested PCR, in H. pylori positive tested patients, because all the patients (48 tested patients, 100%) who were H. pylori positive in bile, were also PDH positive in bile at the same time.

Figure 1. The DNA H. pylori detection from the bile sample using the nested PCR method and electrophoresis on 2% agarose gel

DISCUSSION

Although it seems impossible for H. pylori to grow in the medium containing bile, some of the Helicobacter species were proved to live in gallbladder (H. hepaticus, H. bilis, H. pullorum and others) (7). Fox et al. have detected H. bilis, H. pullorum, and H. rappini using the PCR method in 23 patients diagnosed with the chronic cholecystitis, although there have not been any cases in which the microorganism grew from the bile culture. On the basis of these facts it is possible that H. pylori causes some of the idiopathic hepatobiliary diseases. H. pullorum is known to cause the infection in humans and poultry, which is manifested by diarrhoea or increased hepatic enzymes or the liver enlargement (8). H. rappini infection, causing abortion in sheep, and acute liver insufficiency in sheep fetus, was isolated in humans suffering from diarrhoea. Finally, H. bilis was proved to cause hepatitis in mice (9).

In 1994, Offner et al. reeeported the presence of proteins of 130 kDa which caused the crystallisation of cholesterol (10). It was also found that CagA protein H. pylori was of identical molecular weight and that both these proteins cross reacted with the human leucinaminopeptidase (11). Finally, Figura et al. have found anti-CagA antibodies in bile samples of 15 out of 16 patients who had undergone cholecystectomy due to gallbladder stones, and who were positive on the CagA H. pylori species ( 12). All these findings support the role of this microorganism in the initiation of cholesterol crystallisation, which induces cholelithiasis.

Recent studies on Helicobacter sp. in different diseases of bile ducts and liver have shown that Helicobacter can survive in human bile, and not only in stomach, and that it can be the cause of various hepatobiliary diseases. For that reason Roe et al. have investigated the survival of Helicobacter sp. in the bile of the patients with various diseases of biliary system, whose bile contained modified bile acids, or bile acids H. pylori was resistant to. Twenty patients with intrahepatic lithiasis, 3 with the pancreas cancer, and 2 with common bile duct carcinoma were included in the study. The bile was obtained by the PTBD method, and checked on the presence of 16rs rRNA specific H. pylori gene by the PCR method, by which the products of 375bp were obtained. After the sequencing and the analysis of the sequences, 20 (80%) of the PCR products corresponded to H. pylori genome, while the remaining 5 (20%) corresponded to H. bilis genome. On the basis of these results, the authors have found that H. pylori is the most important and the commonest cause of the infection of all species of Helicobacter sp. in bile duct diseases in humans. However, the authors do not give an explanation how the infection of bile duct by this bacteria occurs, but suggest its further studying in the future.

It has been pointed out earlier that Helicobacter spp. exist in biliary system and gallbladder of the patients of the Chilean population and that they are one of the main causes of chronic inflammation which can stimulate the forming of gallstones (7). Ponzetto et al. have studied the presence of H.pylori sp. in the bile and mucous membrane of the gallbladder of the patients with cholelithiasis and the prevalence of H. pylori antibodies in their serum (14). The study included 64 patients who had undergone cholecystectomy and from which the bile for the analysis was collected during the operations, together with the mucous membrane of the gallbladder. The serum samples were compared to the samples of 610 blood donours. All serums were tested on the presence the specific IgC antibodies against the H. pylori (ELISA). The bile and the mucous membrane of the gallbladder were tested using the PCR method on the presence of the genetic sequence 16s rRNA Helicobacter sp. In 22 of 64 tested samples of bile, PCR was positive on Helicobacter sp. The infection with H. pylori was significantly more common in patients suffering from cholelithiasis than in control patients, as was also the case with H. pylori DNA in the bile of the diseased.

On the basis of our clinical and laboratory testings, it is possible to conclude that the PCR method is highly specific and sensitive for determining the H. pylori genetic sequences in bile, where its genetic material is found in extremely low concentrations. The PCR for the PDH gene is an excellent triage test for checking the regularity of the subsequent PCR on the target gene. Further, it can also be concluded that some pathological conditions of bile ducts, especially tumours (adenocarcinoma), are possibly related to the presence of H. pylori infection of biliary tracts.

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