Detekcija gena virulencije otoka patogenosti cag i gena dupA izolata Helicobacter pylori nakon višestruke neuspjele eradikacijske terapije

Varda Brkić, Dijana (2016) Detekcija gena virulencije otoka patogenosti cag i gena dupA izolata Helicobacter pylori nakon višestruke neuspjele eradikacijske terapije. PhD thesis, Sveučilište u Zagrebu.

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Abstract

BACKGROUND Certain virulence genes including cagA located in the pathogenicity island cagPAI (eng. citotoxin pathogenicity island) of Helicobacter pylori are associated with gastroduodenal diseases and thus the variation of cagPAI might influence various clinical syndroms. According to some studies dupA gene located in plastic region (eng. plasticity region) of H. pylori is associated with development of duodenal ulcer and has protective role against development of atrophy and gastric metaplasia. AIM The aim of this study was to determine the frquency of virulence genes within cagPAI island (Apcag, cagA1, cagA2, cagA3, cagM, cagT, cagE, LEC, tnpA i tnpB) and dupA gene in Helicobacter pylori isolates obtained from patients after multiple unsuccessful antimicrobial therapy, and to analyze the correlation between the presence of these genes and endoscopic diagnosis and pathohistological alterations of gastric mucosa. MATERIALS/METHODS Virulence genes of 103 H. pylori isolates were detected by PCR. According to endoscopid diagnosis the patients were classified into three groups: non-ulcer dyspepsia (NUD) (n=69), erosio/ulcus ventriculi (EUV) (n=22) and erosio/ulcus duodeni (EUD) (n=12). Pathohistological findins were interpreted according to Sydney classification sheme. RESULTS Single prevalence of cagPAI gene was as follows: Apcag 63.1%, cagA1 71.8%, cagA2 69.9%, cagA3 5.8%, cagM 71.8%, cagE 75.7%, cagT 68% tnpA 9.7%, tnpB 6.3% i LEC 48.5%. The frequency of dupA was 34. There was no correlation between 10 analysed genes of cagPAI island with endoscopic diagnosis (P>0.16). The presence of CagA, Apcag i cagM gene was associated with higher grade of pathohistological parameters of gastritisa (p<0.05). DupA gene was not found in patients with duodenal ulcer disease from Nortwest Croatia. DupA gene was significantly more frequent in patients with non-ulcer dyspepsia and gastric ulcer disease (P=0.016). There was no statistically significant difference in gastritis intensity score either in antrumu (P=0.434), or in corpus (P=0.084) in relation to the presence of dupA gene. More then 50% of the isolates were resistant to both macrolides and metronidazol. CONCLUSION Our study demonstrated high frequency of cagA, Apcag i cagM genes in patient’s isolates and their correlation with high grade of pathohistological alterations in gastric mucosa, representing a risk factor for development of ulcer, premalignant and malignant diseases. There was no correlation between the presence of dupA gene and duodenal ulcer. Moreover, no significant difference in the frequency of dupA gene according to the type of gastritis was found. Repeated attempt to eradicate H. pylori seems to be the best choice for the patient. Concerning the unsatisfactory efficiency of triple therapy based on clarithromycine due to increasing resistance, new therapeutic options based on the new treatment strategies should be implemented in order to increase the level of eradication (new antibiotics or combination of antibiotics).

Abstract in Croatian

UVOD Nekoliko gena virulencije, uključujući i cagA smještenih u otoku patogenosti cagPAI (eng. citotoxin pathogenicity island) Helicobacter pylori povezuje se s gastroduodenalnim bolestima i stoga varijacije u genskoj strukturi cagPAI mogu biti odgovorne za različite kliničke ishode. Prema nekim istraživanjima dupA gen smješten u plastičnoj regiji (eng. plasticity region) H. pylori povezuje se s razvojem duodenalnog ulkusa i ima protektivnu ulogu u razvoju atrofije i intestinalne metaplazije. CILJ Cilj ovog istraživanja bio je utvrditi učestalost gena virulencije cagPAI otoka (Apcag, cagA1, cagA2, cagA3, cagM, cagT, cagE, LEC, tnpA i tnpB) i dupA gena Helicobacter pylori izolata bolesnika nakon višestruke neuspjele eradikacijske terapije, te ustanoviti njihovu povezanost s endoskopskom dijagnozom i patohistološkim promijenama želučane sluznice. MATERIJALI/METODE Geni virulencije 103 H. pylori DNA izolata detektirani su PCR metodom. Bolesnici su prema endoskopskoj dijagnozi svrstani u tri grupe: neulkusna dispepsija (NUD) (n=69), erozije/ulkus želuca (EUV) (n=22) i erozije/ulkus duodenuma(EUD) (n=12). Patohistološki nalazi skorirani su po Sydney-skoj klasifikaciji. REZULTATI Pojedinačna učestalost cagPAI gena bila je slijedeća Apcag 63.1%, cagA1 71.8%, cagA2 69.9%, cagA3 5.8%, cagM 71.8%, cagE 75.7%, cagT 68% tnpA 9.7%, tnpB 6.3% i LEC 48.5%. Učestalost dupA bila je 34.0%. Nije utvrđena statistički značajna povezanost prisutnosti niti jednog od 10 detektiranih gena cagPAI otoka s endoskopskom dijagnozom (P>0.16). Za CagA, Apcag i cagM gene utvrđena je statistički značajna povezanost s višim stupnjem patohistoloških parametara kroničnog gastritisa (p<0.05). Nije utvrđena prisutnost dupA gena u bolesnika s duodenalnom ulkusnom bolesti iz sjeverozapadne Hrvatske. Nađena je značajno veća učestalost dupA gena u bolesnika s neulkusnom dispepsijom te s ulkusnom bolešću želuca (P=0.016). Nije utvrđena statistički značajna razlika u skoru intenziteta gastritisa ni u antrumu (P=0.434), ni u korpusu (P=0.084) prema zastupljenosti dupA gena. Više od 50% izolata pokazivalo je rezistenciju i na makrolide i na metronidazol. ZAKLJUČAK Naše istraživanje ukazalo je na visoku frekvenciju cagA, Apcag i cagM gena u izolatima bolesnika i njihovu povezanost s višim stupnjem patohistoloških promjena u želučanoj sluznici, što može ukazati na pojačani rizik za razvoj ulkusne, premaligne i maligne bolesti u bolesnika s bezazlenim endoskopskim nalazom. Nije nađena povezanosti dupA gena i duodenalnog ulkusa u bolesnika, kao ni statistički značajna zastupljenosti dupA gena prema dominaciji gastritisa. Ponovna eradikacija H. pylori infekcije kod tih bolesnika nameće se kao jedini pravilan izbor. S obzirom na nezadovoljavajuću djelotvornost uobičajene trojne terapije zasnovane na klaritromicinu zbog rastuće antibiotske rezistencije, nameće se potreba za promjenama strategije liječenja koje bi povećale stupanj eradikacije (novi antibiotici ili nove kombinacije lijekova).

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