HELICOBACTER PYLORI'DE KLARİTROMİSİN DİRENCİNİN MOLEKÜLER YÖNTEMLE SAPTANMASI

Helicobacter pylori gastrik kronik bakteriyel infeksiyona en sık neden olan bakteridir. Eradikasyonda en sık kullanılan tedavi rejimi proton pompa inhibitörü (PPİ) ile amoksisilin, klaritromisin veya metronidazolü içeren iki antibakteriyel ajanın kombinasyonudur. Klaritromisin, H.pylori eradikasyonunda önerilen birçok tedavi protokolünde yer almaktadır ancak, günü- müzde klaritromisin direncinin giderek artması eradikasyon tedavisinin başarı oranını azaltmaktadır. Klaritromisin, antibakteriyel etkisini bakterinin 23 rRNA gen bölgesine bağlanarak göstermektedir. Direnç bakterinin ribozomal komponentindeki 23rRNA peptidil transferaz kodlayan gen bölgesindeki nokta mutasyonlar (A2115G, G2141A, A2141G, A2142G, A2143G, A2144G, A2143C, A2142T, T2717C, T2182C) ile olmaktadır. Dirençten sorumlu olan en sık mutasyonlar A2143G ve A2144G olup bu çalışmada gerçek zamanlı polimeraz zincir reaksiyonu (PZR) metodu ile bu mutasyonların tespiti amaçlanmıştır. Gastroenteroloji polikliniğine dispeptik şikayetler ile başvuran ve endoskopi yapılması kararlaştırılan 66 kadın, 55 erkek olmak üzere toplam 121 hasta araştırmaya alınmıştır. Hastaların midesinde antrum bölgesinden üç adet biyopsi alınmıştır. Dokudan DNA ekstraksiyonu yapılmıştır. Dokuda PZR yöntemi ile H.pylori pozitif bulunan 78 örnekte gerçek zamanlı PZR yöntemi ile 23S rRNA’daki A2143G ve A2144G nokta mutasyonlarına bağlı klaritromisin direnci araştırılmıştır. Yirmi iki örnekte direnç mutasyonu belirlenmiştir. Beş örnekte 57 ve 65±2ºC’de iki pik görülmesi nedeniyle hastaların iki farklı H.pylori suşu ile infekte oldukları düşünülmüştür. Ayrıca beş örnekte 83±2ºC pik görülmesine karşın (H.pylori pozitif), 57 ve 65±2ºC’de pik belirlenememiştir. Bu nedenle bu beş örnek istatistiksel değerlendirmelere dahil edilmemiştir.

Determination of Clarithromycin Resistance in Helicobacter pylori by Molecular Method

Helicobacter pylori is the most common cause of gastric bacterial infection. Among numerous treatment regimens that have been used to eradicate this microorganism, the most recent recommendation is triple therapy, including a proton pump inhibitor and either amoxicillin and clarithromycin or metronidazole and clarithromycin. However, the development of macrolide resistance, mainly clarithromycin, in H.pylori is the main risk factor for failure of the standard triple therapy. Given that clarithromycin acts to halt bacterial growth by physically interacting with the 23S rRNA component of the bacterial ribosome, resistance to clarithromycin in clinical H. pylori isolates is caused predominantly by distinct point mutations within the peptydil transferase-coding region of 23S rRNA gene. While A2144G, A2143G, A2142G and A2143C are the four most commonly observed mutations, others like A2142C, A2115G, G2141A, A2142T and T2717C have been described but appear to be infrequent. This study aimed to detect the most common mutations A2142G and A2143G by real-time polimerase chain reaction (PCR). A total of 121 patients who admitted to gastroenterology outpatient clinic with dyspeptic complaints and underwent endoscopy for investigation of dyspeptic symptoms were included in the present study conducted. Three antral biopsy specimens were obtained from each patient. DNA extraction from tissue was performed. H.pylori was detected in 78 patientsby PCR. Clarithromycin resistance due to point mutations (A2142G and A2143G) in 23s rRNA gene were investigated via realtime PCR combined with melting curve analysis in H.pylori positive samples. Resistance mutations to clarithromycin were detected in 22 isolates. Based on identification of two peaks at 57 and 65±2ºC in five samples, patients were considered to be infected with two different H.pylori strains. Additionally, while 83±2ºC peak was observed in five samples (H.pylori positive), no peaks were determined at 57 ve 65±2ºC. For this reason, these five samples were not included in the statistical analysis.

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