An alarmingly increasing number of outbreaks caused by contaminated gastrointestinal (GI) endoscopes are being reported as a particularly concerning issue. This study is the first large-scale multicenter survey to evaluate the contamination of GI endoscopes in Tehran, Iran. This multicenter study was conducted among 15 tertiary referral and specialized gastrointestinal settings. Reprocessed GI endoscopes were sampled by the sequence of the flush-brush-flush method. Bacterial and viral contamination, as well as antimicrobial resistance, were explored by culture and molecular assays. A total of 133 reprocessed and ready-to-use GI endoscopes were investigated. In phase I and phase II, 47% and 32%, respectively, of the GI endoscopes were determined to be contaminated. GI flora was the most prevalent contaminant isolated from GI endoscopes, in which the most predominant bacteria were Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae, in both phase I and II evaluations. The majority of the isolated bacteria in the current study were considered multidrug-resistant organisms (MDROs). More importantly, we recovered carbapenem-resistant nonfermentative Gram-negative bacilli (CRNFGNB), carbapenem-resistant Enterobacterales (CRE), extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (ESBL-E), multidrug-resistant Clostridioides difficile, vancomycin-resistant Enterococcus (VRE), and drug-resistant Candida spp. Disconcertingly, our molecular assays revealed contamination of some reprocessed GI endoscopes with hepatitis B virus (HBV), hepatitis C virus (HCV), and even HIV. This multicenter study indicates a higher-than-expected contamination rate among reprocessed and ready-for-patient-use GI endoscopes, which suggests a higher-than-expected endoscopy-associated infection (EAI) risk, and potentially, morbidity and mortality rate, associated with endoscopy procedures in Tehran, Iran. IMPORTANCE In the light of severe outbreaks caused by multidrug-resistant microorganisms due to contaminated GI endoscopes, understanding to what extent GI endoscopes are inadequately reprocessed is crucial. Several studies assessed contamination of GI endoscopes with various outcomes across the world; however, the prevalence and risk factors of contaminated GI endoscopes and potential subsequent nosocomial spread are still unknown in Iran. The present study is the first large-scale multicenter survey to evaluate the microbial contamination of repossessed and ready-to-use GI endoscopes in Tehran, Iran. Our study showed a higher-than-expected contamination rate among reprocessed GI endoscopes, which suggests potential seeding of deadly but preventable outbreaks associated with endoscopy procedures in Iran. These results suggest that the current reprocessing and process control guidelines do not suffice in Iran. The current study is of particular importance and could provide insights into unrecognized and unidentified endoscopy-associated outbreaks in Iran.

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