Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 5 2026 10 05 765 772 Zahra Rouhipanah Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Seyed Sajjad Alavi Kakhki Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Mehdi Fatahi-Bafghi Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Javad Charostad Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Akram Astani Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; Hematology and Oncology Research Center, Non-Communicable Diseases Research Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 2025 10 07 2026 09 08 Background and Objectives: Gastric cancer remains a leading cause of cancer mortality and exhibits significant geographic variation. Although Helicobacter pylori (H. pylori) is a well-established carcinogen, the contributions of Epstein–Barr virus (EBV) and human papillomavirus (HPV), individually or in conjunction with H. pylori, are not fully understood. This study assessed the prevalence and co-detection of these pathogens in gastric tissues from Yazd, Iran. Materials and Methods: An archive-based cross-sectional study was conducted using 130 formalin-fixed, paraffin-embedded (FFPE) gastric specimens collected between 2019 and 2022, comprising 83 gastric adenocarcinomas and 47 non-malignant tissues. H. pylori was detected by conventional PCR targeting 16S rRNA, while EBV and HPV were identified by nested PCR targeting EBNA-1 and L1, respectively. PCR products were confirmed by agarose gel electrophoresis, and selected amplicons were verified by Sanger sequencing. Results: H. pylori was detected in 74 of 130 specimens (56.9%), with a prevalence of 62.7% in malignant tissues and 46.8% in non-malignant tissues (p=0.080). EBV was identified in three malignant cases (3.6%) and HPV in one malignant case (1.2%), with no positive findings in non-malignant samples (p=0.187 and p=0.450, respectively). Three co-detections were observed in malignant tissues, including two cases of H. pylori with EBV and one case of H. pylori with HPV. Due to the limited number of EBV- and HPV-positive cases, association analyses lacked statistical power. Conclusion: H. pylori was the predominant microorganism detected, while EBV and HPV were rare and co-detections were infrequent. Larger prospective studies employing complementary molecular techniques and H. pylori virulence profiling are required to elucidate the clinical significance of these infections in gastric carcinogenesis. https://ijm.tums.ac.ir/index.php/ijm/article/view/5846 https://ijm.tums.ac.ir/index.php/ijm/article/download/5846/1937