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Pneumocystis is an opportunistic infection that mainly affects immature patients. The introduction of molecular biology in the diagnosis of pneumocystis has improved the sensitivity of pathogen detection. The aim of our study is to demonstrate the contribution of real-time PCR compared with staining techniques. Our study is retrospective (January 2022-August 2023), and includes 6 cases of pneumocystis. In 4 cases (67%), co-infections involving Pneumocystis jirovecii were detected in association with HIV, CMV and SARS-CoV-2. Co-infection with Mycobacterium tuberculosis was observed in 1 case (17%). Conventional staining with MGG and Musto-modified Gomori Grocott was negative in all cases. However, real-time PCR analysis showed positive results for all samples, demonstrating 100% sensitivity. Parasite load levels ranged from 10 to106 copies/mL. Quantitative PCR is a highly sensitive technique, with less risk of contamination, and helps clinicians distinguish between colonization and infection.