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Background: Pneumocystis jirovecii is an opportunistic fungal pathogen that causes Pneumocystis pneumonia (PCP) in immunocompromized hosts. PCP is associated with substantial morbidity, and mortality rates range from 10% to 40%. The diagnosis of PCP relies on the microscopic detection of P. jirovecii in stained clinical samples. Polymerase chain reaction (PCR) may provide better sensitivity than microscopy; therefore, evaluation and implementation of PCR assays are required for the detection of Pneumocystis infection. P. jirovecii is not cultivatable, therefore molecular tools are used for characterizing P. jirovecii genotypes; common targets are the dihydropteroate synthase (DHPS) and mitochondrial large subunit rRNA (mtLSU rRNA) genes. DHPS is a therapeutic target; mutations may be associated with co-trimoxazole prophylaxis and treatment failure. Polymorphisms in mtLSUrRNA have been used for phylogenetic studies.