Quantitative real-time PCR in the diagnosis of opportunistic infections in immunocompromised patients: Diagnostic performance, clinical utility, and future directions

Abstract Opportunistic infections (OIs) remain a major cause of morbidity and mortality among immunocompromised individuals, and delayed or inaccurate diagnosis can rapidly lead to adverse clinical outcomes. This narrative review critically evaluates the diagnostic performance, clinical utility, and evolving applications of quantitative real-time polymerase chain reaction (qPCR) for detecting opportunistic pathogens in vulnerable patient populations. Evidence across bacterial, fungal, viral, and parasitic infections demonstrates that qPCR consistently surpasses conventional culture- and serology-based methods in sensitivity, specificity, and turnaround time, particularly in cases of low pathogen burden or prior antimicrobial exposure. Its quantitative capability enables dynamic pathogen load monitoring and facilitates timely, targeted therapeutic interventions. However, limitations such as assay variability, incomplete standardization, and challenges in distinguishing colonization from active infection in polymicrobial contexts persist. Ongoing advances in multiplex technologies, digital PCR, and integrative data-driven diagnostics are expected to further refine clinical implementation. Collectively, qPCR has evolved from a supplemental assay to a cornerstone technology in precision diagnostics for OIs in immunocompromised patients.