Advances in the detection of deadly free-living amoebae (FLA).

Free-living amoebae (FLA), including Naegleria fowleri, Acanthamoeba castellanii, Balamuthia mandrillaris, and Sappinia pedata, are ubiquitous protozoa capable of causing severe infections such as primary amoebic meningoencephalitis (PAM), granulomatous amoebic encephalitis (GAE), and Acanthamoeba keratitis (AK). Early diagnosis remains challenging due to disease rarity, nonspecific clinical presentation, and limited access to specialized laboratory methods. Rapid and accurate detection is critical for patient management and public health response, particularly amid changing environmental exposures. This review summarizes current diagnostic approaches in clinical and environmental contexts, including specimen handling, microscopy, culture, immunohistochemistry, antigen detection, and molecular methods such as conventional PCR, real-time PCR, multiplex qPCR, LAMP, and metagenomic next-generation sequencing. Environmental surveillance, biomarker discovery, quality assurance, and standardized protocols are also discussed. By evaluating strengths and limitations of available tools, this review highlights diagnostic gaps and future priorities to enhance sensitivity, turnaround time, and global accessibility.