Phage-based diagnostic assay for melioidosis using multiplex PCR targeting Burkholderia phage terminase genes

Conventional diagnostic approaches involve identifying the bacterial pathogen from patient samples, which can delay results and miss low-bacteremia cases. Herein, we demonstrate that <i>Burkholderia</i> phages circulating in patient blood can serve as a powerful diagnostic biomarker. Building on this, a multiplex PCR targeting the <i>Burkholderia pseudomallei</i> phage terminase gene was developed to improve melioidosis diagnosis. The assay provided 97.2% sensitivity and 100% specificity, with results obtainable within 6 h. The strength of this PCR assay is the amplification of four different multicopy <i>Burkholderia</i> phage terminase genes from serum samples, resulting in increased sensitivity under low-bacteremia conditions. To our knowledge, this study reports a phage-based PCR assay that detects phage DNA directly from melioidosis patients' blood, representing a shift from molecular pathogen-based to phage-based diagnostics. This approach not only improves sensitivity but also opens avenues for integrating phage biology into the diagnostic landscape of infectious diseases.