<p>Sources and clinical specimen types.</p>

<div><p>Effective control of tuberculosis, nontuberculous mycobacterial (NTM) disease, and melioidosis depends on rapid and accurate laboratory diagnosis. <i>Mycobacterium tuberculosis</i> complex (MTBC) remains a health priority, whereas NTM infections, particularly <i>Mycobacterium avium</i> complex (MAC), are increasingly recognized and may be misdiagnosed as tuberculosis. Melioidosis, caused by <i>Burkholderia pseudomallei</i>, is underdiagnosed in endemic regions.

These infections have overlapping clinical presentations, and conventional diagnostics methods may not accurately differentiate among these diseases. We developed and evaluated a quadruplex real-time PCR assay (TAM-mPCR) for simultaneous detection of MTBC, MAC, and <i>B. pseudomallei</i>, targeting <i>IS6110</i>, <i>rrs</i>, and <i>BPSS1187</i>. A total of 607 pulmonary and extrapulmonary specimens from patients with suspected tuberculosis were collected at two sites in Thailand between 2024 and 2025.