Comparative evaluation of three commercial real-time PCR kits for direct detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria from pulmonary and extrapulmonary clinical samples: experiences from a low-income and high-burden setting.

Publication date: May 18, 2026

The accurate and timely detection of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) remains a diagnostic challenge, particularly in extrapulmonary samples and low-resource settings. This study aimed to evaluate and compare the clinical performance of three commercial real-time polymerase chain reaction (PCR) assays, VIASURE M. tuberculosis complex + non-tuberculous mycobacteria Real-Time PCR Detection Kit (“Viasure”), Mycobacterium Multiplex Nucleic Acid Diagnostic Kit (“Sansure”), and Anyplex MTB/NTMe Real-Time Detection (“Seegene”), against conventional diagnostic methods (smear microscopy and culture) for the detection of MTBC and NTM in pulmonary and extrapulmonary samples. We analyzed 407 clinical samples from patients with suspected tuberculosis (TB) or other mycobacterioses. All samples were tested by smear microscopy, mycobacterial culture, and three real-time PCR assays. First, we compared the kits against smear and culture separately (scheme 1). Second, we used a composite reference standard (CRS) that classified a sample as positive if any of the five tests were positive and evaluated smear, culture, and the three PCRs for MTBC and NTM (scheme 2). Third, we compared Viasure and Sansure to Seegene as a reference, excluding PCR positives with Ct ≥ 35 to focus on high-confidence results (scheme 3). Overall positivity was lowest for smear (14. 8%; 41/278) and culture (32. 7%; 48/147), intermediate for Sansure (18. 7%; 52/278), and highest for Viasure (60. 1%; 167/278) and Seegene (53. 2%; 148/278), both in pulmonary and extrapulmonary samples. In scheme 1, using smear or culture as references, Viasure and Seegene showed high sensitivity but modest specificity, which is, in fact, attributable to the poorer clinical performance of the reference methods. Under the CRS in scheme 2, smear and culture detected only about 20% and 46% of CRS-positive cases, respectively, whereas Viasure and Seegene captured most MTBC infections (total MTBC sensitivities of 0. 73 and 0. 71, respectively). For both schemes 1 and 2, Sansure was clearly less sensitive than the other kits to detect MTBC. For NTM, Viasure markedly outperformed the other kits (total sensitivity 0. 88; specificity 0. 98), whereas Sansure and Seegene showed very low NTM sensitivity (0. 19 and 0. 13) despite near-perfect specificity. In scheme 3, restricting analyses to Ct

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