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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

Year: 2026

Type: article

Abstract: 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 < 35 and using Seegene as reference yielded very high MTBC agreement between kits: Viasure was more sensitive (0.97) but slightly less specific (0.89), while Sansure was less sensitive (0.84) but almost perfectly specific (0.99); NTM performance could not be reliably assessed in this scheme due to the scarcity of Seegene NTM positives after applying the Ct filter. Our findings show that Viasure and Seegene substantially outperform smear microscopy and culture for MTBC detection in both pulmonary and extrapulmonary specimens, with Viasure offering the best overall balance and clearly superior NTM detection. Sansure behaves as a very specific but under-sensitive option, contributing comparatively few additional diagnoses beyond conventional methods. In medium to high-burden settings where rapid and accurate differentiation of TB and NTM from clinical samples is crucial, these results support the use of highly sensitive PCR assays (particularly Viasure and Seegene) as central tools within diagnostic algorithms, complemented by culture for drug susceptibility testing and full species characterization. IMPORTANCE: and non-tuberculous mycobacteria, comparing them with standard methods. This evaluation was made with an extensive population of more than 400 pulmonary and extrapulmonary clinical samples. We found that two of the commercial kits are equivalent in diagnostic performance and outperformed traditional methods for mycobacterial diagnosis, while the third kit had a very poor performance. To the best of our knowledge, for two of the three commercial kits included in the study, there are no publications evaluating their clinical performance. This study provides useful information for the clinical microbiology community related to mycobacterial diagnosis, especially for low- and middle-income settings where smear microscopy and culture are still widely used, and where automatic systems like Gene Xpert still are economically challenging.

Source: Microbiology Spectrum

DOI: 10.1128/spectrum.00158-26