Comparison of diagnostic accuracy and sensitivity of Kinyoun and auramine-rhodamine stains in detecting mycobacteria from specimen smears

ABSTRACT Smear microscopy using carbolfuschin- or auramine-based staining is widely employed for rapid mycobacterial detection from patient specimens. While auramine stains are preferred to detect M. tuberculosis complex (MTBC), there are little data on the relative performance of these stains for the detection of nontuberculous mycobacteria (NTM). This retrospective study evaluated the diagnostic accuracy of Kinyoun and auramine-rhodamine (AR) stains on clinical specimens from a tertiary cancer center (2017–2025), using culture-based identification as the gold standard. Of 16,294 specimens, 651 (4.0%) recovered mycobacteria, predominantly Mycobacterium avium complex (MAC) (56.2%) and MTBC (14.4%). Both stains demonstrated high specificity (>99.9%) but limited sensitivity (17.4% overall, 11.2%–11.8% among index cases) for mycobacteria, with higher sensitivity for MTBC (40.4%–43.6%) than NTM (12.9%–13.5%) ( P < 0.0001). 1.7% of mycobacteria-positive specimens had discordant qualitative AR vs Kinyoun smear results: AR detected three MTBC and three MAC cases missed by Kinyoun stain, while Kinyoun stain detected five rapid-growing mycobacteria (RGM) cases not detected by AR, though differences were not statistically significant ( P > 0.05). Semi-quantitatively (1-4+), of 107 AR+/Kinyoun+ smears, AR was one grade higher in 9 specimens (3 MTBC, 5 MAC, 1 RGM), while Kinyoun was one grade higher in 3 specimens (2 MAC, 1 RGM). A significant increase in frequency of NTM isolation in culture over the study period was observed (P = 0.02), but MTBC frequency remained stable. Given time and resource considerations, even where NTM predominates in prevalence, AR is appropriate as the primary mycobacterial stain, but carbolfuschin-based staining should be considered when RGM is suspected. IMPORTANCE Accurate and rapid detection of mycobacteria is essential for timely patient management, especially in vulnerable immunocompromised patients. This study analyzes more than 16,000 clinical specimens over 8 years and provides a direct comparison of auramine rhodamine and Kinyoun stains for mycobacterial detection on smear microscopy. Both stains show high specificity and comparable overall performance, but Kinyoun exhibits a modest trend toward improved the detection of rapid growing mycobacteria. These findings offer guidance for clinical laboratories performing smear microscopy for both tubercular and non-tuberculous mycobacteria.