Between January 2016 and March 2019, data from inpatients (≥18 years) undergoing evaluation for PTB (having PTB-related symptoms and/or signs, or unexplained cough lasting ≥2 weeks, or unexplained findings on chest radiographs suggestive of PTB) in Tongji Hospital (Wuhan, China) were included. Tongji hospital is the sixth largest hospital (with 5000 beds) in China, and has been certified by both ISO 15189 (Medical Laboratories-Particular Requirements for Quality and Competence) and CAP (College of American Pathologists).

Bronchoscopy-derived BALF and expectoration-derived unconcentrated sputum were used for AFB smear, Xpert, and culture tests. About 40 ml of BALF was collected after instilling 30-50 ml of sterile saline (0.9%) into the airway of the affected lung segment. AFB smears and mycobacterial cultures were conducted as previously described (Forbes et al., 2018), but with minor modifications. Briefly, AFB smears on unconcentrated sputum and concentrated BALF (pelleted after centrifugation) were screened using the auramine fluorescence staining method (Baso Diagnostics Inc. Zhuhai, China). Auramine positive AFB smears were also confirmed by Ziehl–Neelsen staining (Baso Diagnostics Inc. Zhuhai, China), a method that appears to have a high specificity for diagnosing ATB (Tarhan et al., 2003; Lee et al., 2018). As for cultures, all sputum and BALF samples were mixed with an equal volume of a 0.5% N-acetyl-l-cysteine-2.0% NaOH and incubated at 37°C for 15-20 min. The mixture was then neutralized by the addition of phosphate buffer (pH 6.8), followed by centrifugation at 3,000 × g for 15 min. After resuspending the pellet in 2 ml of the phosphate buffer, 0.5 ml of the suspension was inoculated into liquid medium (BACTEC 960/MGIT, Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) and 0.2 ml of the suspension was inoculated onto solid medium (Lowenstein-Jensen, Baso Diagnostics Inc. Zhuhai, China). Cultures were grown for 8 weeks. To distinguish between MTB and NTM, positive cultures were tested using the TBAg MPT64 assay (a MPT64-based rapid immunochromatographic kit, GENESIS, Kaibili, China). Cultures negative for TBAg MPT64 were reported as NTM, or subjected to 16S rRNA sequencing to identify the mycobacterial species.

PTB was defined as at least one of the BALF and/or sputum specimens having a positive culture result for M. tuberculosis from liquid and/or solid media. A similar approach was used to define active NTM and Nocardia infections.

Xpert was conducted according to the manufacturer’s instructions (Cepheid, Sunnyvale, California). Briefly, untreated sputum samples or BALF samples that were pelleted after centrifugation were mixed with the sample reagent at 1:2 ratio (vol/vol), and incubated at 20–30°C for about 15 min (the mixtures were vortexed for at least 10 seconds between 5 and 10 minutes). About 2 ml of the sample reagent-treated sample was then transferred into the sample chamber of the Xpert cartridge. Xpert results were reported according to the manufacturer’s recommended semi-quantitative classification of the cycle-threshold (Ct) values: high (Ct ≤ 16), medium (16<Ct ≤ 22), low (22<Ct ≤ 28), and very low (Ct>28). If initial Xpert results were non-determinate (error, invalid or no result), testing was repeated with the leftover sample reagent-treated sample (at least 2 ml). In case there was less than 2 ml of sample-reagent-treated sample left, the leftover from the original sample was treated with sample reagent and re-tested as above.

Peripheral blood mononuclear cell (PBMC) T-SPOT.TB assay was performed with the T-SPOT ELISpot assay according to the manufacturer’s instructions (Oxford Immunotec Ltd., Oxford, England). Briefly, 2.5 ×10⁵ PBMCs were added to 96-well plates pre-coated with anti-IFN-γ antibody. After incubation for 16–20 h at 37°C with 5% CO2, plates were washed with phosphate buffered saline and developed using an anti-IFN-γ antibody conjugate and substrate, and detected for the presence of secreted IFN-γ. Spot-forming cells (sfc) were counted with an automated ELISpot reader (CTL Analyzers, Cleveland, OH, USA). To report a case of PTB, we used two different methods. One was to use the manufacturer-defined cut-off (T-SPOT^MDC), and the other was to use ratios of Mycobacterium tuberculosis-specific antigens (TBAg) to phytohaemagglutinin (PHA) sfc (TBAg/PHA) as previously described (Wang et al., 2016). Briefly, the ratios of ESAT-6 sfc to PHA sfc and CFP-10 sfc to PHA sfc were calculated, with the larger of the two values representing the TBAg/PHA ratio of one sample.

AFB smear-positive (AFB⁺) status was based on per-person results (defined as at least one of the BALF and/or sputum specimens having a positive AFB smear), unless otherwise stated. Culture-confirmed PTB and NTM infections were defined as at least one of the BALF and/or sputum specimens having a positive MTB or NTM culture. A positive mycobacterial culture from solid and/or liquid media was used as the reference standard. Comparisons of sensitivities and specificities between independent subgroups of interest were assessed using χ2 test. The kappa coefficients were calculated to determine the agreement between BALF and sputum. The agreement of the results (kappa value) was categorized as near perfect (0.8–1.0), substantial (0.6–0.8), moderate (0.4–0.6), fair (0.2–0.4), slight (0–0.2), or poor (<0) (Roberts, 2008). All analyses were performed using SPSS version 19 (IBM, Chicago, Illinois), with results considered significantly different at p<0.05.

A total of 28,192 inpatients were screened for eligibility. 21 patients received TB treatment 1 month before hospitalization and were not included ( Supplementary Table S1 ). Sputum and/or BALF culture results were available for 7,528 patients, with 8.9% and 1.2% being positive for MTB and NTM, respectively. Among the cultured NTM strains, 25 were identified to species level: 12 M. avium-intracellulare complex, 8 M. fortuitum, 4 M.abscessus, and 1 M. kansasii.

TB tests ordered by clinicians were variable, including 8,866 AFB, 9,388 AFB/T-SPOT, and many other combinations of tests ( Supplementary Figure S1 and Table S2 ). While AFB and T-SPOT were the first and second most frequently ordered tests, respectively, the percentage of patients undergoing Xpert increased rapidly from 0.8% in 2016 to 17.3% in 2019.

In a real-world setting, very few patients had their sputum and BALF collected simultaneously for single PTB diagnostic assay. We determined the consistency between sputum and BALF when they were used for AFB smear, culture, and Xpert. Patients having both sputum and BALF collected within one week of hospitalization for AFB smear (n=3,975), culture (n=109), and Xpert (n=181) analysis were included ( Supplementary Table S3 ). Sputum and BALF showed moderate to substantial consistency when used for AFB smear, culture, and Xpert. The positive detection rate by BALF was higher than that by sputum, when they were used for AFB smear or Xpert. The positive detection rate by sputum culture was slightly but insignificantly higher than that by BALF culture.

A total of 7,155 patients had 1-8 BALF and/or sputum AFB smears tested within one week of hospitalization ( Supplementary Table S4 ). The overall sensitivity of 1-4 AFB smears was 24.6%, 33.4%, 36.2%, and 37.3%, respectively ( Table 1 ). While one AFB smear was able to detect 64.7% of AFB⁺ patients with positive MTB culture, two AFB smears increased the detection rate to 88.0% ( Supplementary Table S5 ). Three AFB smears detected a further 7.4% of AFB⁺ TB patients as compared to two AFB smears. Four smears detected 98.3% of AFB⁺ TB patients.

A total of 2,044 patients had their respiratory samples tested for AFB smear, culture, Xpert, and T-SPOT ( Table 2 ). Both AFB smear and Xpert showed great specificity (>95%), but the sensitivity of AFB smear was much lower than that of Xpert (19.8% vs. 79.7%). Depending on AFB smear status, Xpert performance was different. Xpert was able to identify 97.8% of AFB⁺/culture-positive (culture⁺) TB patients, but only 75.3% of AFB smear-negative (AFB^-)/culture⁺ TB patients ( Supplementary Table S6 ). Despite these findings, Xpert was not performed in 4,252 patients who had both AFB smear and culture results available ( Supplementary Table S7 ). Of these patients, 326 (7.7%) were MTB culture⁺, including 212 (65.0%) that were AFB^- ( Supplementary Table S8 ).

In addition to AFB smear and Xpert, T-SPOT performance was analyzed. We used two different methods in the T-SPOT assay to define a PTB case, with one method using the manufacturer-defined cut-off (T-SPOT^MDC), and the other using the TBAg/PHA ratios as previously described (Wang et al., 2016). While T-SPOT^MDC and Xpert demonstrated similar sensitivity ( Table 2 ), T-SPOT^MDC had much lower specificity (69.1%) than Xpert (95.3%). When TBAg/PHA ratios were used, the specificity increased significantly, but at the expense of reduced sensitivity. For instance, TBAg/PHA ≥0.3 demonstrated an overall sensitivity of 37.3% and specificity of 94.8% ( Table 2 ). TBAg/PHA ≥0.5 gave an overall sensitivity of 17.3% and specificity of 97.1%. Increasing the TBAg/PHA cut-off to 1.0 decreased the sensitivity to 9.1%, but increased the specificity to 99.0% ( Supplementary Table S9 ).

While combining AFB smear and Xpert did not further increase their sensitivity and specificity in diagnosing PTB compared to Xpert alone ( Table 2 ), they were able to differentiate PTB and NTM cases more effectively ( Table 3 ). The majority (44/51) of AFB⁺/Xpert-positive (Xpert⁺) patients were MTB culture⁺, and the remaining seven patients were culture^- but diagnosed as having TB disease based on clinical presentations. Of the 216 AFB^-/Xpert⁺ patients, 137 and 73 were MTB culture⁺ and culture^-/clinically active TB, respectively. Six of seven AFB⁺/Xpert^- patients were NTM culture⁺. Of 1,770 AFB^-/Xpert^- patients, the majority (1,710) were negative for both MTB and NTM culture. Together, a combination of AFB and Xpert was able to detect 80.2% of patients with culture-proven PTB, and 28.6% of patients with culture-proven NTM.

We asked if combining T-SPOT with AFB smear and/or Xpert would improve PTB diagnosis. The sensitivity and specificity of AFB/T-SPOT^MDC combination was comparable to those of T-SPOT^MDC alone, suggesting this combination does not improve PTB diagnosis ( Table 2 ). However, when T-SPOT^MDC was used together with Xpert, the sensitivity and negative predictive value (NPV) increased to 95.0% and 99.1%, respectively, much higher than those of Xpert or T-SPOT^MDC alone ( Table 2 ). Adding AFB smear into Xpert/T-SPOT^MDC combination did not further increase the sensitivity and NPV. Notably, although combining T-SPOT^MDC with Xpert or AFB/Xpert greatly increased sensitivity, it was at the expense of reduced specificity (<67.6%). When TBAg/PHA≥0.3 ( Table 2 ) (compared to T-SPOT^MDC) was used in conjunction with AFB smear and/or Xpert, the specificity increased significantly. These results suggest that TBAg/PHA≥0.3 have some added values for PTB diagnosis when combined with AFB smear and/or Xpert.

While the above results analyzed the performance of T-SPOT in the overall population, it remained unclear if T-SPOT would perform differently among patients with different AFB smear and Xpert status. We first defined T-SPOT performance based on AFB smear or Xpert results. For AFB⁺ or Xpert⁺ patient populations, T-SPOT^MDC showed suboptimal sensitivities (84.1% vs. 83.1%) and very low NPVs (30.0% vs. 47.4%) ( Table 4 ). For AFB^- or Xpert^- patient populations, T-SPOT^MDC also showed suboptimal sensitivities (74.4-80.7%), but much higher NPVs (97.3-99.1%). When TBAg/PHA≥0.3 was used, the specificities increased significantly but at the cost of decreased sensitivities ( Table 2 ).

We then defined T-SPOT performance based on the status of both AFB smear and Xpert. Accordingly, patients were grouped into four populations: AFB^-/Xpert^-, AFB⁺/Xpert⁺, AFB⁺/Xpert^-, and AFB^-/Xpert⁺ ( Table 4 ). For AFB^-/Xpert^- patients, T-SPOT^MDC demonstrated a high NPV (99.1%) and a suboptimal sensitivity (74.4%) and specificity (71.0%). When TBAg/PHA≥0.3 was used, the specificity was significantly increased to 95.7% but with a decreased sensitivity (16.3%). In contrast, T-SPOT^MDC and TBAg/PHA showed no added values in 51 AFB⁺/Xpert⁺ patients and 211 AFB^-/Xpert⁺ patients who were either MTB culture⁺ or clinically diagnosed as having PTB. T-SPOT performance was inconclusive in AFB⁺/Xpert^- patients (n=5), although it ruled out PTB in three NTM culture⁺ cases.