Pathogen profile of all patients with pulmonary infections according to tNGS
In the 262 patients enrolled, 47 potential pathogens were identified in the BALF samples via tNGS including 21 bacteria, 13 viruses, 11 fungi, 1 parasite, and 1 mycoplasma (Figure 1). The most frequently detected bacteria were Streptococcus pneumoniae (56 cases), Pseudomonas aeruginosa (55 cases), and Haemophilus influenzae (47 cases). The most frequently identified virus was Epstein–Barr virus (EBV), which was detected in 78 patients. The top three fungi identified by tNGS were Candida albicans (59 cases), Aspergillus fumigatus (20 cases), and Candida tropicalis (14 cases).
The overall microbial detection rate for tNGS was 99.2% (260/262), and 21.9% (57/260) of it accounted for single-pathogen infection and 78.1% (203/260) accounted for polymicrobial infections.
Comparison of tNGS and bacterial culture
The bacterial detection rates of tNGS and ordinary bacterial culture were 74.0% (194/262) and 28.2% (74/262), respectively, indicating that the detection rate of tNGS was significantly superior to that of bacterial culture (P < 0.001). Among the 262 patients, 64 (24.4%) patients showed positive results on both tNGS and ordinary bacterial culture, whereas 58 (22.1%) patients showed negative results on both methods. Furthermore, 130 (49.6%) patients tested positive solely by tNGS, and 10 (3.8%) patients tested positive exclusively by bacterial culture, indicating that the sensitivity of tNGS was significantly higher than that of bacterial culture. Among the 69 patients showing positive results on tNGS and bacterial culture, 21 patients exhibited complete consistency, 43 patients showed partial consistency, and 4 patients displayed complete inconsistency between the tNGS and bacterial culture results. Besides, seven microorganisms, namely Klebsiella oxytoca, Pseudomonas otitidis, Abiotrophia defective, Corynebacterium striatum, Staphylococcus haemolyticus, Shewanella algae, and Aeromonas caviae, were outside the detection range of tNGS in the bacterial culture-positive results (Figure 2). The rates of negative, positive, and total consistent and kappa value between tNGS and bacterial culture were 30.8%, 86.4%, 46.4%, and 0.116, respectively (Table 2).
The positive rate of tNGS for fungal identification was slightly higher than that of fungal culture (31.7 % (83/262) and 22.9 % (60/262), respectively). In total, 42 patients showed positive results on both tNGS and fungal culture, whereas 160 patients showed negative results on both. Additionally, 42 patients tested positive solely by tNGS, and 19 patients tested positive solely by fungal culture (Table 2). Among the 42 patients showing positive results on tNGS and fungal culture, 28 patients exhibited complete consistency and 14 patients exhibited partial consistency between the tNGS and bacterial culture results. The rates of positive, negative, and total consistent and kappa value between tNGS and fungal culture were 68.9%, 79.1%, 76.7%, and 0.424, respectively.
Consistency between tNGS and Xpert® MTB/RIF assay
Herein, 42 patients suspected of tuberculosis infection or with a history of tuberculosis infection were detected by tNGS and Xpert® MTB/RIF assay. The results showed that 23 patients showed positive results for Mycobacterium tuberculosis on both methods. In the Xpert® MTB/RIF assay negative group, 13 patients tested positive for Mycobacterium tuberculosis when detected by tNGS. The rates of positive, negative, and total consistent and kappa value between tNGS and pert® MTB/RIF assay were 100.0%, 68.4%, 85.7%, and 0.704, respectively (Table 2).
TNGS and GM test in identifying Aspergillus infection
Of the 262 patients, 28 patients tested positive for Aspergillus infection by tNGS, including 20 patients infected with Aspergillus fumigatus and 8 with Aspergillus flavus. Among these 28 patients, 60.7% (17/28) showed positive results and 39.3% (11/28) showed negative results when subjected to the GM test. For proven Aspergillus infection fungal culture, tNGS exhibited a sensitivity of 57.1%, a specificity of 90.6%, a positive predictive value (PPV) of 14.3%, and a negative predictive value (NPV) of 98.7%, whereas the GM test showed a sensitivity of 71.4%, a specificity of 82.7%, a PPV of 71.4%, and an NPV of 99.1%. When clinically diagnosed Aspergillus infection was used as a gold standard, the final sensitivity and specificity of tNGS versus the GM test increased to 86.2% and 98.7% versus 69.0% and 87.6%, respectively (Table 3). Spearman’s correlation coefficient between the read counts of Aspergillus detected by tNGS and the OD value of the GM test was 0.120 (P > 0.05). Logistic regression analysis showed no significant correlation between the quantitative results of the two methods (Figure 3).