Establishment of a performance validation method for mNGS DNA workflow to detect BALF specimens / 中华检验医学杂志

ABSTRACT

Objective:To establish a performance validation method for mNGS applied in BALF samples.Method:Hela cells were used as a representative of host cells, and simulated BALF samples were prepared by adding different concentrations of Hela cells, seven species of isolated pathogens (including Streptococcus pneumonia, Hemophilus influenza, Klebsiella pneumonia, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Adenovirus), and interfering substances to sterile normal saline. Clinical BALF samples were collected simultaneously, and the results of mNGS were evaluated using traditional detection methods as a reference. The limit of detection (LOD), precision, anti-interference ability, stability, and accuracy of mNGS were determined. Results:In the simulated samples, the LOD of Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Adenovirus were 150, 262, 102, 67, 96, 83 CFU/ml, and 439 copies/ml, respectively. The repeatability of the detection results for all pathogens of simulated positive BALF samples was 100%. The anti-interference test showed that the higher the concentration of human DNA, the fewer pathogen sequences detected by mNGS. Escherichia coli and Shigella sonnei were used to evaluate the ability of mNGS to distinguish closely related species. The results showed that the system could stably distinguish Escherichia coli and Shigella sonnei when the concentration of Shigella sonnei was 4, 000 CFU/ml. The stability test results showed that there was no significant change in the number of pathogen sequences detected whether after 1 to 3 freeze-thaw cycles or storage at 4 ℃, -20 ℃, or -80 ℃ for 36 h. Compared with traditional detection methods, the accuracy of 17 clinical samples was 82.4%(14/17). Continuous evaluation of clinical BALF samples simultaneously tested by mNGS and traditional methods at Tongji Hospital from October 25, 2021, to September 14, 2022, showed that the accuracy of mNGS compared to bacterial culture, fungal culture, mycobacterial culture, Mycobacterium tuberculosis culture, and conventional PCR techniques was 67.5%(472/699), 81.5%(570/699), 92.3%(335/363), 96.4%(350/363), and 86.8%(132/152), respectively. Compared with conventional PCR techniques, the accuracy of mNGS for detecting Pneumocystis jirovecii, Adenovirus, and Mycoplasma pneumoniae was 89.4%(84/94), 93.3%(56/60), and 87.1%(61/70), respectively. Conclusion:By preparing simulated BALF samples and using traditional detection methods as a reference, the performance characteristics of mNGS in detecting BALF samples can be preliminarily evaluated.