Illumina and Nanopore sequencing in culture-negative samples from suspected lower respiratory tract infection patients.

OBJECTIVE: To evaluate the diagnostic value of metagenomic sequencing technology based on Illumina and Nanopore sequencing platforms for patients with suspected lower respiratory tract infection whose pathogen could not be identified by conventional microbiological tests.

METHODS: Patients admitted to the Respiratory and Critical Care Medicine in Shanghai Ruijin Hospital were retrospectively studied from August 2021 to March 2022. Alveolar lavage or sputum was retained in patients with clinically suspected lower respiratory tract infection who were negative in conventional tests. Bronchoalveolar lavage fluid (BALF) samples were obtained using bronchoscopy. Sputum samples were collected, while BALF samples were not available due to bronchoscopy contraindications. Samples collected from enrolled patients were simultaneously sent for metagenomic sequencing on both platforms.

RESULTS: Thirty-eight patients with suspected LRTI were enrolled in this study, consisting of 36 parts of alveolar lavage and 2 parts of sputum. According to the infection diagnosis, 31 patients were confirmed to be infected with pathogens, while 7 patients were diagnosed with non-infectious disease. With regard to the diagnosis of infectious diseases, the sensitivity and specificity of Illumina and Nanopore to diagnose infection in patients were 80.6% vs . 93.5% and 42.9 vs . 28.6%, respectively. In patients diagnosed with bacterial, Mycobacterium , and fungal infections, the positive rates of Illumina and Nanopore sequencer were 71.4% vs . 78.6%, 36.4% vs . 90.9%, and 50% vs . 62.5%, respectively. In terms of pathogen diagnosis, the sensitivity and specificity of pathogens detected by Illumina and Nanopore were 55.6% vs . 77.8% and 42.9% vs . 28.6%, respectively. Among the patients treated with antibiotics in the last 2 weeks, 61.1% (11/18) and 77.8% (14/18) cases of pathogens were accurately detected by Illumina and Nanopore, respectively, among which 8 cases were detected jointly. The consistency between Illumina and diagnosis was 63.9% (23/36), while the consistency between Nanopore and diagnosis was 83.3% (30/36). Between Illumina and Nanopore sequencing methods, the consistency ratio was 55% (22/42) based on pathogen diagnosis.

CONCLUSION: Both platforms play a certain value in infection diagnosis and pathogen diagnosis of CMT-negative suspected LRTI patients, providing a theoretical basis for clinical accurate diagnosis and symptomatic treatment. The Nanopore platform demonstrated potential advantages in the identification of Mycobacterium and could further provide another powerful approach for patients with suspected Mycobacterium infection.