Detecting circulating microbial cell-free DNA by next-generation sequencing in patients with Mycobacterium avium complex-lung disease: A pilot study

Objectives: Determining a diagnosis for non-Tuberculous mycobacterium (NTM)-lung disease (LD) remains difficult. The value of circulating cell-free DNA (cfDNA) secreted from microbes has been established in the detection of pathogens in septic patients. However, it is unknown whether NTM-derived cfDNA is detectable in plasma from patients with NTM-LD and whether this is associated with the disease status of NTM-LD, especially in patients with Mycobacterium avium complex (MAC)-LD. Materials and Methods: In this pilot study, from 2018 to 2019, we enrolled adult patients with MAC-LD at Taipei Veterans General Hospital in Taiwan for the detection of circulating cfDNA. We performed cfDNA extraction from plasma, next-generation sequencing (NGS) for nonhuman cfDNA, and sequence matching to a microbial database and then assessed the association between pathogen cfDNA and MAC-LD. Results: Two (40%) plasma samples from MAC-LD patients had detectable MAC-specific cfDNA, namely one instance of DNA polymerase III alpha subunit and one instance of ATP-binding cassette transporters permease. The plasma samples from the three other MAC-LD cases and the one tuberculosis control were negative for either NTM-derived cfDNA or tuberculosis-related cfDNA. In addition to MAC-specific cfDNA, Ralstonia solanacearum, Staphylococcus aureus, and Pasteurella multocida were the most observed bacteria in our patients. The two patients with MAC-cfDNA positivity yielded higher radiographic scores (P = 0.076) and presented a higher number of nonhuman reads than those without MAC-cfDNA positivity (P = 0.083). Conclusion: Using NGS method, we demonstrated MAC-cfDNA was detectable in patients with MAC-LD. Further large-scale research is warranted to assess the clinical value of detecting MAC-specific cfDNA in MAC-LD patients.