Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

MS Zinter; CC Dvorak; MY Mayday; K Iwanaga; NP Ly; ME McGarry; GD Church; LE Faricy; CM Rowan; JR Hume; ME Steiner; ED Crawford; C Langelier; K Kalantar; ED Chow; S Miller; K Shimano; A Melton; GA Yanik; A Sapru; JL DeRisi

Surprisingly, Aspergillus RNA equivalent to ≥1 CFU was detected in 34.1% of samples and no water controls, suggesting that the lungs of immunocompromised children are frequently exposed to low levels of Aspergillus. The 100-fold increase in Aspergillus nucleic acid after optimized mechanical homogenization suggests that the origin of the Aspergillus RNA is likely from intact fungal organisms in the lower respiratory tract of immunocompromised children, rather than from migration of extracellular nucleic acid down the respiratory tree. However, as only 10% of samples originated from patients with suspected IPA, we speculate that patient-specific factors such as immune reconstitution, alloreactive inflammation, and impaired mucociliary clearance are as important as inoculum exposure in determining which child might develop IPA (28) . Although Aspergillus growth in culture appeared to be confounded by the frequent use of prophylactic and empiric antifungals, the quantity of Aspergillus sequencing reads was correlated with BAL galactomannan (29, 30) . As nutrient availability and the kinetics of Aspergillus growth may affect both galactomannan release and bioavailability of Aspergillus nucleic acid, we advocate . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/291864 doi: bioRxiv preprint that RNA/DNA-based assays might complement but should not replace antigen-based assays in the diagnosis of IPA (31, 32) . While our mNGS assay was highly sensitive for Aspergillus detection, specificity for differentiating infection from colonization was limited; future studies pairing mNGS with host susceptibility and immune function may improve discrimination of colonization and invasive mycosis (33) (34) (35) (36) .