Diagnostic Value of Microbial Cell-free DNA Sequencing for Suspected Invasive Fungal Infections: A Retrospective Multicenter Cohort Study

Abstract

Background: An early diagnosis and treatment of invasive fungal disease (IFD) is associated with improved outcome, but the moderate sensitivity of noninvasive diagnostic tests makes this challenging. Invasive diagnostic procedures such as bronchoalveolar lavage (BAL) have a higher yield but are not without risk. The detection and sequencing of microbial cell-free DNA (mcfDNA) may facilitate a noninvasive diagnosis.

Materials: In a prospective observational study, we collected plasma in the 120 hours preceding or following a BAL in patients with hematological malignancies suspected for a pulmonary IFD. The EORTC/MSGERC2020 criteria were used for IFD classification. Sequencing was performed by Karius (Redwood City, CA) using their Karius Test (KT) on plasma and a "research use only test" on BAL fluid if available. Cases with a probable/proven IFD were identified based on standard diagnostic tests on serum and BAL (microscopy, polymerase chain reaction, galactomannan, culture) and used to calculate the sensitivity, specificity, and additional diagnostic value of the KT.

Results: Of 106 patients enrolled, 39 (37%) had a proven/probable invasive aspergillosis, 7 (7%) a non-Aspergillus IFD, and 4 (4%) a mixed IFD. The KT detected fungal mcfDNA in 29 (28%) patients. Compared with usual diagnostic tests, the sensitivity and specificity were 44.0% (95% confidence interval [CI], 31.2-57.7) and 96.6% (95% CI, 88.5%-99.1%). Sensitivity of the KT was higher in non-Aspergillus IFD (Mucorales:2/3, Pneumocystis jirovecii: 3/5). On BAL, the sensitivity was 72.2% (95% CI, 62.1-96.3), and specificity 83.3% (95% CI, 49.1-87.5).

Conclusions: Sequencing of mcfDNA may facilitate a noninvasive diagnosis of IFD in particular non-Aspergillus IFD. However, on plasma and similar to currently available diagnostics, it cannot be used as a "rule-out" test.

Keywords: hematological malignancy; invasive fungal disease; invasive fungal infection; microbial cell-free DNA; next generation sequencing.

Figure 1.

Additional diagnostic value (ADV) of the Karius Test on plasma and research-only-use test on BAL fluid compared to the EORTC/MSGERC definitions. A: Diagnostic value of the Karius test (mcfDNA) on plasma compared with the modified EORTC/MSGERC consensus definitions (Mucorales PCR added to the definitions for IM). NGS on plasma was successful in 105 patients. In 60 patients, EORTC/MSGERC criteria were not fulfilled for an invasive fungal infection disease (IFID). In 3 (2.9%) of these patients, the KT (mcfDNA test) showed a fungal pathogen with potential to cause an IFIIFD. This was categorized as a possible ADV. Importantly, 3 patients had a co-infection next to an IA; however, only the co-infection could be confirmed by the KT test (Pneumocystis in 2 patients, Mucorales in 1 patient). Additionally, 1 patient had a probable IM, but the KT only showed Aspergillus in plasma. These patients was also categorized in the “UC + mcfDNA+” group. B: Diagnostic value of the research-use-only Karius test (mcfDNA) on bronchoalveolar fluid (BALf) compared with the modified EORTC/MSGERC consensus definitions (Mucorales PCR added to the definitions for IM). NGS on BALf was successful in 33 patients. In 17 patients, EORTC/MSGERC criteria were not fulfilled for an invasive fungal infection disease (IFDI). In 2 (6.1%) of these patients, the KT (mcfDNA test) showed a fungal pathogen with potential to cause an invasive fungal infection disease (IFID). This was categorized as a possible ADV. Importantly, 1 patient was suspected for an Aspergillus and Mucorales co-infection based on the modified EORTC/MSGERC definitions. The KT test could only confirm the Aspergillus species. Abbreviations: ADV, additional diagnostic value; KT, Karius test; mcfDNA, microbial cell-free DNA; UC, usual care.