Abstract
Background
A diverse spectrum of invasive molds and fungi cause serious opportunistic infections in immunocompromised (IC) children. Their overlap in clinical presentation can make it challenging to differentiate among etiologies and optimally tailor antifungal therapy. Current methods to identify these pathogens lack sensitivity, are limited by long turnaround times and require an array of individual tests on invasively obtained specimens. The delay or lack of a pathogen diagnosis in combination with the reliance on invasive procedures leads to a dependence on broad empiric therapy, the development of antimicrobial resistance and increases in morbidity and mortality. Rapid, non-invasive diagnosis of invasive fungal infections through next-generation sequencing (NGS) of plasma microbial cell-free DNA (mcfDNA) offers a means to overcome these limitations.
Methods
Karius® Test (KT) results were reviewed for detections of Aspergillus, non-Aspergillus molds and Pneumocystis jirovecii (PJP) in children. KT, developed and validated in Karius’ CLIA certified/CAP accredited lab, detects microbial cell-free DNA (mcfDNA) can assist with the diagnosis of invasive infections. McfDNA is extracted, NGS is performed, human sequences removed and remaining sequences aligned to a curated pathogen database of >1400 organisms. Organisms present above a statistical threshold are reported and quantified. For > 85% of tests the time to result reporting is 24 hours from sample receipt. Clinical information was included from data submitted with the requisition or obtained at the time of reporting from clinical consultations with the provider.
Results
KT detected 7 different species of Aspergillus in 61 patients (74% IC, 40% with a pulmonary focus). KT detected 15 different non-Aspergillus molds in 51 patients (80% IC, 36% with a pulmonary focus). KT detected PJP in 37 patients (73% IC, 76% with a pulmonary focus, 54% with a DNA virus co-detection and 32% with a herpesvirus co-detections). There were 31 subjects with serial monitoring (97% IC, 70% with a pulmonary focus) including 48% with Aspergillus, 39% with non-Aspergillus molds and 12% with PJP (Figure 1). 71% of subjects demonstrated a decline in the quantitative mcfDNA signal over time; the duration of a positive mcfDNA signal ranged from 3–92 days (median 16 days, SD 22.4).
Conclusions
Plasma mcfDNA NGS offers a rapid, non-invasive means of detecting a broad diversity of invasive pathogens that overlap in their clinical presentations and are difficult to identify in immunocompromised children. The rapid turnaround time, non-invasive sampling and 1-sample-1000+test-solution may lead to a faster time to pathogen diagnosis, faster time to targeted therapy and obviate the need for invasive diagnostic procedures. The ability with a single test to concomitantly diagnose co-pathogens including reactivating herpesviruses that modulate the progression of principal infecting fungal pathogens (i.e. cytomegalovirus modulation of PJP) can help optimize care. Additionally, this convenient non-invasive means of serial testing of invasive fungal infections may serve as an indicator of burden of infection, provide insight into treatment efficacy and ultimately help define the length and mode (medical/surgical) of therapy required to improve outcomes. Additional studies correlating the mcfDNA signal with individual patient clinical and radiographic parameters will be important to further define the utility of serial mcfDNA monitoring.
Budget Impact of Microbial Cell-Free DNA Testing Using the Karius® Test as an Alternative to Invasive Procedures in Immunocompromised Patients with Suspected Invasive Fungal Infections
