Cell‐free DNA next‐generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease

Abstract

Background We sought to determine if next‐generation sequencing (NGS) of microbial cell‐free DNA (cfDNA) in plasma would detect pathogens in pediatric patients at risk for invasive fungal disease (IFD). Procedures Pediatric hematology, oncology, and stem cell transplant patients deemed at risk for new IFD had blood samples drawn at three time‐points separated by 1‐month intervals. The primary outcome measure was detection of fungal pathogens compared to standard clinical testing. Secondary outcomes included identification of other infectious pathogens, relationship to European Organization for Research and Treatment of Cancer's Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases’ Mycoses Study Group (EORTC/MSG) guidelines, and assessment of antifungal therapy. Results NGS identified fungal pathogens in seven of 40 at‐risk patients for IFD and results were identical in four of six proven cases, including Aspergillus fumigatus by lung biopsy, Candida albicans by blood or pancreatic pseudocyst cultures, and Rhizopus delemar by skin biopsy. Rhizopus oryzae identified on skin biopsy and A. fumigatus isolated on day 27 of 28 of culture from lung biopsy were not detected by cfDNA NGS, possibly due to lack of bloodstream penetration and questionable pathogenicity, respectively. Numerous DNA viruses were detected in patients with prolonged febrile neutropenia or abnormal imaging. Extended antifungal therapy was used in 73% of patients. Follow‐up cfDNA sequencing in patients who were positive at enrollment was negative at 1 and 2 months. Conclusions cfDNA NGS detected fungal pathogens from blood confirming its potential to guide treatment decisions in pediatric patients at risk for IFD and limit excessive empiric antifungal use. Future studies are needed to better understand the sensitivity and specificity of this approach.

Cell-free DNA Profiling Informs Major Complications of Hematopoietic Cell Transplantation

“…2 ). Interestingly, all of the identified species are well documented intestinal commensal organisms, in agreement with results from a recent study by Armstrong et al 37 that suggest a loss of the integrity of the gut vascular barrier associated with GVHD ( supplementary fig. 2 ).…”

Next-generation sequencing of microbial cell-free DNA for rapid noninvasive diagnosis of infectious diseases in immunocompromised hosts

“…In a retrospective case-control study of 57 HCT recipients with proven/probable pulmonary invasive mold infections, the cfDNA NGS test identified 83% (5/6) of molds among patients with non-Aspergillus infections; but among those with Aspergillus proven/probable disease, Aspergillus fumigatus was only identified in 13.7% (7/51) of cases 38 . In the report by Armstrong et al 37 , in a cohort of 40 pediatric hematology-oncology and HCT patients, sequencing of circulating cfDNA detected fungal pathogens in five of seven cases with proven and probable invasive fungal disease, and correlated with microbiological diagnosis in four of six proven cases. In a recent report by Hong et al 24 , in seven out of nine subjects (including seven immunocompromised hosts) with proven invasive fungal infection, plasma NGS testing detected the same fungus identified from the biopsy tissue at the genus level.…”

“…Thus, this test is a promising diagnostic tool in neutropenic fever, a clinical scenario where conventional work up fails to identify an etiological agent in a majority of cases 5 . Another study evaluated 40 pediatric patients with prolonged neutropenia and fever (>96h) despite administration of antibiotics for suspected fungal infection (the authors excluded patients who had received antifungal therapy for >4 days); in this study cfDNA NGS identified fungal pathogens including Aspergillus fumigatus, Rhizopus spp., Candida albicans, Candida glabrata and Pneumocystis jirovecii 37 .…”

Special Staining of the Liquid-Based Cytopathology Test in Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis with Nonneutropenic Patients

“…Currently, the morbidity and mortality rates of IPA are gradually increasing, which are related to the delay in early diagnosis of IPA caused by the lack of typical clinical manifestations [22]. Culture and microscopic examination remain the "gold standard" but requires potentially invasive procedures to obtain infected tissue and are insensitive [23][24][25]. In addition, as mentioned above, attention should be paid to the differentiation of colonization and invasion during IPA diagnosis.…”