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Background Standard testing fails to identify a pathogen in most patients with febrile neutropenia (FN). We evaluated the ability of the Karius microbial cell-free DNA sequencing test (KT) to identify infectious etiologies of FN and its impact on antimicrobial management. Methods This prospective study (ClinicalTrials.gov; NCT02912117) enrolled and analyzed 55 patients with FN. Up to 5 blood samples were collected per subject within 24 hours of fever onset (T1) and every 2 to 3 days. KT results were compared with blood culture (BC) and standard microbiological testing (SMT) results. Results Positive agreement was defined as KT identification of ≥1 isolate also detected by BC. At T1, positive and negative agreement were 90% (9/10) and 31% (14/45), respectively; 61% of KT detections were polymicrobial. Clinical adjudication by 3 independent infectious diseases specialists categorized Karius results as: unlikely to cause FN (N = 0); definite (N = 12): KT identified ≥1 organism also found by SMT within 7 days; probable (N = 19): KT result was compatible with a clinical diagnosis; possible (N = 10): KT result was consistent with infection but not considered a common cause of FN. Definite, probable, and possible cases were deemed true positives. Following adjudication, KT sensitivity and specificity were 85% (41/48) and 100% (14/14), respectively. Calculated time to diagnosis was generally shorter with KT (87%). Adjudicators determined real-time KT results could have allowed early optimization of antimicrobials in 47% of patients, by addition of antibacterials (20%) (mostly against anaerobes [12.7%]), antivirals (14.5%), and/or antifungals (3.6%); and antimicrobial narrowing in 27.3% of cases. Clinical Trials Registration NCT02912117 Conclusion KT shows promise in the diagnosis and treatment optimization of FN. [ABSTRACT FROM AUTHOR]

Background Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA has emerged as an attractive diagnostic modality allowing broad-range pathogen detection, noninvasive sampling, and earlier diagnosis. However, little is known about its real-world clinical impact as used in routine practice. Methods We performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 United States institutions over 1.5 years. Comprehensive records review was performed, and standardized assessment of clinical impact of the mNGS based on the treating team's interpretation of Karius results and patient management was established. Results A total of 82 Karius tests were evaluated from 39 (47.6%) adults and 43 (52.4%) children and a total of 53 (64.6%) immunocompromised patients. Karius positivity rate was 50 of 82 (61.0%), with 25 (50.0%) showing 2 or more organisms (range, 2–8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), and no impact in 71 (86.6%), and was indeterminate in 2 (2.4%). Cases with positive Karius result and clinical impact involved bacteria and/or fungi but not DNA viruses or parasites. In 10 patients who underwent 16 additional repeated tests, only 1 was associated with clinical impact. Conclusions The real-world impact of the Karius test as currently used in routine clinical practice is limited. Further studies are needed to identify high-yield patient populations, define the complementary role of mNGS to conventional microbiological methods, and discern how best to integrate mNGS into current testing algorithms. [ABSTRACT FROM AUTHOR]

Background. Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD. Methods. Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified. Results. Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases. Conclusions. The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing. [ABSTRACT FROM AUTHOR]