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 (mcfDNA) 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 24h of fever onset (T1) and every 2-3 days. KT results were compared to 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. Conclusion KT shows promise in the diagnosis and treatment optimization of FN.

Allogeneic hematopoietic stem cell transplant patients are at risk for common and atypical infections. Superior diagnostics can decrease infection-related morbidity and mortality. A novel plasma cell–free DNA next-generation sequencing test detected an uncommon presentation of Chlamydia trachomatis and recurrent and metastatic complications of Staphylococcus aureus bacteremia before standard microbiology.

Background Microbial cell-free DNA (mcfDNA) sequencing of plasma can identify presence of a pathogen in a host. This study evaluated the duration of pathogen detection by mcfDNA sequencing vs. conventional blood culture in patients with bacteremia. Methods Blood samples from patients with culture-confirmed bloodstream infection were collected within 24 hours of the index positive blood culture and 48 to 72 hours thereafter. mcfDNA was extracted from plasma and next-generation sequencing (NGS) applied. Reads were aligned against a curated pathogen database. Statistical significance was defined with Bonferroni adjustment for multiple comparisons (p < 0.0033). Results A total of 175 patients with Staphylococcus aureus bacteremia (SAB; n=66), Gram-negative bacteremia (GNB; n=74), or non-infected controls (n=35) were enrolled. The overall sensitivity of mcfDNA sequencing compared to index blood culture was 89.3% (125/140) and the specificity was 74.3%. Among patients with bacteremia, pathogen specific mcfDNA remained detectable for significantly longer than conventional blood cultures (median 15 days vs. 2 days; p<0.0001). Each additional day of mcfDNA detection significantly increased the odds of metastatic infection (Odds Ratio [OR]: 2.89; 95% Confidence Interval [CI]: 1.53-5.46; p=0.0011). Conclusions Pathogen mcfDNA identified the bacterial etiology of bloodstream infection for a significantly longer interval than conventional cultures, and its duration of detection was associated with increased risk for metastatic infection. mcfDNA could play a role in the diagnosis of partially treated endovascular infections.

BackgroundThe variable clinical presentation of IE requires a diagnostic tool that accurately detects a wide range of organisms, including in culture-negative (CN) scenarios. A sensitive molecular diagnostic assay that quantitates pathogen DNA could be a useful tool to diagnose IE and evaluate response to antimicrobial therapy.MethodsWe prospectively enrolled 30 hospitalized adult patients evaluated for acute IE classified using the Duke Criteria. Residual plasma samples within 24 hours and/or fresh whole blood within 48–72 hours of enrollment blood culture were collected. Additional samples were collected every 2–3 days for up to 7 time points until discharge. Samples were shipped to the Karius laboratory (Redwood City, California) for testing. Cell-free DNA was extracted and NGS was performed. Human sequences were removed and remaining sequences were aligned to a curated pathogen database of over 1,000 organisms. Organisms present above a predefined statistical threshold were reported. Quantity of DNA for each reported pathogen was expressed as molecules per microliter.ResultsOf 29 patients eligible for analysis, 18 had prosthetic valves and 7 had implanted cardiac devices. Twenty-four patients had Definite IE. Twenty patients had positive blood cultures (including S. aureus, S. epidermidis, E. faecalis, S. agalactiae, Pantoea ananatis, S. sanguinis, C. albicans); NGS identified the same organism isolated in all 20 patients as well as E. cloacae complex, and E. faecalis in 2 of 4 CN Definite IE patients. For 1 CN patient with Possible IE, NGS identified E. coli. NGS and BC were negative for 4 patients with Rejected IE. NGS identified the IE etiology in patients pretreated with antibiotics up to 20 days prior to sample collection. Pathogen DNA signal was often observed in both initial and repeat plasma samples, while repeat blood cultures remained negative.ConclusionThis novel, cell-free pathogen quantitative NGS plasma assay accurately identified causative organisms in patients with IE, even when blood cultures were negative due to pretreatment with antibiotics. Pathogen DNA, detected in plasma longer than blood culture, is a promising biomarker to aid in the diagnosis and monitoring of IE, particularly culture-negative IE.Disclosures P. Shah, Karius Inc.: Collaborator and Research Contractor, Salary. F. Ruffin, Karius Inc.: Collaborator and Research Contractor, Salary. H. Seng, Karius Inc.: Employee, Salary. D. Hollemon, Karius Inc.: Employee, Salary. L. Winn, Karius Inc.: Collaborator and Research Contractor, Salary. C. Drennan, Karius Inc.: Collaborator and Research Contractor, Salary. K. L. Chan, Karius Inc.: Employee, Salary. H. Quach, Karius Inc.: Employee, Salary. T. Blauwkamp, Karius Inc.: Employee, Salary. G. Meshulam-Simon, Karius Inc.: Employee, Salary. D. Hong, Karius Inc.: Employee, Salary. V. G. Fowler Jr., Merck, Cerexa/Actavis, Pfizer, Advanced Liquid Logis, NIH, MedImmune, Basilea, Karius, Contrafect, Regneron, Genentech, Affinergy, Locus, Medical Surface, Inc., Achaogen, Astellas, Arsa...

BackgroundBlood cultures can have low sensitivity if a patient is pretreated with antibiotics. A molecular diagnostic for bloodstream infection (BSI) that can also quantitate pathogen DNA could be a useful tool in detecting and monitoring culture-negative infections.MethodsWe prospectively enrolled 75 patients (50 with culture confirmed BSI due to Staphylococcus aureus [n = 21] or Gram-negative bacilli [n = 29] at baseline, and 25 with negative blood cultures) to evaluate the Karius plasma next generation sequencing (NGS) test to detect BSI. Blood samples from patients with confirmed BSI were collected for the study within one day of positive blood culture and then every 2–3 days. Cell-free DNA (cfDNA) was extracted from plasma and underwent NGS in the Karius CLIA/CAP laboratory (Redwood City, CA). After removal of human sequences, remaining reads were aligned against a curated pathogen database. Organisms present at a significance-level above a predefined threshold were reported. Quantity of cfDNA for each reported pathogen was expressed as molecules per microliter (MPM).ResultsWhen compared with baseline blood culture, the plasma NGS test had a positive agreement of 80% (40/50) and negative agreement of 84% (21/25). Overall, serially collected samples were positive by plasma NGS testing significantly longer than blood culture (mean 6.0 days vs. 2.4 days, respectively; P < 0.0001. Figure). Patients with BSI were positive longer by NGS testing than blood culture for both S. aureus (mean 6.9 days vs. 4.0 days, respectively; P < 0.005) and gram-negative bacilli (mean 5.4 days vs. 1.3 days, respectively; P < 0.001). Pathogen cfDNA in BSI patients, quantified as MPM, declined over time during treatment. S. aureus MPM declined more slowly than gram-negative MPM and was significantly higher than gram-negative MPM at day 6 (P < 0.001).ConclusionThe Karius plasma NGS test can directly detect pathogens in patients with BSI. Pathogen cfDNA signal in plasma remains positive longer than blood culture and combined with quantification of pathogen cfDNA, could be a useful biomarker to aid in diagnosis and monitoring of infections, particularly in those with sterile blood cultures.Disclosures L. Wanda, Karius, Inc: Collaborator and Employee, Salary; F. Ruffin, Karius, Inc.: Collaborator and Research Contractor, Salary; J. Hill-Rorie, Karius, Inc: Collaborator, Research support and Salary; D. Hollemon, Karius, Inc: Employee, Salary; H. Seng, Karius, Inc: Employee, Salary; D. Hong, Karius, Inc.: Employee, Salary; 
T. Blauwkamp, Karius, Inc: Employee, Salary; M. Kertesz, Karius, Inc: Employee, Salary; V. Fowler Jr., Karius, Inc: Grant Investigator, Grant recipient Pfizer, Novartis, Galderma, Novadigm, Durata, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., Cerexa, Tetraphase, Trius, MedImmune, Bayer, Theravance, Cubist, Basilea, Affinergy, Janssen, xBiotech, Contrafect: Consultant, Consulting fee NIH, MedImmune, Cerexa/Forest/Actavis/Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Cubist/Merck; Med...