661. Clinical Utility and Impact of the Metagenomic Microbial Plasma Cell-Free DNA Next-Generation Sequencing Assay on Treatment Decision: a Single-Center Retrospective Study

Abstract Background Metagenomic next-generation sequencing (mNGS) of microbial cell-free DNA (mcfDNA) allows for non-invasive broad-range pathogen detection from plasma. The Karius® test that emerged in 2016 made mNGS widely available. However, there is little data describing the optimal role for this assay in clinical decision making. Methods We performed a single-center retrospective cohort study of adult patients for whom a Karius test was sent between May 2019 and February 2021 to assess clinical utility. We predefined criteria for clinical impact categories (Table 1) and stratified data by patient comorbidities, infectious syndromes, duration of antimicrobial therapy prior to Karius testing, reasons for sending the test, and final clinical diagnoses. Clinical impact was arbitrated by all authors after review and discussion of each case. Results A total of 80 patients were included. 45 patients (56%) were immunocompromised, and 14 (18%) had prosthetic hardware or grafts. The most common clinical syndrome was pneumonia/respiratory failure (31%), followed by sepsis/septic shock (15%) and endocarditis (13%). 72 patients (90%) received antibiotics prior to sending the Karius assay. The most common reason for sending the assay was unknown microbiologic diagnosis (78%), followed by avoiding invasive procedures (14%). The test was consistent with the final diagnosis in 65% of cases and had a positive impact in 34 (43%), a negative impact in 2 (3%), and uncertain or no impact in 44 (55%) (Table 2). A positive impact was observed in solid organ transplant recipients (SOTR, 71.4%, p=0.003), sepsis (71.4%, p=0.003), and those receiving antimicrobial agents for less than 7 days prior to Karius testing (i.e., 61.8%, p=0.004) (Table 3). Conclusion In our cohort, clinical utility of Karius testing was highest in SOTR and in patients with sepsis. Prolonged antimicrobial use ( > 7 days) prior to Karius testing limited the utility of the assay. Prospective studies evaluating the utility of mNGS mcfDNA assays should be performed to further clarify its role in clinical management. Disclosures All Authors: No reported disclosures

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Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study

10.1101/19008755 ◽

2019 ◽

Author(s):

Catherine A. Hogan ◽

Shangxin Yang ◽

Omai B. Garner ◽

Daniel A. Green ◽

Carlos A. Gomez ◽

...

Keyword(s):

Cohort Study ◽

Next Generation Sequencing ◽

Real World ◽

Retrospective Cohort Study ◽

Retrospective Cohort ◽

Positive Impact ◽

Clinical Impact ◽

Next Generation ◽

Free Dna ◽

Generation Sequencing

AbstractBackgroundMetagenomic 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.MethodsWe performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 U.S. institutions over 1.5 years. Comprehensive chart 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.ResultsA 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 50/82 (61.0%), with 24 (48.0%) showing two or more organisms (range, 2-8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), no impact in 70 (85.4%), and was indeterminate in 3 (3.7%). 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 one was associated with clinical impact.ConclusionsThe 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 how best to integrate mNGS into current testing algorithms.SummaryIn a multicenter retrospective cohort study, we show that the real-world clinical impact of plasma metagenomic next-generation sequencing (mNGS) for the noninvasive diagnosis of infections is limited (positive impact 7.3%). Further studies are needed to optimize the impact of mNGS.

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673. Unbiased Microbial Cell-free DNA Next-Generation Sequencing Resolves HHV6 Variant Specificity and Demonstrates a Predominance of HHV6B over HHV6A in Real-World Clinical Experience

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.870 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S438-S439

Author(s):

Jose Alexander ◽

Sivan Bercovici ◽

Nicholas R Degner ◽

Ricardo Castillo-Galvan ◽

Aparna Arun ◽

...

Keyword(s):

Next Generation Sequencing ◽

Human Herpesvirus ◽

Severe Infection ◽

Microbial Cell ◽

Solid Organ ◽

Metagenomic Sequencing ◽

Next Generation ◽

Cell Free Dna ◽

Free Dna ◽

Generation Sequencing

Abstract Background Human herpesvirus 6 (HHV6) has been classified in two distinct variants, HHV6A and HHV6B. Although distinct epidemiology, disease association, biological and immunological properties, their genomes are 90% homologous. Less is known about HHV6A which is typically asymptomatic but can cause severe infection in patients with neurological disorders or HIV. HHV6B infection occurs during childhood, but can reactivate after solid organ and stem cell transplantation. Antibody assays may indicate previous, recent or current infection. Quantitative PCR cannot differentiate active from latent infections and some available qualitative PCR assays are unable to differentiate the two variants. A single open-ended test through plasma-based microbial cell-free DNA (mcfDNA) metagenomic next-generation sequencing (NGS) may overcome these limitations. Methods Karius TestTM (KT) developed and validated in Karius’s CLIA certified/CAP accredited lab, detects mcfDNA from plasma. mcfDNA is extracted, NGS performed, human sequences removed and remaining sequences aligned to a curated pathogen database of > 1500 organisms. Organisms present above a statistical threshold are reported and quantified. For > 85% of tests the time to result reporting is the next day from sample receipt. KT results were reviewed from November-2018 to May-2021 for detections of HHV6A and HHV6B. The comparative incidence of HHV6A and HHVB detections, their age distributions and their quantitative viral concentration in molecules/µL (MPM) were assessed. Results KT detected 322 cases of HHV6; 10% (n=32) were HHV6A and 90% (n=290) HHV6B (Table 1). HHV6B had a higher relative abundance in children (with a distribution into adolescence) compared to adults (Figure 1). The average HHV6A MPM was 860 (range 27 - 10,472); the average HHV6B MPM was 3,361 (range 21 - 131,518). Table 1. Summary of HHV-6 Detections Figure 1. Distribution of HHV6A and HHV6B by Age Group Conclusion The distribution of the HHV6 variants detected through KT shows an overwhelming 9:1 predominance of HHV6B to HHV6A. The application of mcfDNA metagenomic sequencing for open-ended detection, variant determination and quantification of HHV6 provides more specific resolution than serological and PCR methods. KT may lend important insights into the association of specific HHV6 variants with clinical syndromes affecting vulnerable populations. Disclosures Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Sivan Bercovici, PhD, Karius (Employee) Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Aparna Arun, MD, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Matthew Smollin, PharmD, Karius, Inc. (Employee)

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CapnocytophagacanimorsusMeningitis Diagnosed Using Next- Generation Sequencing of Microbial Cell-Free DNA

IDCases ◽

10.1016/j.idcr.2021.e01126 ◽

2021 ◽

pp. e01126

Author(s):

Abuzar A. Asif ◽

Moni Roy ◽

Benjamin R. Tellier ◽

Sharjeel Ahmad

Keyword(s):

Next Generation Sequencing ◽

Microbial Cell ◽

Next Generation ◽

Cell Free Dna ◽

Free Dna ◽

Generation Sequencing

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670. Rapid, Non-invasive Detection of Invasive Mycoplasma hominis Infection using the Karius Test, A Next-Generation Sequencing Test for Microbial Cell-free DNA in Plasma

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.863 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S390-S390

Author(s):

Priya Edward ◽

William V La Via ◽

Mehreen Arshad ◽

Kiran Gajurel

Keyword(s):

Next Generation Sequencing ◽

Case Series ◽

Mycoplasma Hominis ◽

Microbial Cell ◽

Next Generation ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Hominis Infection ◽

Generation Sequencing

Abstract Background Mycoplasma hominis is typically associated with genital infections in women and is a rare cause of musculoskeletal infections often in immunocompromised hosts. Diagnosis of invasive Mycoplasma hominis infections are difficult due to challenges in culturing these organisms. Molecular diagnostics require an index of suspicion which may not be present at the time of tissue sampling. Accurate, rapid diagnosis of Mycoplasma hominis infections are important for antibiotic management. Methods Two cases of invasive Mycoplasma hominis infections are presented in which the Karius test (KT) was used to make the diagnosis. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell-free DNA (mcfDNA). After mcfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of > 1400 organisms. Organisms present above a statistical threshold are reported. Case review was performed for clinical correlation. Results A young woman with lupus nephritis status post renal transplant developed persistent fever with progressive multifocal culture-negative osteoarticular infection despite empiric ceftriaxone. An adolescent female presented with an ascending pelvic infection progressing to purulent polymicrobial peritonitis (see table) requiring surgical debridement and cefipime, metronidazole and micafungin therapy; her course was complicated by progressive peritonitis/abscesses. Karius testing detected high-levels of Mycoplasma hominis mcfDNA in both cases – at 3251 molecules/microliter (MPM) in the first case and 3914 MPM in the second case. The normal range of Mycoplasma hominis mcfDNA in a cohort of 684 normal adults is 0 MPM. The patients rapidly improved with atypical coverage with doxycycline and levofloxaxin. Clinical findings in 2 patients with M. hominis infection detected by the Karius Test Conclusion Open-ended, plasma-based NGS for mcfDNA provides a rapid, non-invasive method to diagnose invasive Mycoplasma hominis infection. This case series highlights the potential to diagnose infections caused by fastidious pathogens to better inform antimicrobial therapy and achieve favorable outcomes. Disclosures William V. La Via, MD, Karius (Employee)

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Early solid tumor diagnosis through next-generation sequencing of cell-free DNA

Biomarkers in Medicine ◽

10.2217/bmm-2018-0269 ◽

2018 ◽

Vol 12 (11) ◽

pp. 1197-1201

Author(s):

Demosthenes E Ziogas ◽

Ioannis D Kyrochristos ◽

Efstathios G Lykoudis ◽

Dimitrios H Roukos

Keyword(s):

Next Generation Sequencing ◽

Solid Tumor ◽

Tumor Diagnosis ◽

Next Generation ◽

Cell Free Dna ◽

Free Dna ◽

Generation Sequencing

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

F1000Research ◽

10.12688/f1000research.19766.3 ◽

2019 ◽

Vol 8 ◽

pp. 1194 ◽

Cited By ~ 2

Author(s):

Jose F. Camargo ◽

Asim A. Ahmed ◽

Martin S. Lindner ◽

Michele I. Morris ◽

Shweta Anjan ◽

...

Keyword(s):

Next Generation Sequencing ◽

Invasive Aspergillosis ◽

Noninvasive Diagnosis ◽

Cell Transplant ◽

Next Generation ◽

Cell Free Dna ◽

Hematopoietic Stem ◽

Free Dna ◽

Immunocompromised Hosts ◽

Generation Sequencing

Background: Cell-free DNA (cfDNA) sequencing has emerged as an effective laboratory method for rapid and noninvasive diagnosis in prenatal screening testing, organ transplant rejection screening, and oncology liquid biopsies but clinical experience for use of this technology in diagnostic evaluation of infections in immunocompromised hosts is limited. Methods: We conducted an exploratory study using next-generation sequencing (NGS) for detection of microbial cfDNA in a cohort of ten immunocompromised patients with febrile neutropenia, pneumonia or intra-abdominal infection. Results: Pathogen identification by cfDNA NGS demonstrated positive agreement with conventional diagnostic laboratory methods in 7 (70%) cases, including patients with proven/probable invasive aspergillosis, Pneumocystis jirovecii pneumonia, Stenotrophomonas maltophilia bacteremia, Cytomegalovirus and Adenovirus viremia. NGS results were discordant in 3 (30%) cases including two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant in whom cfDNA testing identified the etiological agent of sepsis; and one kidney transplant recipient with invasive aspergillosis who had received >6 months of antifungal therapy prior to NGS testing. Conclusion: These observations support the clinical utility of measurement of microbial cfDNA sequencing from peripheral blood for rapid noninvasive diagnosis of infections in immunocompromised hosts. Larger studies are needed.

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2139. Rickettsia typhi Detection in Clinical Infections by the Karius Test, a Plasma Microbial Cell-free DNA Next-Generation Sequencing Test

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1819 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S725-S725

Author(s):

Fernando H Centeno ◽

Asim A Ahmed ◽

David K Hong ◽

Sudeb Dalai ◽

Laila Woc-Colburn

Keyword(s):

Next Generation Sequencing ◽

Medical Center ◽

Severe Disease ◽

Laboratory Data ◽

Microbial Cell ◽

Next Generation ◽

Rickettsia Typhi ◽

Cell Free Dna ◽

Free Dna ◽

Generation Sequencing

Abstract Background Rickettsia typhi typically causes a nonspecific syndrome characterized by fever, rash, and headache but can rarely progress to severe disease. R. typhi is transmitted by the rat flea and there has been an increased incidence in Houston, TX. Establishing the diagnosis can be challenging and is often made by serological studies. Prompt therapy with doxycycline is important especially in severe disease. Methods Karius Test results from the prior 2 years (Redwood City, CA) were reviewed for detections of R. typhi. The Karius Test is a CLIA-certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell free DNA (mcfDNA). After mcfDNA is extracted and NGS performed, human sequences are removed and remaining sequences are aligned to a curated pathogen database of >1,000 organisms. Organisms present above a statistical threshold are reported. Chart review was conducted on the cases of R. typhi identified by the Karius Test. Results The Karius Test detected R. typhi in 6 adult patients, 4 women and 2 men, from a medical center in Houston, TX. In 2 patients, R. typhi mcfDNA was present in the raw sequencing data but at an abundance below validated statistical thresholds. R. typhi mcfDNA was not found in negative controls run simultaneously with the samples. All patients presented with fever, 4 presented with headache, 3 presented with gastrointestinal symptoms, 3 developed rash, one presented with hypotension. Laboratory data were available for 5 patients. Four patients developed thrombocytopenia, 5 had anemia, 4 patients had WBC < 5, 4 had transaminase elevation and 3 developed hyponatremia. 3 out of 5 had R. typhi serologies sent; all 3 were positive (including two of the patients with R. typhi mcfDNA levels below threshold). In the two other patients the Karius test was the means of establishing the diagnosis. 3 out of 5 patients where data were available were treated with doxycyline. Conclusion The Karius test was able to detect R. typhi in a cluster of 6 patients in one medical center in Houston, TX. NGS for mcfDNA offers a rapid means of detecting R. typhi infection. Accurate, rapid diagnosis of R. typhi has important public health implications given its vector-borne mechanism of transmission. Disclosures All authors: No reported disclosures.

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