Cell-free DNA diagnostics in transplantation utilizing next 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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