scholarly journals Donor-Derived Cell-Free DNA to Diagnose Graft Rejection Post-Transplant: Past, Present and Future

2021 ◽  
Vol 2 (3) ◽  
pp. 348-361
Author(s):  
Naveen Kumar ◽  
Rashmi Rana ◽  
Devender Singh Rana ◽  
Anurag Gupta ◽  
Mohinder Pal Sachdeva
Keyword(s):  
Digital Pcr ◽  
Cell Free Dna ◽  
Post Transplant ◽  
Non Invasive ◽  
Free Dna ◽  
Assay Methods ◽  
Transplanted Organs ◽  
Published Research ◽  
Donor Sample ◽  
Generation Sequencing

Donor-derived cell-free DNA (dd-cfDNA) is a non-invasive biomarker that is more sensitive and specific towards diagnosing any graft injury or rejection. Due to its applicability over all transplanted organs irrespective of age, sex, race, ethnicity, and the non-requirement of a donor sample, it emerges as a new gold standard for graft health and rejection monitoring. Published research articles describing the role and efficiency of dd-cfDNA were identified and scrutinized to acquire a brief understanding of the history, evolution, emergence, role, efficiency, and applicability of dd-cfDNA in the field of transplantation. The dd-cfDNA can be quantified using quantitative PCR, next-generation sequencing, and droplet digital PCR, and there is a commendatory outcome in terms of diagnosing graft injury and monitoring graft health. The increased levels of dd-cfDNA can diagnose the rejection prior to any other presently used biochemistry or immunological assay methods. Biopsies are performed when these tests show any signs of injury and/or rejection. Therefore, by the time these tests predict and show any unusual or improper activity of the graft, the graft is already damaged by almost 50%. This review elucidates the evolution, physiology, techniques, limitations, and prospects of dd-cfDNA as a biomarker for post-transplant graft damage and rejection.

scholarly journals 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

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)

scholarly journals Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3541 ◽  
Author(s):  
Md Ismail Hosen ◽  
Nathalie Forey ◽  
Geoffroy Durand ◽  
Catherine Voegele ◽  
Selin Bilici ◽  
...  
Keyword(s):  
Urothelial Cancer ◽  
Digital Pcr ◽  
Kappa Coefficient ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Promoter Mutations

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

scholarly journals COMP-20. THE NON-INVASIVE DETECTION OF GLIOBLASTOMA-DERIVED CELL-FREE DNA IN PLASMA USING NEXT-GENERATION SEQUENCING AND AN UNTARGETED VARIANT SEARCH

2018 ◽  
Vol 20 (suppl_6) ◽  
pp. vi67-vi68
Author(s):  
Hunter Underhill ◽  
Sabine Hellwig ◽  
David Nix ◽  
Preetida Bhetariya ◽  
Carrie Fuertes ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Generation Sequencing

scholarly journals Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

Oncotarget ◽  
2015 ◽  
Vol 6 (31) ◽  
pp. 30850-30858 ◽  
Author(s):  
Hidenobu Ishii ◽  
Koichi Azuma ◽  
Kazuko Sakai ◽  
Akihiko Kawahara ◽  
Kazuhiko Yamada ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasma Cell ◽  
Digital Pcr ◽  
Resistance Mechanisms ◽  
Pcr Analysis ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Egfr Mutant

scholarly journals Identification of novel somatic cell-free DNA variants by next-generation sequencing in breast cancer patients

2021 ◽  
Vol 6 ◽  
pp. 16-26
Author(s):  
Aarthy Raghu ◽  
Meenakumari Balaiah ◽  
Sridevi Veluswami ◽  
Shirley Sundersingh ◽  
Rajkumar Thangarajan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Breast Cancer Patients ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Pathogenic Variants ◽  
Free Dna ◽  
Potential Applications ◽  
Tumor Dna ◽  
Generation Sequencing

Objectives: Breast cancer is a heterogeneous disease affecting women worldwide and is one of the leading causes of mortality in India. Sampling bias due to tumor heterogeneity and invasive nature of biopsies necessitate noninvasive methods for comprehensive tumor profiling. Circulating cell-free DNA presents a complete mutation profile of the tumor, enabling the non-invasive monitoring of disease in real-time. This study aimed to identify tumor-specific variants in cfDNA with potential applications in the liquid-biopsy based testing of breast cancer. Material and Methods: Next-generation sequencing was performed for cell-free DNA, lymphocyte DNA, and tumor DNA from 21 breast cancer patients. Variant calling was performed using Torrent Suite Server v.5.0 and somatic variants were annotated using web-based tools. Pathogenic variants detected in cell-free DNA and tumor DNA of three patients were validated by Sanger sequencing. Results: Fifty-nine somatic variants were detected in the cell-free DNA of 10 breast cancer patients. Hotspot variants were detected in PIK3CA, TP53, and KRAS genes. In addition, previously unreported missense variants in ABL1 and PIK3CA genes were predicted to be pathogenic and potential driver mutations. Several frameshift indels were detected in two triple negative breast cancer patients. Conclusion: Sequencing of cell-free DNA from breast cancer patients identified somatic variants including several potentially pathogenic variants which have not been reported previously. These variants may have potential applications as non-invasive biomarkers for breast cancer.

scholarly journals 663. Two (Plus) Birds, One Stone: The Rapid, Comprehensive, Non-invasive Detection of Co-Pathogens of Critical Importance Using A Plasma-based Microbial Cell-free DNA Next-generation Sequencing Test

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S433-S434
Author(s):  
Matthew Smollin ◽  
Martin S Lindner ◽  
Nicholas R Degner ◽  
Ricardo Castillo-Galvan ◽  
Jose Alexander ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rapid Identification ◽  
Microbial Cell ◽  
Next Generation ◽  
Critical Importance ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Aspergillus Sp ◽  
Generation Sequencing

Abstract Background Immunocompromised (IC) patients are at risk for infections by a spectrum of invasive pathogens. The overlap in presentation makes it challenging to differentiate among infectious etiologies and critical co-infections (CI) may remain undiagnosed. Open-ended, comprehensive assessment of infection through microbial cell-free DNA (mcfDNA) next-generation sequencing (NGS) of plasma offers the potential for the rapid identification of multiple co-infecting pathogens of critical importance (CI-POCI) with one test. Methods Karius TestTM (KT) results from patients who underwent clinical testing from December 2016 to April 2021 were reviewed for detections of two or more CI-POCI including parasites, fungi (Pneumocystis jirovecii, Trichosporon sp, endemic mycoses, Aspergillus sp., Mucorales, Non-Aspergillus/Non-Mucorales molds), mycobacteria, Legionella sp., Nocardia sp. and Listeria. KT, developed and validated in Karius’ 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 &gt; 1500 organisms. Organisms present above a statistical threshold are reported and quantified. For &gt; 85% of tests the time to result reporting is the next day from sample receipt. Results KT detected CI of two or more POCI in 59 samples (75% adults, 25% children). The most common partnering co-pathogens of critical importance were Aspergillus sp (38), Mucorales (17) and PJP (14); the most common combinations were two or more distinct Aspergillus sp (14) followed by an Aspergillus sp and a Mucorales (12). There were 17 samples with the detection of three or more CI-POCI (29%). Figure 1. Chord Plot of Co-infections with Pathogens of Critical Importance The outer circle sections represent Karius Test detections belonging to different taxonomic groups. The length of each circle section is proportional to the total number of detections of a taxon belonging to that group. The chords connecting a pair of circle sections are proportional to the number of times two taxa from those groups were observed together, weighted by the total number of taxa detected. Conclusion Plasma mcfDNA NGS offers a rapid, comprehensive non-invasive means of detecting CI-POCI in IC patients with one test. Although rare, co-infections with POCI can greatly increase mortality. The KT can provide important insights into pathogen-pathogen interactions in complex hosts and help optimize therapy. Disclosures Matthew Smollin, PharmD, Karius, Inc. (Employee) Martin S. Lindner, PhD, Karius, Inc. (Consultant) Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Aparna Arun, MD, Karius (Employee)

scholarly journals 390. Non-invasive Detection of Co-infections in Hospitalized Patients with COVID-19 using the Karius Test, A Plasma-based Next-Generation Sequencing Test for Microbial Cell-free DNA

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S264-S264
Author(s):  
William V La Via ◽  
Sudeb Dalai ◽  
Christiaan R de Vries ◽  
Ann Macintyre ◽  
Asim A Ahmed
Keyword(s):  
Next Generation Sequencing ◽  
Critically Ill ◽  
Antimicrobial Therapy ◽  
Next Generation ◽  
Invasive Procedures ◽  
Independent Contractor ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Generation Sequencing

Abstract Background Patients hospitalized with SARS-CoV2 infections (Covid-19) are frequently febrile and can become critically ill quickly often leading to intervention with antimicrobial therapy. An etiologic diagnosis of superinfections can be difficult to obtain through the usual invasive procedures because of patient instability and the desire to avoid them because they may not be tolerated by the patient. Providers may also be hesitant to embark on such interventions in order to avoid healthcare personnel (HCP) exposure to aerosols. Methods Karius Test (KT) results are presented from 30 patients who presented with Covid-19. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects pathogen cell free DNA (cfDNA). After cfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of &gt; 1400 organisms. Organisms present above a statistical threshold are reported. Results The KT detected pathogens in the majority of patients (n=20) with COVID-19. The most common infections were herpesviruses in 60% of patients. The most common bacterial pathogen was E. coli seen in 25% of patients. 15 out of 20 patients had more than one pathogen detected. 15% of patients had fungal pathogens, including one detection of Lichtheimia ramosa, in an immunocompromised patient. The results are summarized in the table. Co-infections detected by the Karius Test in patients hospitalized with COVID-19 Conclusion Open-ended, plasma-based NGS for mcfDNA provides a non-invasive method to assess for co-infections in critically ill patients with COVID-19. This report highlights the potential to increase diagnostic yield as well as to decrease the need for invasive procedures – and their attendant risks to patients and HCP – to obtain etiologic diagnoses to better inform antimicrobial therapy for superinfection. It also serves to highlight the variety of pathogens affecting these patients during the COVID-19 pandemic. Disclosures William V. La Via, MD, Karius (Employee) Sudeb Dalai, MD, Karius (Employee) Christiaan R. de Vries, MD, PhD, Karius (Consultant, Independent Contractor)Stanford University (Employee) Ann Macintyre, DO, Karius (Employee) Asim A. Ahmed, MD, Karius (Employee)

scholarly journals 1177. Rapid, Non-invasive Detection of Invasive Mucormycosis Caused by Syncephalastrum monosporum Using Next-Generation Sequencing of Circulating Microbial Cell-free DNA in Plasma

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S613-S614
Author(s):  
Melissa Sanacore ◽  
Melhem M Solh ◽  
H Kent Holland ◽  
Asad Bashey ◽  
Samuel Webster ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Cell ◽  
Ct Scans ◽  
Cell Transplant ◽  
Next Generation ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Serial Ct ◽  
Generation Sequencing

Abstract Background Improving diagnostics have led to newly identified causes of invasive fungal infection (IFI) in immunocompromised hosts. Syncephalastrum spp. are Zygomycetes more commonly associated with skin infections and have only rarely been implicated as a cause of IFI(1). Next generation sequencing (NGS) for circulating microbial cell-free DNA (mcfDNA) in plasma offers a unique tool to diagnose rare causes of IFI (2,3). Methods Karius results were reviewed for Syncephalastrum detections with 2 identified at the same institution. McfDNA was extracted from plasma and NGS was performed by Karius, Inc. (Redwood City, California). Human sequences were removed and remaining sequences were aligned to a database of over 1,400 pathogens. Organisms present above a predefined statistical significance threshold were quantified in DNA molecules per microliter (MPM). Chart review was performed for clinical correlation. Results A 66 y/o male one month out of induction therapy for acute myeloblastic leukemia (AML) developed pneumonia. Although BAL was negative for mold and despite empiric antifungals, plasma NGS for mcfDNA showed S. monosporum at 562 MPM; the reference range is 0 MPM. Amphotericin was added to empiric posaconazole. The patient was discharged 10 days later and serial CT scans showed improvement. Repeat NGS mcfDNA 11 days later was negative. He underwent stem cell transplant (SCT) 4 months later. In a second case, a 66 y/o female with acute prolymphocytic leukemia was admitted for fever with neutropenia. A CT chest showed new multifocal, bilateral, nodular opacities. Despite negative BAL fungal culture and pretreatment with fluconazole, plasma NGS mcfDNA revealed S. monosporum at 575 MPM. She was treated with micafungin, amphotericin, and posaconazole with clinical improvement. Repeat NGS mcfDNA 8 weeks later was negative. Serial CT scans showed improvement over 5 months. She proceeded to SCT. Conclusion Plasma-based NGS for mcfDNA enabled rapid, non-invasive detection of pulmonary mucormycosis caused by S. monosporum despite antifungal pretreatment and unrevealing invasive procedures in 2 patients with leukemia. The rapid identification of the specific etiology of IFI enabled targeted anti-fungal therapy and resumption of definitive oncological care including SCT. Table 1: Clinical Parameters Disclosures Christiaan R. de Vries, MD, PhD, Karius (Consultant, Independent Contractor)Stanford University (Employee)

Sign in / Sign up

Export Citation Format

Share Document