scholarly journals Plasma Microbial Cell-Free DNA Sequencing Technology for the Diagnosis of Sepsis in the ICU

2021 ◽  
Vol 8 ◽  
Author(s):  
Lili Wang ◽  
Wenzheng Guo ◽  
Hui Shen ◽  
Jian Guo ◽  
Donghua Wen ◽  
...  
Keyword(s):  
Blood Culture ◽  
Central Nervous System Infection ◽  
The Body ◽  
Microbial Cell ◽  
Sepsis Group ◽  
Acid Extraction ◽  
Sequencing Analysis ◽  
Cell Free Dna ◽  
Free Dna ◽  
Significant Difference

Sepsis is a common life-threatening disease in the intensive care unit (ICU) that is usually treated empirically without pathogen identification. As a non-invasive and high-throughput technology, plasma microbial cell-free DNA (mcfDNA) sequencing can detect unknown pathogens independent of previous clinical or laboratory information. In this study, a total of 199 cases suspected of bloodstream infection (BSI) from January 2020 to June 2020 were collected, and potential pathogens were detected by simultaneous blood culture and plasma mcfDNA sequencing. Other clinical microbiological assays were performed within 7 days of plasma mcfDNA sequencing, including smear, culture of samples taken from relevant infected sites, and β-D-glucan/galactomannan (BDG/GM) tests, among others. The diagnoses were classified as sepsis [94 (47.2%)], non-sepsis [87 (43.7%)], and non-infectious disease [18 (9.0%)]. The sensitivity and specificity of plasma mcfDNA sequencing for diagnosing sepsis were 68.1 and 63.2%, respectively, which were significantly better than those of blood culture, especially for the common bacteria that cause hospital-acquired infection, namely, Acinetobacter baumannii (p < 0.01) and Klebsiella pneumoniae (p < 0.01), and DNA viruses (plasma mcfDNA sequencing only, p < 0.01). However, there was no significant difference in the rate of positivity between plasma mcfDNA sequencing and blood culture for antibiotic-non-exposed cases (43.6 vs. 30.9%, p = 0.17). In the non-sepsis group, 44.8% of cases (13/29) detected only by plasma mcfDNA sequencing showed infections in other parts of the body, such as lower respiratory infection (LRI), intra-abdominal infection (IAI) and central nervous system infection (CNSI). For some common pathogens (not including anaerobes), turnaround time (TAT) 3 (TAT from the initiation of blood sample processing by nucleic acid extraction to the completion of sequencing analysis) was longer than TAT1 (TAT from blood culture bottles in Virtuo to off Virtuo). With disease progression, significant dynamic changes in microbial species were clearly detected by plasma mcfDNA sequencing.

Microbial Cell-Free DNA Identifies Etiology of Bloodstream Infections, Persists Longer Than Conventional Blood Cultures, and its Duration of Detection is Associated with Metastatic Infection in Patients with Staphylococcus aureus and Gram-Negative Bacteremia

2021 ◽  
Author(s):  
Emily M Eichenberger ◽  
Christiaan R de Vries ◽  
Felicia Ruffin ◽  
Batu Sharma-Kuinkel ◽  
Lawrence Park ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Blood Culture ◽  
Bloodstream Infection ◽  
Blood Cultures ◽  
Microbial Cell ◽  
Cell Free Dna ◽  
Gram Negative ◽  
Free Dna ◽  
Metastatic Infection ◽  
Gram Negative Bacteremia

Abstract 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.

scholarly journals Pneumocystis pneumonia and rheumatic disease: diagnostic potential of circulating microbial cell-free DNA sequencing

2021 ◽  
Author(s):  
Jia Li ◽  
Jun Li ◽  
Yuetian Yu ◽  
Rongsheng Wang ◽  
Mi Zhou ◽  
...  
Keyword(s):  
Rheumatic Disease ◽  
Microbial Cell ◽  
Pneumocystis Jirovecii ◽  
Control Group ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Cell Free Dna ◽  
Invasive Approach ◽  
Cell Counts ◽  
Free Dna ◽  
Significant Difference

Abstract Objectives This study aimed to explore the clinical utility of circulating microbial cell-free DNA (cfDNA) sequencing as a non-invasive approach for diagnosing Pneumocystis jirovecii pneumonia (PJP) in immunocompromised patients with rheumatic disease (RD). Methods The study included 72 RD patients with suspected lung infections admitted to Renji hospital. Eighteen individuals were diagnosed with PJP, and 54 patients without PJP were enrolled as control group. All patients had undergone pulmonary computed tomography scans, and blood and respiratory tract specimens had been subjected to metagenomic next-generation sequencing (mNGS) and conventional microbiological tests. The clinical and laboratory parameters were collected and efficacy of circulating microbial cfDNA of PJP was evaluated. Results Of the 18 patients with PJP, the average age was 53.0 years and the median time between RD diagnosis and PJP presentation was 126 days (IQR 84.0–176.3). Low circulating CD4+ cell counts and a lack of PJP prophylaxis were observed in the patients. Metagenomic NGS of circulating microbial cfDNA was performed in 69 patients including 15 cases with PJP and 54 controls. Twelve (80%) of 15 analysed blood samples contained Pneumocystis jirovecii (PJ) sequences in PJP group with PJ not detected among controls. There was a significant difference between PJP and non-PJP groups (p < 0.001) with a sensitivity of 83.3% and specificity of 100% when using plasma cfDNA sequencing. Higher β-D-glucan levels were found in patients with positive results for PJ in plasma cfDNA sequencing. Conclusion Metagenomic NGS of circulating microbial cfDNA is a potential tool for diagnosing PJP in RD patients.

scholarly journals 666. Microbial Cell-Free DNA Sequencing for Evaluation of Response to Antibiotic Therapy in Patients with Relapsed or Refractory Leukemia

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S388-S388
Author(s):  
Joshua Wolf ◽  
Kathryn Goggin ◽  
Amanda griffen ◽  
Christina Kohler ◽  
Kim J Allison ◽  
...  
Keyword(s):  
Blood Culture ◽  
Antibiotic Therapy ◽  
Dna Sequencing ◽  
Microbial Cell ◽  
Response To Treatment ◽  
Research Support ◽  
Cell Free Dna ◽  
Free Dna ◽  
Slow Decay ◽  
Pathogen Dna

Abstract Background In patients with bloodstream infection (BSI), true eradication of infection takes longer than blood culture clearance. Therefore, optimal treatment duration, especially in immunocompromised hosts, is unknown. A sensitive test of microbiological response to treatment could improve care by indicating a time for safe antibiotic discontinuation. Microbial cell-free DNA sequencing (mcfDNA-seq) is a sensitive predictor of BSI, and we hypothesize that it might also be useful to measure response to treatment. Methods Eligible participants were < 25 years of age being treated for leukemia. Remnant plasma samples were collected as part of a prospective study (PREDSEQ), and underwent mcfDNA-seq by Karius Inc. in a CLIA/CAP-accredited laboratory. Pathogen DNA was reported in molecules per microliter (MPM). Testing was batched and blinded. Available samples from Day 1 through Day 7 after onset of bacterial BSI were included. We evaluated decay of the BSI pathogen DNA after initiation of effective antibiotic therapy, from the peak to last available sample, and compared episodes with slow (< 0.5 log10 MPM/day) vs. rapid DNA decay. Results There were 13 evaluable BSI episodes in 9 participants; 7 had slow DNA decay. Persistence of bacteremia or fever ≥1 day after initiation of effective antibiotics occurred in 9/13 episodes (7/7 slow decay and 2/6 rapid decay; P = 0.02). Slow decay persisted beyond resolution of bacteremia and fever in 3/7 of these cases. Figure 1. Pathogen DNA Concentration by mcfDNA-seq During Antibiotic Treatment of Bacteremia; Dashed line, blood culture positive; Red circle, last fever Conclusion In this small convenience sample of patients with leukemia, slow mcfDNA-seq DNA decay correlated with persistent fever or bacteremia. Post-BSI mcfDNA-seq monitoring should be investigated with the goal of decreasing inappropriate antibiotic therapy and preventing treatment failure. Disclosures Joshua Wolf, MBBS, PhD, FRACP, Karius inc (Grant/Research Support) Asim A. Ahmed, MD, Karius (Employee) Desiree D. Hollemon, MSN, MPH, Karius inc (Employee) Charles Gawad, MD PhD, Karius inc (Grant/Research Support)

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)

Sequencing of Circulating Microbial Cell-Free DNA Can Identify Pathogens in Periprosthetic Joint Infections

2021 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
Adriana P. Echeverria ◽  
Ian S. Cohn ◽  
David C. Danko ◽  
Sara Shanaj ◽  
Lily Blair ◽  
...  
Keyword(s):  
Microbial Cell ◽  
Cell Free Dna ◽  
Joint Infections ◽  
Free Dna

Circulating microbial cell-free DNA is associated with inflammatory host-responses in severe pneumonia

Thorax ◽  
2021 ◽  
pp. thoraxjnl-2020-216013
Author(s):  
Haopu Yang ◽  
Ghady Haidar ◽  
Nameer S Al-Yousif ◽  
Haris Zia ◽  
Daniel Kotok ◽  
...  
Keyword(s):  
Large Scale ◽  
Inflammatory Responses ◽  
Severe Pneumonia ◽  
Systemic Circulation ◽  
Microbial Cell ◽  
Metagenomic Sequencing ◽  
Cell Free Dna ◽  
Free Dna ◽  
Culture Positive ◽  
Culture Negative

Host inflammatory responses predict worse outcome in severe pneumonia, yet little is known about what drives dysregulated inflammation. We performed metagenomic sequencing of microbial cell-free DNA (mcfDNA) in 83 mechanically ventilated patients (26 culture-positive, 41 culture-negative pneumonia, 16 uninfected controls). Culture-positive patients had higher levels of mcfDNA than those with culture-negative pneumonia and uninfected controls (p<0.005). Plasma levels of inflammatory biomarkers (fractalkine, procalcitonin, pentraxin-3 and suppression of tumorigenicity-2) were independently associated with mcfDNA levels (adjusted p<0.05) among all patients with pneumonia. Such host–microbe interactions in the systemic circulation of patients with severe pneumonia warrant further large-scale clinical and mechanistic investigations.

scholarly journals Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA

2017 ◽  
Vol 114 (36) ◽  
pp. 9623-9628 ◽  
Author(s):  
Mark Kowarsky ◽  
Joan Camunas-Soler ◽  
Michael Kertesz ◽  
Iwijn De Vlaminck ◽  
Winston Koh ◽  
...  
Keyword(s):  
Human Body ◽  
Sequence Data ◽  
Human Microbiome ◽  
Pcr Amplification ◽  
The Body ◽  
Direct Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Novel Taxa ◽  
Circulating Cell Free Dna

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

scholarly journals Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma

2018 ◽  
Vol 115 (46) ◽  
pp. E10925-E10933 ◽  
Author(s):  
Peiyong Jiang ◽  
Kun Sun ◽  
Yu K. Tong ◽  
Suk Hang Cheng ◽  
Timothy H. T. Cheng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Circulation ◽  
Sequence Information ◽  
Sequencing Analysis ◽  
Plasma Samples ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Dna

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease

2019 ◽  
Vol 4 (4) ◽  
pp. 663-674 ◽  
Author(s):  
Timothy A. Blauwkamp ◽  
Simone Thair ◽  
Michael J. Rosen ◽  
Lily Blair ◽  
Martin S. Lindner ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Dna Sequencing ◽  
Microbial Cell ◽  
Clinical Validation ◽  
Cell Free Dna ◽  
Free Dna
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