Microbial cfDNA Research Articles

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

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.

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

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. Methods: Here we report our experience 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: Among five hematological malignancy patients, for whom a microbiological diagnosis was established, pathogen identification by cfDNA NGS demonstrated 100% positive agreement with conventional diagnostic laboratory methods. Further, cfDNA identified the etiological agent in two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant. Conclusion: These data 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.

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

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 potential 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.

Application of cell-free DNA sequencing in characterization of bloodborne microbes and the study of microbe-disease interactions.

It is an important issue whether microorganisms can live harmoniously with normal cells in the cardiovascular system. The answer to the question will have enormous impact on medical microbiology. To address the issue, it is essential to identify and characterize the bloodborne microbes in an efficient and comprehensive manner. Due to microbial sequence complexity and the composition of significant number of unknown microbial species in the circulatory system, traditional approaches using cell culture, PCR, or microarray are not suitable for the purpose. Recent reports indicate that cell-free DNA (cfDNA) sequencing using next-generation sequencing (NGS) or single-molecule sequencing (SMS), together with bioinformatics approaches, possesses a strong potential enabling us to distinguish microbial species at the nucleotide level. Multiple studies using microbial cfDNA sequencing to identify microbes for septic patients have shown strong agreement with cell culture. Similar approaches have also been applied to reveal previously unidentified microorganisms or to demonstrate the feasibility of comprehensive assessment of bloodborne microorganisms for healthy and/or diseased individuals. SMS using either SMRT (single-molecule real-time) sequencing or Nanopore sequencing are providing new momentum to reinforce this line of investigation. Taken together, microbial cfDNA sequencing provides a novel opportunity allowing us to further understand the involvement of bloodborne microbes in development of diseases. Similar approaches should also be applicable to the study of metagenomics for sufficient and comprehensive analysis of microbial species living in various environments. This article reviews this line of research and discuss the methodological approaches that have been developed, or are likely to be developed in the future, which may have strong potential to facilitate cfDNA- and cfRNA-based studies of cancer and acute/chronic diseases, in the hope that a better understanding of the hidden microbes in the circulatory system will improve diagnosis, prevention and treatment of problematic diseases.

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

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. Methods: Here we report our experience using next-generation sequencing (NGS) for detection of microbial cfDNA in a cohort of ten immunocompromised patients with febrile neutropenia or deep-seated infection. Results: Among five hematological malignancy patients, for whom a microbiological diagnosis was established, pathogen identification by cfDNA NGS demonstrated 100% positive agreement with conventional diagnostic laboratory methods. Further, cfDNA identified the etiological agent in two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant. Conclusion: These data 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.

Next Generation Sequencing of Microbial Cell-Free DNA for Rapid Noninvasive Diagnosis of Infectious Diseases in Immunocompromised Hosts

Background Cell-free DNA (cfDNA) sequencing has emerged as a novel laboratory method for rapid and noninvasive diagnosis in prenatal screening, organ transplant rejection, and tumor detection. We describe a series of immunocompromised patients in which cfDNA sequencing was applied to infectious disease diagnosis. Methods Ten adult patients receiving chemotherapy or status post transplantation followed at the Sylvester Comprehensive Cancer Center with febrile illness or documented infection were enrolled between July 31 and October 2, 2018. As part of the clinical evaluation, 5ml of blood was sent for the Karius® Test (Redwood City, CA) which uses next generation sequencing (NGS) of microbial cfDNA in plasma to identify over 1,000 pathogens including bacteria, DNA viruses and fungi. Results The characteristics of the patients studied are presented in Table 1. The median age was 56 years (range, 20-65) with 60% males. Except for a kidney transplant recipient, all other patients had underlying hematological disease and/or had received a hematopoietic cell transplant. All patients were receiving antimicrobials at the time of plasma sample collection. Three patients had neutropenia (absolute neutrophil count Conclusion These data support the clinical utility of NGS-based detection of microbial cfDNA in peripheral blood for rapid noninvasive diagnosis of infections in immunocompromised hosts. Larger studies are needed to validate these findings.

Analysis of microbial sequences in plasma cell-free DNA for early-onset breast cancer patients and healthy females

BackgroundCell-free circulating DNA (cfDNA) is becoming a useful biopsy for noninvasive diagnosis of diseases. Microbial sequences in plasma cfDNA may provide important information to improve prognosis and treatment. We have developed a stringent method to identify microbial species via microbial cfDNA in the blood plasma of early-onset breast cancer (EOBC) patients and healthy females. Empirically, microbe-originated sequence reads were identified by mapping non-human PE reads in cfDNA libraries to microbial databases. Those mapped concordantly to unique microbial species were assembled into contigs, which were subsequently aligned to the same databases. Microbial species uniquely aligned were identified and compared across all individuals on MCRPM (Microbial CfDNA Reads Per Million quality PE reads) basis.ResultsThe predominant microbial cfDNAs in all plasma samples examined are originated from bacteria and these bacteria were limited to only a few genera. Among those, Acinetobacter johnsonii XBB1 and low levels of Mycobacterium spp. were commonly found in all healthy females, but also present in an EOBC patient. Compared to those in healthy counterparts, bacterial species in EOBC patients are more diverse and more likely to present at high levels. Among these three EOBC patients tested, a patient who has record high titer (2,724 MCRPM) of Pseudomonas mendocina together with 8.82 MCRPM of Pannonibacter phragmitetus has passed away; another patient infected by multiple Sphingomonas species remains alive; while the third patient who has similar microbial species (Acinetobacter johnsonii XBB1) commonly seen in normal controls is having a normal life.ConclusionsOur preliminary data on the profiles of microbial cfDNA sequences suggested that it may have some prognostic value in cancer patients. Validation in larger number of patients is warranted.

Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.

Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10−5, Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10−5) and microbial cfDNA (71.3x, p 10−5). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.