Microbial cfDNA Research Articles

Leveraging circulating microbial DNA for early cancer detection

Microbial cell-free DNA (microbial cfDNA) offers a minimally invasive approach for profiling the microbiome. Despite technical and biological challenges, the potential of microbial cfDNA for cancer detection is increasingly being evaluated using deep sequencing, novel laboratory approaches, and computational methods. Targeting the microbiome using liquid biopsies could improve early cancer detection.

Clinical Accuracy and Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for the Diagnosis of Invasive Aspergillosis in Patients with Hematologic Malignancies or Coronavirus Disease 2019.

We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel non-invasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13

Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.

Fragment Ends of Circulating Microbial DNA as Signatures for Pathogen Detection in Sepsis.

Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.

319. Clinical accuracy and utility of plasma microbial cell free DNA NGS for the diagnosis of invasive aspergillosis in patients with hematologic malignancy and COVID-19

Abstract Background Invasive aspergillosis (IA) is a great threat to the severely immunocompromised and patients with coronavirus disease (COVID-19). However, diagnosis of IA is often difficult due to need for invasive biopsy and low sensitivity of other diagnostic tests. Next-generation sequencing (NGS) of plasma cell free DNA (cfDNA) can be a novel non-invasive diagnostic modality. We evaluated the clinical accuracy and utility of microbial cfDNA NGS for the diagnosis of IA in patients with hematologic malignancy (HM) and COVID-19. Methods A single-center prospective study of plasma microbial cfDNA NGS was conducted in a tertiary-care hospital in South Korea. We enrolled adult patients with HM and COVID-19, who suspected IA and performed conventional diagnostic tests for IA. The results of NGS were compared with the diagnosis of IA through conventional methods. IA cases were diagnosed according to EORTC/MSG definitions in patients with HM, and modified AspICU criteria in patients with COVID-19. (Figure 1). Figure 1.Flow chart for the participant selection method used in this study Results Between March 2021 and January 2022, a total of 33 participants (22 [64.7%] male, median age 66.0 [50.5, 72.0]) were enrolled;19 participants with HM and 15 with COVID-19 were analyzed (Figure1 and Table1). In participants with HM, aspergillus cfDNA was detected in 100% of both proven (1/1) and probable (12/12) IA cases, and 33.3% of both possible (1/3) and no IA (1/3) cases. In participants with COVID-19, 46.2% of probable IA (6/13) showed positive aspergillus cfDNA. Detection rate of aspergillus cfDNA was significantly higher in proven/probable IA cases in participants with HM compared to participants with COVID-19. (100% vs 46.2%, p=0.005) (Figure 2). As shown in Table 2, among proven/probable IA cases, participants with positive aspergillus cfDNA showed significantly higher rate of having uncontrolled hematologic disease, receiving stem cell transplantation and recent chemotherapy. In three participants with HM, non-aspergillus strains confirmed by cfDNA NGS were in accordance with pathogens identified through conventional culture methods. Table 1.Baseline characteristics of participants suspected of invaisve aspergillosis performing microbial cell free DNA NGSFigure 2.cfDNA detection rate in participants with suspected fungal infection according to the EORTC/MSG or modified AspICU diagnostic criteria Conclusion Detection of aspergillus cfDNA showed high concordance in the results of conventional diagnostic methods in proven/probable IA of patients with HM and could be a helpful non-invasive approach to IA diagnosis in those populations. Disclosures All Authors: No reported disclosures.

Peripheral Blood Microbiome Analysis via Noninvasive Prenatal Testing Reveals the Complexity of Circulating Microbial Cell-Free DNA.

ABSTRACTWhile circulating cell-free DNA (cfDNA) is becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a microbial cfDNA baseline in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Because noninvasive prenatal testing (NIPT) shares many similarities with the sequencing protocol of metagenomics, we utilized the standard low-pass whole-genome-sequencing-based NIPT to establish a microbial cfDNA baseline in healthy people. Sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening were retrospectively collected and reanalyzed for microbiome DNA screening. It was found that more than 95% of exogenous cfDNA was from bacteria, 3% from eukaryotes, and 0.4% from viruses, indicating the gut/environment origins of many microorganisms. Overall and regional abundance patterns were well illustrated, with huge regional diversity and complexity, and unique interspecies and symbiotic relationships were observed for TORCH organisms (Toxoplasma gondii, others [Treponema pallidum {causing syphilis}, hepatitis B virus {HBV}, and human parvovirus B19 {HPV-B19}], rubella virus, cytomegalovirus [CMV], and herpes simplex virus [HSV]) and another common virus, Epstein-Barr virus (EBV). To sum up, our study revealed the complexity of the baseline circulating microbial cfDNA and showed that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner.IMPORTANCE While circulating cell-free DNA (cfDNA) has been becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a baseline for microbial cfDNA in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Standard low-pass whole-genome-sequencing-based NIPT shares many similarities with the sequencing protocol for metagenomics and could provide a microbial cfDNA baseline in healthy people; thus, a reference cfDNA data set of the human microbiome was established with sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening. Our study revealed the complexity of circulating microbial cfDNA and indicated that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner, especially with regard to geographic patterns and coexistence networks.

Analysis of the methods and quality assurance of metagenomic next-generation sequencing to detect the microbial cfDNA from blood samples in China

Objective: To investigate the methods and quality assurance of metagenomic next-generation sequencing (mNGS) to detect the microbial cfDNA (mcfDNA) from blood samples in different laboratories across China. Methods: In October 2020, questionnaires about detecting mcfDNA in blood samples with mNGS were distributed to 80 laboratories across the country. The questionnaire included four parts: pre-analysis, during analysis, post-analysis, and carrying out of performance validation for mNGS. (1) Pre-analysis: the requirements for samples quality, such as collection, storage, the transportation conditions of samples; (2) During analysis: the extraction workflows of mcfDNA, the quality requirements of the library, the application of the sequencing platforms and the bioinformatics analysis pipelines; (3) Post-analysis: the standard of interpretation results for mNGS; (4) Carrying out of performance validation: the minimum detection limit for various pathogens. All laboratories are required to fill in the questionnaire according to the actual situation. The feedback data were summarized and analyzed. Results: The 80 laboratories included 20 medical centers and 60 independent medical laboratories. There were 80.0% (64/80) of laboratories indicated that both plasma and serum samples were used to detect mcfDNA in blood, and the rest of the laboratories (16/80, 20.0%) only used plasma samples. The sequencing platforms used by mNGS laboratories involved in the survey included illumina (49), Beijing Genomics Institute (16), Ion Torrent (13) and Nanopore sequencing (2). There were 87.5% (70/80) of laboratories used the integrated analysis tools built by the third-party laboratories, and other laboratories (12.5%, 10/80) independently built the analysis platform by open-source software. The interpretation criteria of mNGS results varied between laboratories, among which the normalized number of pathogen-specific sequences, relative abundance, genome coverage rate, and the detection of the microorganism in the negative control were the main factors considered by laboratories. Most laboratories (76.3%, 61/80) had carried out the performance validation for the mcfDNA mNGS workflows. The limit of detection of the laboratories-developed mNGS workflows for Gram-positive bacteria, Gram-negative bacteria, fungi, parasites, and other pathogens were mainly distributed at 10-100 copies/ml, DNA virus was mainly distributed at 500-1 000 copies/ml. Conclusions: The mNGS workflows of various laboratories are very different. In order to ensure timely and accurate testing results, every laboratory needs to actively optimize the mNGS testing procedures, improve quality assurance measures, and carry out performance validation before mNGS is widely used in clinical settings.

Fragment Size-Based Enrichment of Viral Sequences in Plasma Cell-Free DNA

Sequencing of plasma cell-free DNA (cfDNA) is a promising milieu for broad-based cancer and infectious disease diagnostics. The performance of cfDNA sequencing for infectious disease diagnostics is chiefly limited by inadequate analytical sensitivity. The current study investigated whether the analytical sensitivity of cfDNA sequencing for viral diagnostics could be improved by selective sequencing of short cfDNA fragments, given prior observations of shorter fragment size distribution in microbial and cytomegalovirus-derived cfDNA compared with human-derived cfDNA. It shows that the shorter plasma cfDNA fragment size distribution is a general feature of multiple DNA viruses, including adenovirus [interquartile range (IQR), 87 to 165 bp], herpes simplex virus 2 (IQR, 114 to 195 bp), human herpesvirus 6 (IQR, 145 to 176 bp), and varicella zoster virus (IQR, 98 to 182 bp), compared with human (IQR, 148 to 178 bp). It was used to further optimize a size selection-based cfDNA sequencing method, demonstrating an enrichment of viral sequences up to 16.6-fold, with a median fold enrichment of 6.7×, 4.6×, 2.2×, and 10.3× for adenovirus, herpes simplex virus 2, human herpesvirus 6, and varicella zoster virus, respectively. These findings demonstrate a simple yet scalable method for enhanced detection of DNA viremia that maintains the unbiased nature of cfDNA sequencing.

Utility of liquid biopsy in diagnosing isolated cerebral phaeohyphomycosis: illustrative case.

BACKGROUNDCladophialophora bantiana is a dematiaceous, saprophytic fungus and a rare but reported cause of intracranial abscesses due to its strong neurotropism. Although it predominantly affects immunocompetent individuals with environmental exposure, more recently, its significance as a highly lethal opportunistic infection in transplant recipients has been recognized. Successful treatment requires timely but often challenging diagnosis, followed by complete surgical excision. Next-generation sequencing of microbial cell-free DNA (cfDNA) from plasma is a novel diagnostic method with the potential to identify invasive fungal infections more rapidly and less invasively than conventional microbiological testing, including brain biopsy.OBSERVATIONSThe authors described the case of a recipient of a liver transplant who presented with seizures and was found to have innumerable ring-enhancing intracranial lesions. The Karius Test, a commercially available method of next-generation sequencing of cfDNA, was used to determine the causative organism. Samples from the patient’s plasma identified C. bantiana 6 days before culture results of the surgical specimen, allowing optimization of the empirical antifungal regimen, which led to a reduction in the size of the abscesses.LESSONSThe authors’ findings suggest that microbial cfDNA sequencing may be particularly impactful in improving the management of brain abscesses in which the differential diagnosis is wide because of immunosuppression.

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

ObjectivesThe aim of this study was to explore the clinical utility of circulating microbial cell-free DNA (cfDNA) sequencing as a non-invasive approach for diagnosis of Pneumocystis jirovecii pneumonia (PJP) in immunocompromised patients with rheumatic disease (RD).MethodsThe 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 the control group. All patients had undergone pulmonary CT 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 the efficacy of circulating microbial cfDNA of P. jirovecii was evaluated.ResultsOf the 18 patients with PJP, the average age was 53.0 years, and the median time between RD diagnosis and PJP presentation was 126.0 days (interquartile range 84.0–176.3 days). 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 P. jirovecii sequences in the PJP group, with P. jirovecii 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 P. jirovecii in plasma cfDNA sequencing.ConclusionMetagenomic NGS of circulating microbial cfDNA is a potential tool for diagnosis of PJP in RD patients.

Peritoneal Effluent Cell-Free DNA Sequencing in Peritoneal Dialysis Patients With and Without Peritonitis

Rationale & ObjectiveConventional culture can be insensitive for the detection of rare infections and for the detection of common infections in the setting of recent antibiotic usage. Patients receiving peritoneal dialysis (PD) with suspected peritonitis have a significant proportion of negative conventional cultures. This study examines the utility of metagenomic sequencing of peritoneal effluent cell-free DNA (cfDNA) for evaluating the peritoneal effluent in PD patients with and without peritonitis.Study DesignProspective cohort study.Setting & ParticipantsWe prospectively characterized cfDNA in 68 peritoneal effluent samples obtained from 33 patients receiving PD at a single center from September 2016 to July 2018.OutcomesPeritoneal effluent, microbial, and human cfDNA characteristics were evaluated in culture-confirmed peritonitis and culture-negative peritonitis.Analytical ApproachDescriptive statistics were analyzed and microbial cfDNA was detected in culture-confirmed peritonitis and culture-negative peritonitis.ResultsMetagenomic sequencing of cfDNA was able to detect and identify bacterial, viral, and eukaryotic pathogens in the peritoneal effluent from PD patients with culture-confirmed peritonitis, as well as patients with recent antibiotic usage and in cases of culture-negative peritonitis.LimitationsParallel cultures were not obtained in all the peritoneal effluent specimens.ConclusionsMetagenomic cfDNA sequencing of the peritoneal effluent can identify pathogens in PD patients with peritonitis, including culture-negative peritonitis.

Measurement Biases Distort Cell-Free DNA Fragmentation Profiles and Define the Sensitivity of Metagenomic Cell-Free DNA Sequencing Assays.

BackgroundMetagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing.MethodsWe characterized the fragment length biases inherent to select DNA isolation and library preparation procedures and developed a model to correct for these biases. We analyzed 305 cfDNA sequencing data sets, including publicly available data sets and 124 newly generated data sets, to evaluate the dependence of the sensitivity of metagenomic cfDNA sequencing on pre-analytical variables.ResultsLength bias correction of fragment length distributions measured from different experimental procedures revealed the ultrashort (<100 bp) nature of microbial-, mitochondrial-, and host-specific urinary cfDNA. The sensitivity of metagenomic sequencing assays to detect the clinically reported microorganism differed by more than 5-fold depending on the combination of DNA isolation and library preparation used.ConclusionsSubstantial gains in the sensitivity of microbial and other short fragment recovery can be achieved by easy-to-implement changes in the sample preparation protocol, which highlights the need for standardization in the liquid biopsy field.

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

Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing. In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results. Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen. Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period. Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

711. Rapid, non-invasive detection and monitoring of Bartonella quintana endocarditis by plasma-based next-generation sequencing of microbial cell-free DNA

BackgroundThere are up to 50,000 new cases of infective endocarditis each year in the United States, of which approximately 20% are culture negative endocarditis (CNE). In-hospital mortality remains high at 20 to 30%. Despite advances in diagnostic testing, determining the timing of surgery and duration of treatment in CNE are significant challenges for clinicians. Plasma next-generation sequencing (NGS) for circulating microbial cell-free DNA (mcfDNA) has shown utility in diagnosing and monitoring the response to treatment in endocarditis.MethodsSerial blood samples were obtained prior to and after aortic valve replacement in a patient with culture negative endocarditis. Microbial cfDNA 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 curated database of over 1,400 pathogens. Organisms present above a predefined statistical significance threshold were reported and quantified in DNA molecules per microliter (MPM). Chart review was performed for clinical correlation.ResultsA 53-year old man with history of homelessness, well-controlled HIV infection and a bioprosthetic aortic valve presented with symptomatic severe aortic stenosis and elevated inflammatory markers 3 years following valve surgery. Transesophageal echocardiography showed a paravalvular leak. Bartonella quintana was detected by Karius NGS (in parallel Bartonella henselae serologies were positive). After 4 weeks of parenteral antibiotics, repeat Karius testing demonstrated a 94% (16-fold) decrease in the Bartonella quintana mcfDNA signal to 8813 MPM. He underwent surgical valve replacement; twenty-four hours after removal of the infected valve repeat Karius testing showed a rapid decay of the Bartonella quintana mcfDNA signal to 103 MPM. The patient completed 3 months of oral antibiotics post-operatively, ultimately returning to his former performance status.ConclusionPlasma-based next-generation sequencing assays for circulating microbial cell-free DNA offer a unique means of pathogen detection, assessment of infection burden and monitoring of response to both medical treatment and surgical debridement/definitive source control in a case of Bartonella quintana endocarditis.DisclosuresAsim A. Ahmed, MD, Karius (Employee)

Rare Presentation of Toxoplasma Pneumonitis in the Absence of Neurological Symptoms in an AIDS Patient and Use of Next-Generation Sequencing for Diagnosis.

Next-generation sequencing can help make a correct diagnosis and detect culture-negative opportunistic infections.Recognition of Toxoplasma pneumonitis as a rare presentation of disseminated toxoplasmosis.In cases of Toxoplasma pneumonitis, brain imaging should be conducted to rule out CNS involvement even in the absence of neurological symptoms.

Assessment of the Clinical Utility of Plasma Metagenomic Next-Generation Sequencing in a Pediatric Hospital Population.

Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA (cfDNA) is commercially available, but its role in the workup of infectious diseases is unclear. To understand the clinical utility of plasma mNGS, we retrospectively reviewed patients tested at a pediatric institution over 2 years to evaluate the clinical relevance of the organism(s) identified and the impact on antimicrobial management. We also investigated the effect of pretest antimicrobials and interpretation of molecules of microbial cfDNA per microliter (MPM) of plasma. Twenty-nine of 59 (49%) mNGS tests detected organism(s), and 28/51 (55%) organisms detected were clinically relevant. The median MPM of clinically relevant organisms was 1,533, versus 221 for irrelevant organisms (P = 0.01). mNGS test positive and negative percent agreements were 53% and 79%, respectively, and 50% of negative mNGS tests were true negatives. Fourteen percent of tests impacted clinical management by changing antimicrobial therapy. Immunocompromised status was the only patient characteristic that trended toward a significant clinical impact (P = 0.056). No patients with culture-negative endocarditis had organisms identified by mNGS. There were no significant differences in antimicrobial duration retest between tests with clinically relevant organism(s) and those that returned negative, nor were the MPMs different between pretreated and untreated organisms, suggesting that 10 days of antimicrobial therapy as observed in this cohort did not sterilize testing; however, no pretreated organisms identified resulted in a new diagnosis impacting clinical management. Plasma mNGS demonstrated higher utility for immunocompromised patients, but given the detection of many clinically irrelevant organisms (45%), cautious interpretation and infectious diseases consultation are prudent.

Cell free DNA from respiratory pathogens is detectable in the blood plasma of Cystic Fibrosis patients

Diagnostically informative microbial cell-free DNA (cfDNA) can be detected from blood plasma during fulminant infections such as sepsis. However, the potential for DNA from airway pathogens to enter the circulation of cystic fibrosis (CF) patients during chronic infective states has not yet been evaluated. We assessed whether patient blood contained measurable quantities of cfDNA from CF respiratory microorganisms by sequencing plasma from 21 individuals with CF recruited from outpatient clinics and 12 healthy controls. To account for possible contamination with exogenous microbial nucleic acids, statistical significance of microbe-derived read counts from CF patients was determined relative to the healthy control population. In aggregate, relative abundance of microbial cfDNA was nearly an order of magnitude higher in CF patients than in healthy subjects (p = 8.0×10−3). 15 of 21 (71%) CF patients demonstrated cfDNA from one or more relevant organisms. In contrast, none of the healthy subjects evidenced significant microbial cfDNA for any of the organisms examined. Concordance of cfDNA with standard microbiological culture of contemporaneously collected patient sputum was variable. Our findings provide evidence that cfDNA from respiratory pathogens are present in the bloodstream of most CF patients, which could potentially be exploited for the purposes of noninvasive clinical diagnosis.

Detection of microbial cell-free DNA in maternal and umbilical cord plasma in patients with chorioamnionitis using next generation sequencing.

BackgroundChorioamnionitis has been linked to spontaneous preterm labor and complications such as neonatal sepsis. We hypothesized that microbial cell-free (cf) DNA would be detectable in maternal plasma in patients with chorioamnionitis and could be the basis for a non-invasive method to detect fetal exposure to microorganisms.ObjectiveThe purpose of this study was to determine whether next generation sequencing could detect microbial cfDNA in maternal plasma in patients with chorioamnionitis.Study designMaternal plasma (n = 94) and umbilical cord plasma (n = 120) were collected during delivery at gestational age 28–41 weeks. cfDNA was extracted and sequenced. Umbilical cord plasma samples with evidence of contamination were excluded. The prevalence of microorganisms previously implicated in choriomanionitis, neonatal sepsis and intra-amniotic infections, as described in the literature, were examined to determine if there was enrichment of these microorganisms in this cohort. Specific microbial cfDNA associated with chorioamnionitis was first detected in umbilical cord plasma and confirmed in the matched maternal plasma samples (n = 77 matched pairs) among 14 cases of histologically confirmed chorioamnionitis and one case of clinical chorioamnionitis; 63 paired samples were used as controls. A correlation of rank of a given microorganism across maternal plasma and matched umbilical cord plasma was used to assess whether signals found in umbilical cord plasma were also present in maternal plasma.ResultsMicrobial DNA sequences associated with clinical and/or histological chorioamnionitis were enriched in maternal plasma in cases with suspected chorioamnionitis when compared to controls (12/14 microorganisms, p = 0.02). Analysis of the microbial cfDNA in umbilical cord plasma among the 1,251 microorganisms detectable with this assay identified Streptococcus mitis, Ureaplasma spp., and Mycoplasma spp. in cases of suspected chorioamnionitis. This assay also detected cfDNA from Lactobacillus spp. in controls. Comparison between maternal plasma and umbilical cord plasma confirmed these signatures were also present in maternal plasma. Unbiased analysis of microorganisms with significantly correlated signal between matched maternal plasma and umbilical cord plasma identified the above listed 3 microorganisms, all of which have previously been implicated in patients with chorioamnionitis (Mycoplasma hominis p = 0.0001; Ureaplasma parvum p = 0.002; Streptococcus mitis p = 0.007). These data show that the pathogen signal relevant for chorioamnionitis can be identified in both maternal and umbilical cord plasma.ConclusionThis is the first report showing the detection of relevant microbial cell-free cfDNA in maternal plasma and umbilical cord plasma in patients with clinical and/or histological chorioamnionitis. These results may lead to the development of a specific assay to detect perinatal infections for targeted therapy to reduce early neonatal sepsis complications.

Separating the signal from the noise in metagenomic cell-free DNA sequencing

BackgroundCell-free DNA (cfDNA) in blood, urine, and other biofluids provides a unique window into human health. A proportion of cfDNA is derived from bacteria and viruses, creating opportunities for the diagnosis of infection via metagenomic sequencing. The total biomass of microbial-derived cfDNA in clinical isolates is low, which makes metagenomic cfDNA sequencing susceptible to contamination and alignment noise.ResultsHere, we report low biomass background correction (LBBC), a bioinformatics noise filtering tool informed by the uniformity of the coverage of microbial genomes and the batch variation in the absolute abundance of microbial cfDNA. We demonstrate that LBBC leads to a dramatic reduction in false positive rate while minimally affecting the true positive rate for a cfDNA test to screen for urinary tract infection. We next performed high-throughput sequencing of cfDNA in amniotic fluid collected from term uncomplicated pregnancies or those complicated with clinical chorioamnionitis with and without intra-amniotic infection.ConclusionsThe data provide unique insight into the properties of fetal and maternal cfDNA in amniotic fluid, demonstrate the utility of cfDNA to screen for intra-amniotic infection, support the view that the amniotic fluid is sterile during normal pregnancy, and reveal cases of intra-amniotic inflammation without infection at term.7ESvei6brGwyYZ7yuuRbtxVideo abstract.

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.