Microbial Cell-free DNA Research Articles

Analytical and clinical validation of direct detection of antimicrobial resistance markers by plasma microbial cell-free DNA sequencing.

This manuscript is ideally suited for the Innovative Diagnostic Methods sections as it reports the analytical and clinical validation of a novel application of plasma microbial cell-free DNA sequencing for direct detection of seven selected antimicrobial resistance markers in 18 target pathogens. Clearly, it has potential clinical utility in optimizing therapy and was incorporated into the Karius test workflow in September 2023. In addition, the workflow could readily be adapted to expand the number of target bacteria and antimicrobial resistance markers as needed.

Clinical Utility of Metagenomic Next-Generation Sequencing of Plasma Microbial Cell-Free DNA: a Single-Center Retrospective Study

Abstract Infectious diseases account for significant morbidity and mortality in healthcare, and metagenomic next-generation sequencing of plasma microbial cell-free DNA has emerged as a promising non-invasive diagnostic tool in this scenario. However, the available data on the real impact of this test in clinical practice is limited and conflicting. In this context, we performed a retrospective evaluation of the utilization of this test at our institution since February 17, 2019. A total of 76 tests were performed in this period in 68 different patients. The mean age of the patients was 9 years old, with 40 being male (58.82%) and 28 female (41.18%). The ordering specialties included infectious diseases (82.90%), hematology/oncology (9.21%), neurology (3.95%), pulmonology (2.63%), and dermatology (1.31%). The vast majority of the tests were ordered as inpatient (73 tests, 96.05%). 45 tests (59.21%) resulted as positive, of which 19 (42.22%) were polymicrobial. The tests were concordant with conventional microbiological studies 47 times (61.84%). Most tests (67 tests, 88.16%) had no significant clinical impact, 8 tests (10.53%) had a positive impact, and one test (1.31%) had an indeterminate impact. No tests were determined to have a negative clinical impact. Overall, our data is consistent with most studies which assessed the clinical utility of metagenomic next-generation sequencing of plasma microbial cell-free DNA in the literature, showing that testing did not have a significant impact on patient care for the majority of cases. There were some cases, however, in which testing showed a positive impact in escalating or de-escalating antibiotic therapy. In conclusion, despite being mostly a bystander, in selected cases and in the right clinical context, the test can be useful to guide antimicrobial treatment without causing significant harm. More studies are needed to further investigate the real utility of the test.

Bugs and Brains: The Microbiome & Dementia

Interest in the microbiome and its correlation with different neurological diseases has grown significantly over the past decade. The gut microbiome contains a vast array of diverse organisms living in the gastrointestinal (GI) tract. Predominantly composed of bacteria, the gut microbiome helps regulate homeostasis and metabolism in the host, aiding in immune system development as well as vitamin and nutrient absorption. Studies suggest that, in addition to providing humans with these physiological benefits, the gut microbiome is also linked to and communicates with the brain via the gut-brain axis (GBA). Long-standing research on the GBA has demonstrated that the GI tract and the central nervous system are interconnected through both neurological and immunological signaling pathways. Recent research is expanding our understanding of the microbiome and the GBA, revealing correlations between the microbiome impact on the GBA and neurological conditions such as Alzheimer’s dementia and Lewy body dementia, going as far as to identify and highlight specific microbial species that may serve as risk or protective factors for various forms of dementia. This letter aims to highlight the current findings and recommend the use of tools such as microbial cell-free DNA testing to gain a better understanding of the role of gut dysbiosis in dementia patients.

Microbial cell-free DNA-sequencing as an addition to conventional diagnostics in neonatal sepsis.

Bloodstream infections remain a challenge for neonatologists, as traditional culture-based methods are time-consuming and rely on adequate blood volume. Next-generation sequencing (NGS) offers an alternative, as it can identify microbial cell-free DNA (mcfDNA) in a small blood sample, providing rapid pathogen detection. This study aimed to assess the diagnostic performance of DISQVER®-NGS compared to blood cultures in neonatal patients with suspected sepsis. In neonates with suspected sepsis, blood cultures and samples for NGS were prospectively collected. Patients were divided into four categories: 1) sepsis, blood culture positive, 2) clinical sepsis, culture negative, 3) suspected sepsis, 4) validation cohort. NGS detected bacterial, viral or fungal mcfDNA in 24 of 82 samples. Blood cultures were collected in 46 of 84 patients (15/46 positive). DISQVER® correctly identified pathogens in 9/15 patients with a positive blood culture, two with intrinsic resistance to their antibiotic regimen. In seven samples NGS reported the mcfDNA of bacteria that could have theoretically grown in culture but did not. NGS may enhance sensitivity in sepsis diagnostics by detecting mcfDNA in neonates with suspected sepsis. Interpreting NGS results requires correlation with clinical data, laboratory values, and routine microbiological tests for a comprehensive understanding of the patient's condition. Conventional blood culture methods have limitations in accuracy and turnaround time. The study aimed to investigate the diagnostic performance of the Next-Generation Sequencing method DISQVER® compared to traditional blood cultures in neonatal patients with suspected sepsis. Our findings suggest that NGS has the potential to augment the precision of conventional diagnostic techniques, can lead to improved detection of pathogens and targeted treatment approaches in neonatal sepsis. It is emphasized that further validation and integration with clinical and microbiological data are required to ensure optimal clinical utility.

Clinical Utility of Plasma Microbial Cell-Free DNA Sequencing Among Immunocompromised Patients With Pneumonia.

Plasma microbial cell-free DNA (mcfDNA) sequencing can establish the etiology of multiple infectious syndromes by identifying microbial DNA in plasma. However, data are needed to define the clinical scenarios where this tool offers the highest clinical benefit. We conducted a prospective multicenter observational study that evaluated the impact of plasma mcfDNA sequencing compared with usual care testing among adults with hematologic malignancies. This is a secondary analysis of an expanded cohort that evaluated the clinical utility of plasma mcfDNA sequencing across prespecified and adjudicated outcomes. We examined the percentage of participants for whom plasma mcfDNA sequencing identified a probable cause of pneumonia or clinically relevant nonpneumonia infection. We then assessed potential changes in antimicrobial therapy based on plasma mcfDNA sequencing results and the potential for early mcfDNA testing to avoid bronchoscopy and its associated adverse events. Of 223 participants, at least 1 microbial detection by plasma mcfDNA sequencing was adjudicated as a probable cause of pneumonia in 57 (25.6%) and a clinically relevant nonpneumonia infection in 88 (39.5%). A probable cause of pneumonia was exclusively identified by plasma mcfDNA sequencing in 23 (10.3%) participants. Antimicrobial therapy would have changed for 41 (18.4%) participants had plasma mcfDNA results been available in real time. Among the 57 participants with a probable cause of pneumonia identified by plasma mcfDNA sequencing, bronchoscopy identified no additional probable cause of pneumonia in 52 (91.2%). Plasma mcfDNA sequencing could improve management of both pneumonia and other concurrent infections in immunocompromised patients with suspected pneumonia.

Utility of Serial Microbial Cell-free DNA Sequencing for Inpatient and Outpatient Pathogen Surveillance Among Allogeneic Hematopoietic Stem Cell Transplant Recipients.

This study characterizes the clinical utility and validity of the Karius test (KT), a plasma microbial cell-free DNA sequencing platform, as an infection surveillance tool among hematopoietic stem cell transplant (HCT) recipients, including monitoring for cytomegalovirus (CMV) and detecting infections relative to standard microbiologic testing (SMT). A prospective, observational cohort study was performed among adult HCT recipients as inpatients and outpatients. Serial KTs were performed starting with 1 sample within 14 days before HCT, then weekly from 7-63 days posttransplant then monthly from 3-12 months post-HCT. Diagnostic performance of KT versus CMV polymerase chain reaction was evaluated with positive percent agreement and negative percent agreement. Infectious events (<12 months post-HCT) were extracted from medical records. For infectious events without positive SMT, 2 clinicians adjudicated KT results to determine if any detections were a probable cause. Difference in time from KT pathogen detection and infection onset was calculated. Of the 70 participants, mean age was 49.9 years. For CMV surveillance, positive percent agreement was 100% and negative percent agreement was 90%. There was strong correlation between CMV DNA and KT molecules per microliter (r 2: 0.84, P < .001). Of the 32 SMT+/KT+ infectious events, KT identified 26 earlier than SMT (median: -12 days) and an additional 5 diagnostically difficult pathogens identified by KT but not SMT. KT detected CMV with high accuracy and correlation with quantitative polymerase chain reaction. Among infectious events, KT demonstrated additive clinical utility by detecting pathogens earlier than SMT and those not detected by SMT.

Cost-Effectiveness of Plasma Microbial Cell-Free DNA Sequencing When Added to Usual Care Diagnostic Testing for Immunocompromised Host Pneumonia.

Immunocompromised host pneumonia (ICHP) is an important cause of morbidity and mortality, yet usual care (UC) diagnostic tests often fail to identify an infectious etiology. A US-based, multicenter study (PICKUP) among ICHP patients with hematological malignancies, including hematological cell transplant recipients, showed that plasma microbial cell-free DNA (mcfDNA) sequencing provided significant additive diagnostic value. The objective of this study was to perform a cost-effectiveness analysis (CEA) of adding mcfDNA sequencing to UC diagnostic testing for hospitalized ICHP patients. A semi-Markov model was utilized from the US third-party payer's perspective such that only direct costs were included, using a lifetime time horizon with discount rates of 3% for costs and benefits. Three comparators were considered: (1) All UC, which included non-invasive (NI) and invasive testing and early bronchoscopy; (2) All UC & mcfDNA; and (3) NI UC & mcfDNA & conditional UC Bronch (later bronchoscopy if the initial tests are negative). The model considered whether a probable causative infectious etiology was identified and if the patient received appropriate antimicrobial treatment through expert adjudication, and if the patient died in-hospital. The primary endpoints were total costs, life-years (LYs), equal value life-years (evLYs), quality-adjusted life-years (QALYs), and the incremental cost-effectiveness ratio per QALY. Extensive scenario and probabilistic sensitivity analyses (PSA) were conducted. At a price of $2000 (2023 USD) for the plasma mcfDNA, All UC & mcfDNA was more costly ($165,247 vs $153,642) but more effective (13.39 vs 12.47 LYs gained; 10.20 vs 9.42 evLYs gained; 10.11 vs 9.42 QALYs gained) compared to All UC alone, giving a cost/QALY of $16,761. NI UC & mcfDNA & conditional UC Bronch was also more costly ($162,655 vs $153,642) and more effective (13.19 vs 12.47 LYs gained; 9.96 vs 9.42 evLYs gained; 9.96 vs 9.42 QALYs gained) compared to All UC alone, with a cost/QALY of $16,729. The PSA showed that above a willingness-to-pay threshold of $50,000/QALY, All UC & mcfDNA was the preferred scenario on cost-effectiveness grounds (as it provides the most QALYs gained). Further scenario analyses found that All UC & mcfDNA always improved patient outcomes but was not cost saving, even when the price of mcfDNA was set to $0. Based on the evidence available at the time of this analysis, this CEA suggests that mcfDNA may be cost-effective when added to All UC, as well as in a scenario using conditional bronchoscopy when NI testing fails to identify a probable infectious etiology for ICHP. Adding mcfDNA testing to UC diagnostic testing should allow more patients to receive appropriate therapy earlier and improve patient outcomes.

A One-year Hospital System Review of Plasma Next-Generation Sequencing in a Mixed Population

Objective: To describe whether detecting plasma microbial cell-free DNA by next-generation sequencing (NGS) provided additional information compared to routine cultures or led to change in antimicrobial management. Design and setting: This is a retrospective cohort study evaluating NGS tests performed on patients who were admitted to an 11-acute care hospital health system in the greater Houston area between May 2022 and May 2023. Repeat tests on the same patient encounter were included if &gt;7 days from previous test. Routine microbiology data was compared if test was collected within 7 days before or after NGS testing. Results: During the study period there were 135 unique patient encounters identified with an NGS order. Of which, 74.1% were ≥18 years of age and 46.7% were immunocompromised. A total of 143 NGS tests were ordered, with 4 not being run due to quality control issues. Out of 139 NGS tests completed, 76 (54.7%) were positive for at least one organism. When compared to routine testing, NGS alone was positive in 29 (20.9%) instances, routine testing alone was positive in 17 (12.2%) instances, both were positive in 44 (31.7%) instances, and both were negative in 49 (35.3%) instances. In the 44 instances that both NGS and routine testing were positive, 15 (34.1%) were concordant for all organisms. In total, NGS identified 92 more organisms (69 bacterial, 8 fungal, and 15 viral) compared to routine testing and routine testing identified 42 more organisms (28 bacterial, 6 fungal, 11 viral, and 1 parasite) compared to NGS. Fifty-six changes to antibiotic therapy were made within 48 hours of the NGS test resulting, with 16 of these changes being directly attributed to NGS test. Nine of these changes being escalations and seven being de-escalations. Conclusion: NGS may aid in determining further testing, earlier detection of pathogens, and detection of pathogens outside of routine testing resulting in direct changes to antimicrobials. However, results need to be interpreted with caution. NGS can miss pertinent pathogens and is difficult to interpret when commensal organisms are detected, both of which can lead to unnecessary testing or treatment. There is an absence of a clear algorithm for the use of NGS testing and the test comes with a high price and unclear utility. Further studies are needed to determine which patients may most benefit from NGS testing.

Plasma Microbial Cell-free DNA Next-generation Sequencing Can Be a Useful Diagnostic Tool in Patients With Osteoarticular Infections.

Recent advances in shotgun metagenomic sequencing (sMGS) for detecting microbial cell-free DNA (mcfDNA) in peripheral blood have shown promise across various patient populations. This study evaluates the application of sMGS for diagnosing osteoarticular infections (OAIs), a condition with significant diagnostic challenges. We conducted a retrospective analysis on 73 patients suspected of OAIs at the Mayo Clinic from 2019 to 2023, incorporating mcfDNA sMGS (Karius test [KT]) into their diagnostic evaluation. We categorized the clinical impact of KT on OAI diagnoses and management into 4 distinct outcomes. (1) KT was able to confirm an established diagnosis, (2) KT supported noninfectious diseases diagnosis, (3) KT established an unsuspected diagnosis, (4) KT did not add relevant information. In our cohort, KT was performed in 73 patients. Among the infected individuals, KT yielded positive results in 22 of 43 (51.2%) cases. Of these 22 cases, 11 (50%) showed agreement with conventional diagnostic workup, whereas in 5 (22.7%) cases, the KT established an unsuspected diagnosis. Native vertebral osteomyelitis diagnosis (P < .001) or OAIs with concomitant presence of endocarditis or endovascular infection (P = .005) were statistically associated with a definite, probable, or possible diagnostic certainty of KT result. In complex OAIs, KT enhanced diagnostic accuracy by 11.6%, proving especially beneficial in diagnosing native vertebral osteomyelitis and infections with concurrent endocarditis or endovascular complications. Our findings underscore the utility of KT in the diagnostic workflow for challenging OAI cases, potentially altering clinical management for a significant subset of patients.

A Machine learning model for predicting sepsis based on an optimized assay for microbial cell-free DNA sequencing

ObjectiveTo integrate an enhanced molecular diagnostic technique to develop and validate a machine-learning model for diagnosing sepsis. MethodsWe prospectively enrolled patients suspected of sepsis from August 2021 to August 2023. Various feature selection algorithms and machine learning models were used to develop the model. The best classifier was selected using 5-fold cross validation set and then was applied to assess the performance of the model in the testing set. Additionally, we employed the Shapley Additive exPlanations (SHAP) method to illustrate the effects of the features. ResultsWe established an optimized mNGS assay and proposed using the copies of microbe-specific cell-free DNA per milliliter of plasma (CPM) as the detection signal to evaluate the real burden, with strong precision and high accuracy. In total, 237 patients were eligible for participation, which were randomly assigned to either the training set (70 %, n = 165) or the testing set (30 %, n = 72). The random forest classifier achieved accuracy, AUC and F1 scores of 0.830, 0.918 and 0.856, respectively, outperforming other machine learning models in the training set. Our model demonstrated clinical interpretability and achieved good prediction performance in differentiating between bacterial sepsis and non-sepsis, with an AUC value of 0.85 and an average precision of 0.91 in the testing set. Based on the SHAP value, the top nine features of the model were PCT, CPM, CRP, ALB, SBPmin, RRmax, CREA, PLT and HRmax. ConclusionWe demonstrated the potential of machine-learning approaches for predicting bacterial sepsis based on optimized mcfDNA sequencing assay accurately.

Diagnostic Value of Microbial Cell-free DNA Sequencing for Suspected Invasive Fungal Infections: A Retrospective Multicenter Cohort Study.

An early diagnosis and treatment of invasive fungal disease (IFD) is associated with improved outcome, but the moderate sensitivity of noninvasive diagnostic tests makes this challenging. Invasive diagnostic procedures such as bronchoalveolar lavage (BAL) have a higher yield but are not without risk. The detection and sequencing of microbial cell-free DNA (mcfDNA) may facilitate a noninvasive diagnosis. In a prospective observational study, we collected plasma in the 120 hours preceding or following a BAL in patients with hematological malignancies suspected for a pulmonary IFD. The EORTC/MSGERC2020 criteria were used for IFD classification. Sequencing was performed by Karius (Redwood City, CA) using their Karius Test (KT) on plasma and a "research use only test" on BAL fluid if available. Cases with a probable/proven IFD were identified based on standard diagnostic tests on serum and BAL (microscopy, polymerase chain reaction, galactomannan, culture) and used to calculate the sensitivity, specificity, and additional diagnostic value of the KT. Of 106 patients enrolled, 39 (37%) had a proven/probable invasive aspergillosis, 7 (7%) a non-Aspergillus IFD, and 4 (4%) a mixed IFD. The KT detected fungal mcfDNA in 29 (28%) patients. Compared with usual diagnostic tests, the sensitivity and specificity were 44.0% (95% confidence interval [CI], 31.2-57.7) and 96.6% (95% CI, 88.5%-99.1%). Sensitivity of the KT was higher in non-Aspergillus IFD (Mucorales:2/3, Pneumocystis jirovecii: 3/5). On BAL, the sensitivity was 72.2% (95% CI, 62.1-96.3), and specificity 83.3% (95% CI, 49.1-87.5). Sequencing of mcfDNA may facilitate a noninvasive diagnosis of IFD in particular non-Aspergillus IFD. However, on plasma and similar to currently available diagnostics, it cannot be used as a "rule-out" test.

Utility of microbial cell free DNA next-generation sequencing for diagnosis and management of infectious diseases

Microbial cell-free DNA (mcfDNA) sequencing is a promising tool to identify infectious pathogens when traditional methods fail to identify the causative agent. We performed a retrospective observational cohort study to evaluate clinical outcomes among pediatric and adult patients who underwent mcfDNA testing. 127 mcfDNA tests were reviewed from 112 patients. Baseline characteristics included 61 (54.5 %) adults, 52 (40.9 %) tests were from female patients, and 67 (52.8 %) tests were obtained from patients designated as immunocompromised. Of all tests obtained, 59 (46.4 %) were deemed clinically relevant. 41 (32.3 %) of tests also led to a change in antimicrobial management for the corresponding patient. No statistically significant association was ascertained between patient-specific factors and clinically relevant test results. Testing in certain clinical scenarios or high-risk settings may be useful, however further studies are needed to assess the cost-benefit of this approach.

Plasma microbial cell-free DNA following chimeric antigen receptor T cell therapy in pediatric patients with relapsed/refractory leukemia.

Chimeric antigen receptor (CAR) T cell therapy is a promising treatment for pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia (R/R B ALL). Cytokine release syndrome (CRS) is a common toxicity after CAR T cell therapy and fever is often the first symptom. Differentiating CRS from infection after CAR T cell therapy can be challenging. Plasma microbial cell free DNA (mcfDNA) is a novel diagnostic tool which allows for qualitative and quantitative assessment of over 1000 organisms. This pilot study sought to characterize mcfDNA results in pediatric patients with R/R B ALL in the first 2 months after CAR T cell therapy.

Emerging Role of Plasma Microbial Cell-free DNA in the Diagnosis of Pediatric Mucormycosis.

Mucormycosis is a rare and devastating angioinvasive infection that can be challenging to diagnose due to the low sensitivity of current noninvasive diagnostics and the lack of a "gold standard" reference test. We describe a retrospective case series of children with suspected mucormycosis where plasma microbial cell-free DNA testing was utilized in the diagnostic evaluation to illustrate the ways in which microbial cell-free DNA testing can noninvasively contribute to the evaluation and management of at-risk, immunosuppressed patients suspected of mucormycosis.

Plasma Microbial Cell-Free DNA Sequencing for Pathogen Detection and Quantification in Children With Musculoskeletal Infections.

Nearly half of all pediatric musculoskeletal infections (MSKIs) are culture negative. Plasma microbial cell-free DNA (mcfDNA) sequencing is noninvasive and not prone to the barriers of culture. We evaluated the performance of plasma mcfDNA sequencing in identifying a pathogen, and examined the duration of pathogen detection in children with MSKIs. We conducted a prospective study of children, aged 6 months to 18 years, hospitalized from July 2019 to May 2022 with MSKIs, in whom we obtained serial plasma mcfDNA sequencing samples and compared the results with cultures. A pathogen was recovered by culture in 23 of 34 (68%) participants, and by initial mcfDNA sequencing in 25 of 31 (81%) participants. Multiple pathogens were detected in the majority (56%) of positive initial samples. Complete concordance with culture (all organisms accounted for by both methods) was 32%, partial concordance (at least one of the same organism(s) identified by both methods) was 36%, and discordance was 32%. mcfDNA sequencing was more likely to show concordance (complete or partial) if obtained prior to a surgical procedure (82%), compared with after (20%), (RR 4.12 [95% CI 1.25, 22.93], p = .02). There was no difference in concordance based on timing of antibiotics (presample antibiotics 60% vs no antibiotics 75%, RR 0.8 [95% CI 0.40, 1.46], p = .65]). mcfDNA sequencing was positive in 67% of culture-negative infections and detected a pathogen for a longer interval than blood culture (median 2 days [IQR 1, 6 days] vs 1 day [1, 1 day], p < .01). Plasma mcfDNA sequencing may be useful in culture-negative pediatric MSKIs if the sample is obtained prior to surgery. However, results must be interpreted in the appropriate clinical context as multiple pathogens are frequently detected supporting the need for diagnostic stewardship.

LIQUID-BIOPSY FOR ASSESSING SEVERITY OF INFLAMMATORY BOWEL DISEASE VIA PLASMA MICROBIAL CELL-FREE DNA

Abstract Plasma cell-free DNA (cfDNA) has emerged as a robust biomarker utilized in multiple applications that span cancer screening, diagnosis and monitoring, prenatal testing, and characterization of organ health post-transplantation. Cell-free DNA-based assays leverage the occurrence of processed extracellular DNA that produce unique signatures to discern a variety of biological states, disease manifestations, as well as forms of cell death. Similar to human cells, microbes produce cfDNA when they turnover, providing evident clinical utility in diagnosing and treating deep-seated infections in immunocompromised patients. Here, we describe the use of plasma microbial cell-free DNA (mcfDNA) as a unique means to assess inflammatory bowel disease type and severity. A total of 392 plasma samples encompassing remission and active (mild, moderate, severe) Crohn’s disease (N=196) and ulcerative colitis (N=196) were provided by the Crohn’s and Colitis Foundation for this study. All samples were derived from patients who had an endoscopic assessment within a 30-day window of plasma collection. Our classifiers, based solely on plasma mcfDNA signatures, are able to discriminate between Crohn’s disease, Ulcerative colitis, or otherwise healthy patient populations with an AUC &amp;gt;0.95. Similarly, a second set of classifiers are able to distinguish between endoscopically validated remission, mild, moderate, and severe disease cases (as determined by Mayo Endoscopic score or Crohn’s disease activity index) with AUCs &amp;gt;0.75. These results correspond to greater than 37% (UC) and 18% (CD) increase in sensitivity in comparison to standard of care measurements via fecal-calprotectin and high sensitivity-C-reactive protein for this cohort. Upon investigation, critical features in our classifiers correspond with well-known signatures from microbes associated with IBD or well-curated from the human microbiome. The use of mcfDNA for IBD diagnosis and assessment of disease severity may provide clinicians and patients a valuable non-invasive means to gain additional insight into disease progression in a routine manner. Further opportunities to leverage these signatures to predict response to therapy, and validate classifier performance on an independent cohort are currently being explored.

Use of Advanced Diagnostics for Timely Identification of Travel-associated Leptospira santarosai Infection in Four Adolescents Through Plasma Microbial Cell-free DNA Sequencing With the Karius Test.

Leptospirosis is an important zoonotic infection worldwide. Diagnosis of leptospirosis is challenging given its nonspecific clinical symptoms that overlap with other acute febrile illnesses and limitations with conventional diagnostic testing. Alternative advanced diagnostics, such as microbial cell-free DNA (mcfDNA), are increasingly being used to aid in the diagnosis of infections and can be applied to pathogens with public health importance such as Leptospira , a nationally notifiable disease. The Karius Test uses plasma mcfDNA sequencing to detect and quantify DNA-based pathogens. This test offered through the Karius lab detected 4 cases of Leptospira santarosai during a 5-month period across the United States in 2021 and were clinically reviewed. In our case series, 4 adolescents with recent travel to Central America (Costa Rica, n = 3 and Belize, n = 1) from April to August 2021 were diagnosed with leptospirosis. While a large workup was performed in all cases, mcfDNA testing was the first test to detect L. santarosai as the microbiological diagnosis in all cases. Results of the Karius Test enabled rapid, noninvasive diagnosis of leptospirosis allowing for targeted therapy. Use of mcfDNA can be utilized for diagnosis of pathogens where conventional testing is challenging or limited. This in turn can enable quick diagnosis for targeted treatment and potentially aid in supporting case definitions of reportable diseases of public health concern.

Detection of invasive Bartonella infections with next-generation sequencing of microbial cell-free DNA.

We report 9 patients with invasive Bartonella infections, including 5 with endocarditis, who were diagnosed with microbial cell-free DNA next-generation sequencing and Bartonella serology studies. Diagnosis with plasma mcfDNA NGS enabled a faster clinical and laboratory diagnosis in 8 patients. Prompt diagnosis impacted antibiotic management in all 9 patients.

Next-Generation Sequencing of Microbial Cell-Free DNA to Rapidly Detect Fluoribacter Bozemanae Pneumonia in an Immunocompromised Host

Background: Fluoribacter bozemanae is an uncommon pathogen that accounts for just 3-5% of cases of pneumonia caused by the different Legionella species. The mortality rate has been reported to be up to 40%. This organism is difficult to identify, making diagnosis extremely challenging with current diagnostic modalities. Case Report: We present a case of a 75-year-old female with a history of multiple myeloma undergoing active treatment with immunosuppressive agents who presented with confusion, hypoxemic respiratory failure and sepsis. Her initial presentation was consistent with Legionella infection, however, all routine testing including a Legionella urine antigen test came back negative. A CT scan of the chest was completed which showed near complete left lower lobe consolidation concerning for pneumonia. An infectious workup was initiated. Atypical and viral pneumonia polymerase chain reaction panel was negative. Microbial cell-free DNA sequencing returned positive for Fluoribacter bozemanae, a subspecies of the Legionellaceae family that is known to cause severe pneumonia in immunocompromised hosts. This information allowed a therapy adjustment resulting in rapid improvement of our patient’s symptoms. Conclusion: In cases such as this one, the diagnosis of Legionella species is often missed due to the many challenges associated with the different testing modalities. In these circumstances, next-generation genetic sequencing can be extremely useful. Now with the increasing availability of next-generation sequencing as a microbiological diagnostic tool, we can diagnose many more infections that once would have gone undiagnosed and either untreated or delayed in treatment.

Clinical Analysis of Metagenomic Sequencing of Plasma Microbial Cell-Free DNA for Infected Patients in SAA

Backgroun d: As a high-risk group of clinical infection, patients with severe aplastic anemia (SAA) have high mortality due to agranulocytosis or immune function impairment. The current conventional detection methods such as blood culturetissue chulture etc. often cannot identify pathogenic bacteria. We evaluate the diagnostic performance of metagenomic next-generation sequencing (mNGS), and explore the application value of mNGS in infected patients in SAA, in order to provide help for precision therapy. Methods: From Ocb 2020 to Dec 2022 in our center, we enrolled 70 SAA patients with infection and investigated the usefulness of mNGS of plamsa or tissue DNA for identification of infectious pathogens. The consistency of the results of mNGS and the results of routine etiological detection (blood culture(BC), swab/tissue culture) with clinical infection diagnosis and the influence of the results of mNGS on clinical infection treatment were compared. Results: 70 SAA patients of mNGS were detected, including plasma-mNGS 66 cases and tissue-mNGS 4 cases. Among all patients, 48 cases(68.6%) were positive. In comparison to blood culture, the positive and negative agreement of mNGS were 100% (13/13) and 38.6% (22/57), respectively. In 48 mNGS positive cases, 36 patients (75%) underwent antimicrobials adjustment, resulting in positive impact on 30 patients. Fungi were detected in 29 cases, the detection rate was 41.4% (29/70) by mNGS, otherwise 2.8% (2/70) by BC. Of the 70 patients with SAA, 55 infections eventually impproved (78.6%). Conclusion: The positive rate of mNGS detection and the consistent rate with clinical diagnosis are higher than that of conventional etiological detection, which provides a faster and more sensitive detection method for infected patients in SAA. 【 Key words 】 Metagenomic Sequencing ; Plasma Microbial Cell-free DNA; Aplastic anemia; Infection; pathogen diagnosis