Next Generation Sequencing of free pathogenic DNA in blood samples of a critically-ill pediatric population: A single-center experience Short title: NGS of pathogenic cfDNA in critically ill children

Next Generation Sequencing of free pathogenic DNA in blood samples of a critically-ill pediatric population: A single-center experience Short title: NGS of pathogenic cfDNA in critically ill children | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Next Generation Sequencing of free pathogenic DNA in blood samples of a critically-ill pediatric population: A single-center experience Short title: NGS of pathogenic cfDNA in critically ill children Jonas E. Adolph, Christina Pentek, Thomas Bauch, Clara Held, Thorsten Brenner, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8133441/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2026 Read the published version in Molecular and Cellular Pediatrics → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Rapid and accurate pathogen detection is critical for optimizing outcomes in pediatric sepsis. Next-generation sequencing (NGS) of cell-free DNA (cfDNA) from blood enables culture-independent identification of microbial DNA from bacteria, viruses, fungi, and parasites. We evaluated the diagnostic yield and clinical impact of cfDNA-based NGS in critically ill and predominantly immunocompromised pediatric patients (≤ 18 years) with suspected infection. This retrospective single-center study included pediatric patients who underwent plasma cfDNA-NGS at a tertiary care hospital in Germany. Following computational removal of human DNA, remaining sequences were aligned to curated microbial reference databases. Diagnostic performance was compared with blood cultures and viral PCR, and clinical relevance was assessed by pediatric infectious disease specialists. Results: 111 tests in 78 pediatric patients, mostly with systemic inflammatory response syndrome of unknown etiology, were performed. Overall, 61 tests (54.5%) were positive for pathogenic cfDNA. Compared with conventional microbiological diagnostics, NGS demonstrated a sensitivity of 64.7% and specificity of 88.2% when blood cultures and viral PCR served as the reference standard. NGS identified additional pathogens in a substantial proportion (41.1%) of cases that remained negative by standard testing. Of those pathogens only found by NGS, over 60% were deemed clinically relevant. In 14.8% of positive NGS results, a pathogen-specific therapy was started, while 40.2% of tests led to a discontinuation of therapy (51.0% of negative tests). Out of all positive NGS, 38 (62.3%) were classified as clinically relevant. NGS testing also detected rare infections with fungi and parasites in four cases each. Conclusion: Detection of pathogenic cfDNA through NGS from blood shows promising results as an additional diagnostic tool in critically ill pediatric patients with suspected infections. Clinical utility is currently still limited by its high cost, undetermined diagnostic validity and limitations in testing for resistances and restricted availability of raw sequencing data due to data-protection constraints. cell-free DNA metagenomic next-generation sequencing pediatric sepsis infectious disease diagnostics antimicrobial stewardship Backround Diagnosing suspected infections in pediatric patients is challenging, but crucial for choosing an effective therapy, and subsequently for patient outcome [ 1 , 2 ]. Children and adolescents with complex underlying conditions, impaired immune responses, or ongoing inflammation frequently receive multiple anti-infective treatments prior to or during hospitalisation. These prior exposures may obscure the clinical picture and lead to false-negative microbiological results. Conventional diagnostic approaches include culture-based techniques, nucleic acid amplification methods such as polymerase chain reaction (PCR), and antigen detection assays. Each of these methods has distinct limitations. Culture-based diagnostics are time-consuming and susceptible to pre-analytical errors such as contamination, delayed processing, or insufficient sample volumes—an issue particularly relevant in paediatrics [ 3 , 4 ]. PCR and antigen-based tests, while faster, are typically limited to predefined pathogens and provide little or no information on antimicrobial resistance [ 5 – 7 ]. Consequently, pathogens may remain unidentified or the time to diagnosis may delay effective clinical decision-making. Recent advances in molecular diagnostics have introduced next generation sequencing (NGS) of microbial genetic material circulating freely in the bloodstream as a promising complementary approach. This technology enables culture-independent identification of bacterial, viral, fungal, and parasitic DNA fragments in a single assay. Studies in adult populations with severe infections, including sepsis, have shown that NGS can expand the spectrum of identified pathogens and potentially shorten time to diagnosis compared with standard methods [ 8 – 14 ]. However, data on its diagnostic performance and clinical impact in paediatric patients remain limited. The aim of this study was to evaluate the diagnostic yield, clinical relevance, and potential therapeutic impact of NGS for detection of circulating microbial DNA in paediatric patients with suspected infection at a tertiary care centre. We hypothesised that NGS would identify additional pathogens not detected by conventional methods and thereby influence clinical management decisions. Materials and Methods Study design and participants This retrospective, single-centre study evaluated all results from Next Generation Sequencing (NGS) of circulating cell-free deoxyribonucleic acid (cfDNA) obtained from paediatric patients (≤ 18 years) treated at the University Hospital Essen, Germany, between January 2020 and January 2023. Indications for NGS testing included systemic inflammatory response syndrome (SIRS) of unknown origin, suspected infection by rare or atypical pathogens, and follow-up analysis during ongoing treatment. Testing was performed at the discretion of the attending physician and after consultation with the paediatric infectious disease (ID) team. All NGS results were subsequently reviewed for clinical relevance by the same team. In total, 78 patients and 111 NGS tests were included. Clinical data collected comprised demographic information, primary diagnosis, clinical presentation, results of standard microbiological diagnostics, and inflammatory parameters such as C-reactive protein (CRP) and procalcitonin (PCT) when available. Sample collection and DNA extraction Up to 10 mL of peripheral whole blood was drawn under aseptic conditions into two Streck Cell-Free DNA BCT CE tubes (Streck, Omaha, NE, USA) containing a cell- and cfDNA stabiliser. Samples were stored at 4°C and transported within 24 hours to the laboratory facility of Noscendo GmbH (Reutlingen, Germany) for processing. Plasma was separated by centrifugation at 1,600 × g for 10 minutes at 4°C, followed by centrifugation of the supernatant at 16,000 × g for 10 minutes at 4°C. cfDNA was extracted using the QIAsymphony DSP Circulating DNA Kit on the QIAsymphony SP instrument (Qiagen, Hilden, Germany). DNA concentration and purity were assessed with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Library preparation and sequencing Sequencing libraries were prepared from 1 ng of cfDNA. Single-read sequencing (1 × 75 bp) was performed using Illumina NextSeq 1000/2000 instruments (Illumina, San Diego, CA, USA) with Illumina reagents. Each run included up to 1 4 multiplexed samples and at least 24 million reads per sample were required. Bioinformatic analysis Bioinformatic processing was conducted using the CE-IVD-certified DISQVER® platform (Version R4 and R5, Noscendo GmbH, Duisburg, Germany). Human reads were computationally excluded. Remaining non-human sequences were systematically compared against a proprietary microbial genome database containing over 16,000 microbial species and 1,500 pathogenic taxa (defined as curated “species-level entries and pathogenic taxa” according to vendor documentation). Statistical analysis Statistical analyses were performed using R software (version 4.3.1; R Core Team, 2023). Continuous variables were summarised as medians with interquartile ranges (IQR). The Wilcoxon rank-sum test was used for comparisons of continuous variables between groups. The chi-squared test assessed independence between categorical variables. Correlations between continuous variables were analysed using Pearson’s correlation coefficient, and 95% confidence intervals (CI) were calculated. A two-sided p-value < 0.05 was considered statistically significant. Ethical approval This study was approved by the Ethics Committee of the University of Duisburg-Essen (approval number 23-11277-BO). The requirement for informed consent was waived due to the retrospective design and anonymized data analysis. All procedures were performed in accordance with the Declaration of Helsinki and relevant institutional guidelines. Results Patient Characteristics Between November 2020 and January 2023, a total of 111 tests were performed on 78 patients. Per patient, one to nine tests were performed. The median age was 10.3 years (range: 0.5 – 17.6). Most patients (79.3%, n = 89) had an underlying chronic disease, and 67 (60.4%) were immunocompromised. Patient demographics as well as the results of standard diagnostics are presented in Table 1 . Indications and Testing NGS was requested mostly for systemic inflammatory response syndrome of unknown origin (96/111; 86.5%). Additional indications included suspected infection with rare pathogens (12/111; 10.8%) and follow-up analysis (29/111; 26.1%). Eight patients underwent follow-up testing; overlap between indications was possible. At the time of sampling, 67 patients (60.4%) were receiving at least one antibiotic therapy. NGS Results In total, 60 tests (54.1%) were positive for microbial cfDNA. The median number of pathogens detected per test was one (interquartile range [IQR] 1–2, range 1–5). Among positive samples, at least one bacterium was found in 41 (68.3%), one virus in 24 (40%), one fungus in 4 (6.7%), and one parasite in 4 (6.7%) samples. Mixed bacterial–viral findings occurred in seven tests (11.5%). NGS identified additional pathogens in 41.4% (46/111) of tests where conventional diagnostics in blood were negative. The complete list of microorganisms detected by NGS is presented in Table 2 . Comparison with Standard Diagnostics When compared with conventional blood diagnostics for bacteria and viruses, NGS showed a sensitivity of 64.7%, specificity of 88.2%, positive predictive value (PPV) of 18.0% and a negative predictive value (NPV) of 88.2%. When only clinically relevant pathogens were considered, sensitivity increased to 87.5%, specificity was 66.7%, PPV 18.4%, and NPV 66.7%. Among seven cases with positive blood cultures, NGS detected the same pathogen in four (57.1%), while three were missed. In urine cultures, one of four positive bacterial findings (25%) was confirmed by NGS. For viral pathogens identified by PCR, NGS fully matched four of fifteen (26.7%) and partially matched three of fifteen (20%). All four fungal pathogens were confirmed by conventional diagnostics. Zero cases were found in which NGS was negative but classical diagnostics were positive. Detailed contingency tables comparing NGS and conventional results are provided in Table 3 . Factors influencing NGS positivity The likelihood of a positive NGS result was not significantly associated with serum C-reactive protein (p = 0.13) or procalcitonin (p = 0.97) levels. Antibiotic treatment at the time of testing also did not influence the probability of NGS positivity (p = 0.15). Correlation analysis showed no relationship between inflammatory markers and sequencing read counts (CRP: r = −0.09, 95% CI −0.34 to 0.17, p = 0.49; PCT: r = −0.05, 95% CI −0.37 to 0.28, p = 0.78). Clinical impact of NGS results NGS findings led to initiation of pathogen-specific therapy in 12 cases (10.8%), confirmation of existing diagnoses in 8 cases (7.1%), and discontinuation of antibiotic therapy in 45 cases (40.5%). Among the 60 positive NGS results, 37 (62.7%) were judged clinically relevant by the infectious disease team. Clinically significant organisms included Aspergillus fumigatus, Burkholderia contaminans, Candida dubliniensis, Enterocytozoon bieneusi, Fusobacterium nucleatum, Leishmania infantum, Mycobacterium chimaera , and Pseudomonas protegens. Pathogens classified as clinically irrelevant were mainly Torque teno viruses and Propionibacterium species. Discussion We present a cohort of 78 pediatric patients treated at a tertiary care center with a total of 111 NGS analyses for pathogenic cfDNA, mostly in cases of SIRS of unknown origin. Compared with conventional microbiological diagnostics, NGS demonstrated a sensitivity of 64.7% and specificity of 88.2% when blood cultures and viral PCR served as the reference standard. Taking conventional diagnostics as the gold-standard, the positive predictive value was modest, as NGS identified additional pathogens in a substantial proportion of cases that were negative by standard testing and therefore were calculated as false-positives, lowering the positive predictive value. Of those pathogens only found by NGS, over 60% were deemed clinically relevant. Our results align with previous studies in adult and pediatric populations, which also demonstrated that NGS can broaden the diagnostic yield and expedite pathogen identification [14-20]. This particularly applies to children with chronic diseases or those receiving immunosuppressive therapy, who constituted the main subgroup within our cohort, as also reported elsewhere [21–22]. The turnaround time from drawing of blood to receipt of NGS results was one to two days, with the longest timespan being four days due to weekends or holidays, which is similar to previously reported times in a clinical setting [14, 18, 19]. Thus, NGS results were mostly available faster than blood culture results and were similar in timing to viral PCR assays. Given that the outcome of pediatric sepsis depends critically on prompt and accurate therapy, rapid pathogen detection and identification may be lifesaving [1–3]. Importantly, negative NGS results, if shown to have adequate diagnostic reliability, may support safe de-escalation or discontinuation of antimicrobial treatment, thereby reducing unnecessary exposure, toxicity, and the risk of secondary infections such as Clostridioides difficile [23]. Positive NGS results identified several rare pathogens which were subsequently only detected by conventional diagnostics after targeted investigations based on the NGS findings were initiated. Without NGS overlooked pathogens included Burkholderia contaminans , Enterocytozoon bieneusi , Fusobacterium nucleatum , Leishmania infantum and donovani , Mycobacterium chimaera . Rare pathogens like Enterocytozoon bieneusi , can be challenging to detect using standard microbiological techniques [24]. Similarly, Mycobacterium chimaera often requires prolonged culture and specialized laboratory protocols for identification [25]. Meanwhile, Leishmania infantum , which is endemic to specific regions, may be overlooked in non-endemic settings unless clinical suspicion prompts targeted testing [26]. In the presented patient cohort, the ability of NGS to detect such pathogens not only enhanced diagnostic accuracy but also had direct therapeutic implications, enabling the timely initiation of targeted antimicrobial or antiparasitic therapies. Possible explanations for non-detection in culture-positive cases include low pathogen cfDNA fraction, insufficient sequencing depth in those runs, high host-to-microbe DNA ratio, different sample materials used in conventional diagnostics (such as urine) and timing of sampling after antimicrobial exposure. These interventions significantly impacted patient outcomes and might have otherwise been delayed or entirely missed. Besides its short turnaround time and broad range of detectable pathogens, further advantages of NGS diagnostics include detecting resistance factors within the same sample and workflow [27, 28], as well as its use in different specimens, such as cerebrospinal fluid, lavages or tissues [29,30]. This study has several limitations. First, it was conducted at a single tertiary centre and primarily included chronically ill and immunocompromised patients, introducing selection bias and limiting generalisability. Second, NGS testing was performed at the discretion of treating clinicians, which may have favoured patients with higher pre-test probability of infection [31]. Third, the retrospective design and relatively small sample size restricted the ability to draw definitive conclusions about diagnostic accuracy and clinical outcomes. Finally, NGS, like other sequencing-based methods, remains susceptible to contamination, and data protection regulations prevented the public release of raw sequencing data. Only aggregated results could be made available upon reasonable request. Conclusions In this study, we evaluated the diagnostic yield, clinical relevance, and therapeutic impact of Next Generation Sequencing (NGS) for detection of circulating microbial DNA in paediatric patients with suspected infection at a tertiary care centre. NGS identified additional pathogens not detected by conventional methods and influenced clinical management decisions in a subset of cases, including both initiation and discontinuation of antimicrobial therapy. These findings indicate that NGS can provide meaningful diagnostic and therapeutic value when used as a complementary tool to standard microbiological testing, particularly in complex or critically ill children. However, the clinical utility of NGS remains limited by factors such as high cost, restricted availability, lack of standardized interpretation criteria, and uncertain diagnostic validity in broader populations. Future multicenter, prospective studies should focus on defining patient groups that benefit most from NGS, validating its performance metrics, and integrating resistance gene detection and quantitative analyses into clinical workflows. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the University of Duisburg-Essen, Germany (reference number 23-11277-BO). The requirement for written informed consent was waived due to the retrospective design and anonymised data analysis. All procedures followed the ethical standards of the institutional research committee and the Declaration of Helsinki. Consent for publication Not applicable. The manuscript does not contain any individual person’s identifiable data. Availability of data and materials The datasets generated and analysed during the current study are not publicly available due to patient confidentiality and data protection regulations. De-identified aggregated data may be made available from the corresponding author upon reasonable request and pending approval by the local Ethics Committee and data protection office. Competing interests Silke Grumaz is a co-founder, employee and shareholder of Noscendo GmbH. Petra Horvatek is a former employee of Noscendo GmbH. Thorsten Brenner has received a lecture honorarium from Noscendo GmbH. All other authors declare that they have no competing interests. Funding This study received scientific and logistic support from Noscendo GmbH, Duisburg, Germany, which performed the metagenomic NGS analyses. Additional institutional research funding from the Department of Pediatrics I, University Hospital Essen, supported this work. The funders had no influence on study design, data collection, analysis, interpretation, or the decision to publish the manuscript. Authors’ contributions JEA and SCG conceived the study and developed the study design. JEA, CP, TB, CH, TB, and UFM collected clinical data and reviewed patient cases. SG and PH performed sequencing and bioinformatic analyses. MS, LA, SV and JD contributed microbiological and virological expertise. CDS and UFM provided clinical supervision. SCG and JEA drafted the first manuscript version. All authors critically revised the manuscript, approved the final version, and agree to be accountable for all aspects of the work. Acknowledgements We would like to express our sincere gratitude to all patients and their families for putting their trust in our care. We thank the nursing and medical staff of the Department of Pediatrics I at the University Hospital Essen for their dedicated support in patient care and sample collection. Our appreciation also goes to the team at Noscendo GmbH for their technical assistance with sequencing and data processing. We acknowledge the members of the paediatric infectious diseases team for their valuable clinical input during case evaluations and data interpretation. Authors’ information Sarah C. Goretzki, MD, is a paediatric infectious diseases specialist at the University Hospital Essen, Germany, with a research focus on molecular pathogen diagnostics and antimicrobial stewardship. ORCID: 0000-0001-5218-070X. References Weiss SL, Fitzgerald JC, Balamuth F, Alpern ER, Lavelle J, Chilutti M et al (2014) Delayed antimicrobial therapy increases mortality and organ dysfunction duration in pediatric sepsis. Crit Care Med 42:2409–2417 Kumar A, Ellis P, Arabi Y, Roberts D, Light B, Parrillo JE et al (2009) Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. 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BMC Biol 12:87 Tables Table 1 to 3 are available in the Supplementary Files section. Additional Declarations Competing interest reported. Silke Grumaz is a co-founder, employee and shareholder of Noscendo GmbH. Petra Horvatek is a former employee of Noscendo GmbH. Thorsten Brenner has received a lecture honorarium from Noscendo GmbH. All other authors declare that they have no competing interests. Supplementary Files FiguresNGS.docx Cite Share Download PDF Status: Published Journal Publication published 08 Apr, 2026 Read the published version in Molecular and Cellular Pediatrics → Version 1 posted Editorial decision: Revision requested 13 Jan, 2026 Reviews received at journal 05 Jan, 2026 Reviews received at journal 31 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers invited by journal 25 Nov, 2025 Editor assigned by journal 25 Nov, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 17 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8133441","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":551608348,"identity":"4c1f40b2-96b5-4ba1-aa76-6de813a2d04c","order_by":0,"name":"Jonas E. 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Goretzki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABF0lEQVRIie2PMUvDQBiGv3AQl7OuV6LJX7hyUAfF3+IRiGvHDrXEpV2ErleE/ob+hJSDZom6BioYCWRyObq0KOJF1MXT4CZ4z3R83z287wdgsfxR0MfDiekxkM950qigWol+rYBsVoLx9cO6B+f+4Xgilejd8mmMpNr076CVxkaFZmfME5Cy/UxCZ05X/ArcyMNZBe3MHEMhAg/DkgsSAi+0Mgseu8gZSaD5qbnYpEJPb0pQwqKgN3wGe2u1fdHKfWE+Jo9cnTLQKci5mNNEF8NAduM6xWzQvHKPME0YwSFzBA3ZNHa7Hl5K3M6+KxahFe4PfbKzKNXl88mBSFCpNgPpt1Lz+e9Z8usM//C/Ztiwt1gsln/NK+EoXg5dzAGHAAAAAElFTkSuQmCC","orcid":"","institution":"Essen University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Sarah","middleName":"C.","lastName":"Goretzki","suffix":""}],"badges":[],"createdAt":"2025-11-17 09:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8133441/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8133441/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40348-026-00226-3","type":"published","date":"2026-04-08T15:59:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96988690,"identity":"0f5ab0f7-f7fa-48a7-8084-14721d9607af","added_by":"auto","created_at":"2025-11-28 10:42:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61612,"visible":true,"origin":"","legend":"","description":"","filename":"NGSManuscriptMolCelPed.docx","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/6a1c449114bb2ce457c9592d.docx"},{"id":96988694,"identity":"3cf6944e-6635-47db-82b3-95483e507d82","added_by":"auto","created_at":"2025-11-28 10:42:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23490,"visible":true,"origin":"","legend":"","description":"","filename":"FiguresNGS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/4d0d14a7c3733fbe9c707562.docx"},{"id":96988692,"identity":"7cb788dd-fc09-4bf5-b61f-933f59ee9701","added_by":"auto","created_at":"2025-11-28 10:42:50","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15416,"visible":true,"origin":"","legend":"","description":"","filename":"a767cd5451ad4628b3cc151877af2036.json","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/220f0deb10a8f35258eb3772.json"},{"id":97138793,"identity":"131b7fe0-4548-4c89-bba7-023316a2da6a","added_by":"auto","created_at":"2025-12-01 09:59:20","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102782,"visible":true,"origin":"","legend":"","description":"","filename":"a767cd5451ad4628b3cc151877af20361enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/0c641b5224e797288afdaae9.xml"},{"id":96988695,"identity":"e04e9ef4-0678-4d6f-9722-4dfc8a218b8f","added_by":"auto","created_at":"2025-11-28 10:42:50","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98239,"visible":true,"origin":"","legend":"","description":"","filename":"a767cd5451ad4628b3cc151877af20361structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/341c11f650fc11629c93b786.xml"},{"id":96988697,"identity":"25afb08b-03dc-464b-b870-315cb42cfffd","added_by":"auto","created_at":"2025-11-28 10:42:50","extension":"html","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112032,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/4cf28b4e002b5dfbf1cf3b0b.html"},{"id":106809193,"identity":"9114cf50-85dd-4980-a10a-f3ec9ffe5fa7","added_by":"auto","created_at":"2026-04-13 16:08:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":670491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/ef6aad53-9ae2-439b-b884-0488bb490c4c.pdf"},{"id":97138791,"identity":"5b65b4f5-6fbf-49b5-a337-ba50945036ac","added_by":"auto","created_at":"2025-12-01 09:59:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23490,"visible":true,"origin":"","legend":"","description":"","filename":"FiguresNGS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8133441/v1/0521556390dcb6478e053826.docx"}],"financialInterests":"Competing interest reported. Silke Grumaz is a co-founder, employee and shareholder of Noscendo GmbH.\nPetra Horvatek is a former employee of Noscendo GmbH.\nThorsten Brenner has received a lecture honorarium from Noscendo GmbH.\nAll other authors declare that they have no competing interests.","formattedTitle":"Next Generation Sequencing of free pathogenic DNA in blood samples of a critically-ill pediatric population: A single-center experience Short title: NGS of pathogenic cfDNA in critically ill children","fulltext":[{"header":"Backround","content":"\u003cp\u003eDiagnosing suspected infections in pediatric patients is challenging, but crucial for choosing an effective therapy, and subsequently for patient outcome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Children and adolescents with complex underlying conditions, impaired immune responses, or ongoing inflammation frequently receive multiple anti-infective treatments prior to or during hospitalisation. These prior exposures may obscure the clinical picture and lead to false-negative microbiological results.\u003c/p\u003e\u003cp\u003eConventional diagnostic approaches include culture-based techniques, nucleic acid amplification methods such as polymerase chain reaction (PCR), and antigen detection assays. Each of these methods has distinct limitations.\u003c/p\u003e\u003cp\u003eCulture-based diagnostics are time-consuming and susceptible to pre-analytical errors such as contamination, delayed processing, or insufficient sample volumes\u0026mdash;an issue particularly relevant in paediatrics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PCR and antigen-based tests, while faster, are typically limited to predefined pathogens and provide little or no information on antimicrobial resistance [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, pathogens may remain unidentified or the time to diagnosis may delay effective clinical decision-making.\u003c/p\u003e\u003cp\u003eRecent advances in molecular diagnostics have introduced next generation sequencing (NGS) of microbial genetic material circulating freely in the bloodstream as a promising complementary approach. This technology enables culture-independent identification of bacterial, viral, fungal, and parasitic DNA fragments in a single assay. Studies in adult populations with severe infections, including sepsis, have shown that NGS can expand the spectrum of identified pathogens and potentially shorten time to diagnosis compared with standard methods [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, data on its diagnostic performance and clinical impact in paediatric patients remain limited.\u003c/p\u003e\u003cp\u003eThe aim of this study was to evaluate the diagnostic yield, clinical relevance, and potential therapeutic impact of NGS for detection of circulating microbial DNA in paediatric patients with suspected infection at a tertiary care centre. We hypothesised that NGS would identify additional pathogens not detected by conventional methods and thereby influence clinical management decisions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design and participants\u003c/h2\u003e\u003cp\u003e This retrospective, single-centre study evaluated all results from Next Generation Sequencing (NGS) of circulating cell-free deoxyribonucleic acid (cfDNA) obtained from paediatric patients (≤ 18 years) treated at the University Hospital Essen, Germany, between January 2020 and January 2023.\u003c/p\u003e\u003cp\u003eIndications for NGS testing included systemic inflammatory response syndrome (SIRS) of unknown origin, suspected infection by rare or atypical pathogens, and follow-up analysis during ongoing treatment.\u003c/p\u003e\u003cp\u003eTesting was performed at the discretion of the attending physician and after consultation with the paediatric infectious disease (ID) team. All NGS results were subsequently reviewed for clinical relevance by the same team. In total, 78 patients and 111 NGS tests were included.\u003c/p\u003e\u003cp\u003eClinical data collected comprised demographic information, primary diagnosis, clinical presentation, results of standard microbiological diagnostics, and inflammatory parameters such as C-reactive protein (CRP) and procalcitonin (PCT) when available.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample collection and DNA extraction\u003c/h3\u003e\n\u003cp\u003eUp to 10 mL of peripheral whole blood was drawn under aseptic conditions into two Streck Cell-Free DNA BCT CE tubes (Streck, Omaha, NE, USA) containing a cell- and cfDNA stabiliser. Samples were stored at 4°C and transported within 24 hours to the laboratory facility of Noscendo GmbH (Reutlingen, Germany) for processing. Plasma was separated by centrifugation at 1,600 × g for 10 minutes at 4°C, followed by centrifugation of the supernatant at 16,000 × g for 10 minutes at 4°C. cfDNA was extracted using the QIAsymphony DSP Circulating DNA Kit on the QIAsymphony SP instrument (Qiagen, Hilden, Germany). DNA concentration and purity were assessed with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eLibrary preparation and sequencing\u003c/h3\u003e\n\u003cp\u003eSequencing libraries were prepared from 1 ng of cfDNA. Single-read sequencing (1 × 75 bp) was performed using Illumina NextSeq 1000/2000 instruments (Illumina, San Diego, CA, USA) with Illumina reagents. Each run included up to 1 4 multiplexed samples and at least 24 million reads per sample were required.\u003c/p\u003e\n\u003ch3\u003eBioinformatic analysis\u003c/h3\u003e\n\u003cp\u003eBioinformatic processing was conducted using the CE-IVD-certified DISQVER® platform (Version R4 and R5, Noscendo GmbH, Duisburg, Germany). Human reads were computationally excluded. Remaining non-human sequences were systematically compared against a proprietary microbial genome database containing over 16,000 microbial species and 1,500 pathogenic taxa (defined as curated “species-level entries and pathogenic taxa” according to vendor documentation).\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using R software (version 4.3.1; R Core Team, 2023). Continuous variables were summarised as medians with interquartile ranges (IQR). The Wilcoxon rank-sum test was used for comparisons of continuous variables between groups. The chi-squared test assessed independence between categorical variables. Correlations between continuous variables were analysed using Pearson’s correlation coefficient, and 95% confidence intervals (CI) were calculated. A two-sided p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the University of Duisburg-Essen (approval number 23-11277-BO). The requirement for informed consent was waived due to the retrospective design and anonymized data analysis. All procedures were performed in accordance with the Declaration of Helsinki and relevant institutional guidelines.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatient Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween November 2020 and January 2023, a total of 111 tests were performed on 78 patients. Per patient, one to nine tests were performed. The median age was 10.3 years (range: 0.5 \u0026ndash; 17.6). \u0026nbsp;Most patients (79.3%, n = 89) had an underlying chronic disease, and 67 (60.4%) were immunocompromised. Patient demographics as well as the results of standard diagnostics are presented in \u003cem\u003eTable 1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndications and Testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNGS was requested mostly for systemic inflammatory response syndrome of unknown origin (96/111; 86.5%). Additional indications included suspected infection with rare pathogens (12/111; 10.8%) and follow-up analysis (29/111; 26.1%). Eight patients underwent follow-up testing; overlap between indications was possible. At the time of sampling, 67 patients (60.4%) were receiving at least one antibiotic therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNGS Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn total, 60 tests (54.1%) were positive for microbial cfDNA.\u0026nbsp;The median number of pathogens detected per test was one (interquartile range [IQR] 1\u0026ndash;2, range 1\u0026ndash;5). Among positive samples, at least one bacterium was found in 41 (68.3%), one virus in 24 (40%), one fungus in 4 (6.7%), and one parasite in 4 (6.7%) samples. Mixed bacterial\u0026ndash;viral findings occurred in seven tests (11.5%). NGS identified additional pathogens in 41.4% (46/111) of tests where conventional diagnostics in blood were negative. The complete list of microorganisms detected by NGS is presented in \u003cem\u003eTable 2\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison with Standard Diagnostics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen compared with conventional blood diagnostics for bacteria and viruses, NGS showed a sensitivity of 64.7%, specificity of 88.2%, positive predictive value (PPV) of 18.0% and a negative predictive value (NPV) of 88.2%. When only clinically relevant pathogens were considered, sensitivity increased to 87.5%, specificity was 66.7%, PPV 18.4%, and NPV 66.7%. Among seven cases with positive blood cultures, NGS detected the same pathogen in four (57.1%), while three were missed. In urine cultures, one of four positive bacterial findings (25%) was confirmed by NGS. For viral pathogens identified by PCR, NGS fully matched four of fifteen (26.7%) and partially matched three of fifteen (20%). All four fungal pathogens were confirmed by conventional diagnostics. Zero cases were found in which NGS was negative but classical diagnostics were positive. Detailed contingency tables comparing NGS and conventional results are provided in \u003cem\u003eTable 3\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFactors influencing NGS positivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe likelihood of a positive NGS result was not significantly associated with serum C-reactive protein (p = 0.13) or procalcitonin (p = 0.97) levels. Antibiotic treatment at the time of testing also did not influence the probability of NGS positivity (p = 0.15). Correlation analysis showed no relationship between inflammatory markers and sequencing read counts (CRP: r = \u0026minus;0.09, 95% CI \u0026minus;0.34 to 0.17, p = 0.49; PCT: r = \u0026minus;0.05, 95% CI \u0026minus;0.37 to 0.28, p = 0.78).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical impact of NGS results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNGS findings led to initiation of pathogen-specific therapy in 12 cases (10.8%), confirmation of existing diagnoses in 8 cases (7.1%), and discontinuation of antibiotic therapy in 45 cases (40.5%). Among the 60 positive NGS results, 37 (62.7%) were judged clinically relevant by the infectious disease team. Clinically significant organisms included \u003cem\u003eAspergillus fumigatus, Burkholderia contaminans, Candida dubliniensis, Enterocytozoon bieneusi, Fusobacterium nucleatum, Leishmania infantum, Mycobacterium chimaera\u003c/em\u003e, and Pseudomonas protegens. Pathogens classified as clinically irrelevant were mainly Torque teno viruses and Propionibacterium species.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe present a cohort of 78 pediatric patients treated at a tertiary care center with a total of 111 NGS analyses for pathogenic cfDNA, mostly in cases of SIRS of unknown origin.\u003c/p\u003e\n\u003cp\u003eCompared with conventional microbiological diagnostics, NGS demonstrated a sensitivity of 64.7% and specificity of 88.2% when blood cultures and viral PCR served as the reference standard. Taking conventional diagnostics as the gold-standard, the positive predictive value was modest, as NGS identified additional pathogens in a substantial proportion of cases that were negative by standard testing and therefore were calculated as false-positives, lowering the positive predictive value. Of those pathogens only found by NGS, over 60% were deemed clinically relevant.\u003c/p\u003e\n\u003cp\u003eOur results align with previous studies in adult and pediatric populations, which also demonstrated that NGS can broaden the diagnostic yield and expedite pathogen identification [14-20]. This particularly applies to children with chronic diseases or those receiving immunosuppressive therapy, who constituted the main subgroup within our cohort, as also reported elsewhere [21\u0026ndash;22]. The turnaround time from drawing of blood to receipt of NGS results was one to two days, with the longest timespan being four days due to weekends or holidays, which is similar to previously reported times in a clinical setting [14, 18, 19]. Thus, NGS results were mostly available faster than blood culture results and were similar in timing to viral PCR assays. Given that the outcome of pediatric sepsis depends critically on prompt and accurate therapy, rapid pathogen detection and identification may be lifesaving [1\u0026ndash;3].\u003c/p\u003e\n\u003cp\u003eImportantly, negative NGS results, if shown to have adequate diagnostic reliability, may support safe de-escalation or discontinuation of antimicrobial treatment, thereby reducing unnecessary exposure, toxicity, and the risk of secondary infections such as \u003cem\u003eClostridioides difficile\u003c/em\u003e [23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePositive NGS results identified several rare pathogens which were subsequently only detected by conventional diagnostics after targeted investigations based on the NGS findings were initiated. Without NGS overlooked pathogens included \u003cem\u003eBurkholderia contaminans\u003c/em\u003e, \u003cem\u003eEnterocytozoon bieneusi\u003c/em\u003e, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, \u003cem\u003eLeishmania infantum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;donovani\u003c/em\u003e, \u003cem\u003eMycobacterium chimaera\u003c/em\u003e. Rare pathogens like \u003cem\u003eEnterocytozoon bieneusi\u003c/em\u003e, can be challenging to detect using standard microbiological techniques [24]. Similarly, \u003cem\u003eMycobacterium chimaera\u003c/em\u003e often requires prolonged culture and specialized laboratory protocols for identification [25]. Meanwhile, \u003cem\u003eLeishmania infantum\u003c/em\u003e, which is endemic to specific regions, may be overlooked in non-endemic settings unless clinical suspicion prompts targeted testing [26]. \u0026nbsp; In the presented patient cohort, the ability of NGS to detect such pathogens not only enhanced diagnostic accuracy but also had direct therapeutic implications, enabling the timely initiation of targeted antimicrobial or antiparasitic therapies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePossible explanations for non-detection in culture-positive cases include low pathogen cfDNA fraction, insufficient sequencing depth in those runs, high host-to-microbe DNA ratio, different sample materials used in conventional diagnostics (such as urine) and timing of sampling after antimicrobial exposure. These interventions significantly impacted patient outcomes and might have otherwise been delayed or entirely missed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides its short turnaround time and broad range of detectable pathogens, further advantages of NGS diagnostics include detecting resistance factors within the same sample and workflow [27, 28], as well as its use in different specimens, such as cerebrospinal fluid, lavages or tissues [29,30].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, it was conducted at a single tertiary centre and primarily included chronically ill and immunocompromised patients, introducing selection bias and limiting generalisability. Second, NGS testing was performed at the discretion of treating clinicians, which may have favoured patients with higher pre-test probability of infection [31]. Third, the retrospective design and relatively small sample size restricted the ability to draw definitive conclusions about diagnostic accuracy and clinical outcomes. Finally, NGS, like other sequencing-based methods, remains susceptible to contamination, and data protection regulations prevented the public release of raw sequencing data. Only aggregated results could be made available upon reasonable request.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we evaluated the diagnostic yield, clinical relevance, and therapeutic impact of Next Generation Sequencing (NGS) for detection of circulating microbial DNA in paediatric patients with suspected infection at a tertiary care centre. NGS identified additional pathogens not detected by conventional methods and influenced clinical management decisions in a subset of cases, including both initiation and discontinuation of antimicrobial therapy. These findings indicate that NGS can provide meaningful diagnostic and therapeutic value when used as a complementary tool to standard microbiological testing, particularly in complex or critically ill children. However, the clinical utility of NGS remains limited by factors such as high cost, restricted availability, lack of standardized interpretation criteria, and uncertain diagnostic validity in broader populations. Future multicenter, prospective studies should focus on defining patient groups that benefit most from NGS, validating its performance metrics, and integrating resistance gene detection and quantitative analyses into clinical workflows.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the University of Duisburg-Essen, Germany (reference number 23-11277-BO). The requirement for written informed consent was waived due to the retrospective design and anonymised data analysis. All procedures followed the ethical standards of the institutional research committee and the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. The manuscript does not contain any individual person\u0026rsquo;s identifiable data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are not publicly available due to patient confidentiality and data protection regulations. De-identified aggregated data may be made available from the corresponding author upon reasonable request and pending approval by the local Ethics Committee and data protection office.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSilke Grumaz is a co-founder, employee and shareholder of Noscendo GmbH.\u003c/p\u003e\n\u003cp\u003ePetra Horvatek is a former employee of Noscendo GmbH.\u003c/p\u003e\n\u003cp\u003eThorsten Brenner has received a lecture honorarium from Noscendo GmbH.\u003c/p\u003e\n\u003cp\u003eAll other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received scientific and logistic support from Noscendo GmbH, Duisburg, Germany, which performed the metagenomic NGS analyses. Additional institutional research funding from the Department of Pediatrics I, University Hospital Essen, supported this work. The funders had no influence on study design, data collection, analysis, interpretation, or the decision to publish the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJEA and SCG conceived the study and developed the study design. JEA, CP, TB, CH, TB, and UFM collected clinical data and reviewed patient cases. SG and PH performed sequencing and bioinformatic analyses. MS, LA, SV and JD contributed microbiological and virological expertise. CDS and UFM provided clinical supervision. SCG and JEA drafted the first manuscript version. All authors critically revised the manuscript, approved the final version, and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to all patients and their families for putting their trust in our care. We thank the nursing and medical staff of the Department of Pediatrics I at the University Hospital Essen for their dedicated support in patient care and sample collection. Our appreciation also goes to the team at Noscendo GmbH for their technical assistance with sequencing and data processing. We acknowledge the members of the paediatric infectious diseases team for their valuable clinical input during case evaluations and data interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSarah C. Goretzki, MD, is a paediatric infectious diseases specialist at the University Hospital Essen, Germany, with a research focus on molecular pathogen diagnostics and antimicrobial stewardship. ORCID: 0000-0001-5218-070X.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeiss SL, Fitzgerald JC, Balamuth F, Alpern ER, Lavelle J, Chilutti M et al (2014) Delayed antimicrobial therapy increases mortality and organ dysfunction duration in pediatric sepsis. Crit Care Med 42:2409\u0026ndash;2417\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar A, Ellis P, Arabi Y, Roberts D, Light B, Parrillo JE et al (2009) Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. 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Cells. ;10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF et al (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12:87\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"macp","sideBox":"Learn more about [Molecular and Cellular Pediatrics](http://molcellped.springeropen.com)","snPcode":"40348","submissionUrl":"https://submission.nature.com/new-submission/40348/3","title":"Molecular and Cellular Pediatrics","twitterHandle":"@springeropen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cell-free DNA, metagenomic next-generation sequencing, pediatric sepsis, infectious disease diagnostics, antimicrobial stewardship","lastPublishedDoi":"10.21203/rs.3.rs-8133441/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8133441/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRapid and accurate pathogen detection is critical for optimizing outcomes in pediatric sepsis. Next-generation sequencing (NGS) of cell-free DNA (cfDNA) from blood enables culture-independent identification of microbial DNA from bacteria, viruses, fungi, and parasites. We evaluated the diagnostic yield and clinical impact of cfDNA-based NGS in critically ill and predominantly immunocompromised pediatric patients (≤ 18 years) with suspected infection.\u003c/p\u003e\n\u003cp\u003eThis retrospective single-center study included pediatric patients who underwent plasma cfDNA-NGS at a tertiary care hospital in Germany. Following computational removal of human DNA, remaining sequences were aligned to curated microbial reference databases. Diagnostic performance was compared with blood cultures and viral PCR, and clinical relevance was assessed by pediatric infectious disease specialists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e111 tests in 78 pediatric patients, mostly with systemic inflammatory response syndrome of unknown etiology, were performed. Overall, 61 tests (54.5%) were positive for pathogenic cfDNA. Compared with conventional microbiological diagnostics, NGS demonstrated a sensitivity of 64.7% and specificity of 88.2% when blood cultures and viral PCR served as the reference standard. NGS identified additional pathogens in a substantial proportion (41.1%) of cases that remained negative by standard testing. Of those pathogens only found by NGS, over 60% were deemed clinically relevant. In 14.8% of positive NGS results, a pathogen-specific therapy was started, while 40.2% of tests led to a discontinuation of therapy (51.0% of negative tests). Out of all positive NGS, 38 (62.3%) were classified as clinically relevant. NGS testing also detected rare infections with fungi and parasites in four cases each.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Detection of pathogenic cfDNA through NGS from blood shows promising results as an additional diagnostic tool in critically ill pediatric patients with suspected infections. Clinical utility is currently still limited by its high cost, undetermined diagnostic validity and limitations in testing for resistances and restricted availability of raw sequencing data due to data-protection constraints.\u003c/p\u003e","manuscriptTitle":"Next Generation Sequencing of free pathogenic DNA in blood samples of a critically-ill pediatric population: A single-center experience Short title: NGS of pathogenic cfDNA in critically ill children","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 10:42:45","doi":"10.21203/rs.3.rs-8133441/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T10:44:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T09:45:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T19:25:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28611067315778928606801249055685040803","date":"2025-12-19T21:19:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129522609487519633388999278120014188772","date":"2025-12-12T05:30:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-25T08:48:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-25T08:47:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T00:17:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Pediatrics","date":"2025-11-17T09:27:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"macp","sideBox":"Learn more about [Molecular and Cellular Pediatrics](http://molcellped.springeropen.com)","snPcode":"40348","submissionUrl":"https://submission.nature.com/new-submission/40348/3","title":"Molecular and Cellular Pediatrics","twitterHandle":"@springeropen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"413e5391-440b-4df2-95c7-d4ec05c91123","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:04:54+00:00","versionOfRecord":{"articleIdentity":"rs-8133441","link":"https://doi.org/10.1186/s40348-026-00226-3","journal":{"identity":"molecular-and-cellular-pediatrics","isVorOnly":false,"title":"Molecular and Cellular Pediatrics"},"publishedOn":"2026-04-08 15:59:25","publishedOnDateReadable":"April 8th, 2026"},"versionCreatedAt":"2025-11-28 10:42:45","video":"","vorDoi":"10.1186/s40348-026-00226-3","vorDoiUrl":"https://doi.org/10.1186/s40348-026-00226-3","workflowStages":[]},"version":"v1","identity":"rs-8133441","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8133441","identity":"rs-8133441","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}