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Hematological Disorders and Diagnostics / Orthopedic Infections and Treatments · Journal article
Frontiers in Pediatrics · August 5, 2026
Early or partial results. Treat as a signal, not a conclusion.
This retrospective single-center study compared metagenomic next-generation sequencing (mNGS) to conventional microbiology in 41 hospitalized children with fever of unknown origin, all of whom had already failed conventional testing or showed poor clinical response. mNGS identified 30 microbial isolates from 20 patients (17 confirmed as causative), but showed no statistically significant difference in positivity rates versus conventional assays (all P ≥ 0.05). The authors conclude mNGS cannot replace standard workflows and recommend it as an auxiliary tool only for complicated cases with negative conventional results, pending larger prospective validation.
Retrospective single-center observational study. Hospitalized children at the Department of Infectious Diseases, Affiliated Children's Hospital of Shandong University, diagnosed with FUO who underwent mNGS testing. Enrollment was selective: patients with persistent fever despite comprehensive routine evaluations, suspected infectious etiology, or…. Intervention: Metagenomic next-generation sequencing (mNGS) on sterile body fluid specimens (blood, CSF, tissue fluid). Compared with: Conventional microbiological testing including culture, smear microscopy, and PCR on paired specimens. n = 41. Single center: Department of Infectious Diseases, Affiliated Children's Hospital of Shandong University, China.
mNGS identified 30 microbial isolates from 20 of 41 patients, with 17 ultimately confirmed as causative pathogens No statistically significant difference in positivity rates between mNGS and conventional assays (all P ≥ 0.05) on paired specimens Antimicrobial regimen changes following mNGS results: 4 patients received adjuvant antiviral therapy, 6 escalation, 2 de-escalation, 5 comprehensive modifications
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mNGS showed equivalent (not superior) detection to conventional testing in this enriched cohort of difficult FUO cases already failed by standard methods. Clinicians should not expect mNGS to replace conventional workflows, but may consider it as a supplementary tool for complicated infections with negative conventional results; prospective validation with larger samples is required before routine adoption.
Single-center retrospective analysis of 41 children with uncontrolled selection bias (only tested after conventional failure), no control arm, and equivalence to standard testing rather than superiority; generates descriptive data warranting prospective validation.
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mNGS showed equivalent (not superior) detection to conventional testing in this enriched cohort of difficult FUO cases already failed by standard methods. Clinicians should not expect mNGS to replace conventional workflows, but may consider it as a supplementary tool for complicated infections with negative conventional results; prospective validation with larger samples is required before routine adoption.
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Purpose Infectious diseases constitute the predominant cause of fever of unknown origin (FUO). Conventional microbiological testing is limited by prolonged turnaround times, susceptibility to host/environmental interference, low detection sensitivity, and limited capacity to identify rare pathogens. Metagenomic next-generation sequencing (mNGS) enables parallel broad-spectrum screening for microbial agents. This study aimed to investigate the clinical utility of mNGS in children presenting with FUO, to generate descriptive observational data on pathogen detection and temporally associated anti-infective regimen adjustments. Methods This retrospective single-center analysis enrolled 41 hospitalized children diagnosed with FUO who underwent mNGS testing at the Department of Infectious Diseases, Affiliated Children's Hospital of Shandong University, from June 2022 to July 2025. Initially, all patients underwent comprehensive routine systemic evaluations. For cases where fever persisted despite conventional testing and an infectious etiology was highly suspected, or where there was a poor therapeutic response to empirical anti-infective treatments, mNGS was subsequently performed. All specimens submitted for testing were sterile body fluids. Each sample was divided into two aliquots: one was subjected to conventional microbiological testing (including culture, smear microscopy, and PCR), while the other was cryopreserved for mNGS analysis. The performance of pathogen detection was compared between mNGS and conventional testing modalities using paired specimen data. Results In this study, we analyzed 41 pediatric cases, which included three types of specimens: blood, cerebrospinal fluid (CSF), and tissue fluid (comprising deep pus, postoperative drainage fluid, subdural effusion, and aspirated fluid from the mass). mNGS identified 30 microbial isolates from 20 patients, which included bacteria, viruses, fungi, and mycoplasmas; of these, 17 isolates were ultimately confirmed as causative pathogens. No statistically significant differences in positivity rates were observed between mNGS and conventional assays, as indicated by paired 2 × 2 contingency tables (all P 0.05).The present study also recorded changes to antimicrobial regimens that occurred after pathogen identification by mNGS testing, including adjuvant antiviral therapy for 4 patients, antimicrobial escalation for 6 patients, antimicrobial de-escalation for 2 patients, and comprehensive regimen modifications for an additional 5 patients. Conclusion We analyzed a targeted pediatric FUO subgroup, and the overall pathogen detection positivity rate showed no statistical difference between mNGS and routine microbial testing. Accordingly, mNGS cannot currently replace standard workflows or routinely screen all FUO children. The two testing methods exhibited complementary pathogen detection spectra. mNGS may act as an auxiliary tool for complicated infectious cases with negative conventional test results. This study generates descriptive observational data on pathogen identification and temporally associated anti-infective regimen adjustments in a selected cohort of FUO children. Further prospective studies with larger sample sizes are required to validate these findings.
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