Rodning, Amanda A

Abstract Background Identification of the microorganisms causing infection in children with central nervous system infection is important. Conventional cerebrospinal fluid (CSF) tests may provide limited pathogen detection. CSF metagenomic next-generation sequencing (mNGS) may provide broader pathogen detection in a single assay. Methods Results of CSF mNGS testing performed at Mayo Clinic on patients and nonpatients between January 2024 and June 2025 were retrospectively analyzed. The subcohort of children treated at Mayo Clinic was analyzed to assess test performance as compared with conventional CSF diagnostics and potential clinical impact. Results In total 134 patients (138 CSF mNGS tests) from 22 US states and 2 international sites were included. Overall positivity was 16% with viruses representing most (59%) detections. In the Mayo Clinic subcohort (n = 15), test positivity was 27%: organisms included single detections of Streptococcus mitis group and HIV type 1, as well as 2 detections of Epstein-Barr virus, all confirmed by conventional tests. Positive results were associated with CSF pleocytosis (P = .026). Higher yield was observed in participants whose care was overseen by pediatric infectious diseases (PID) specialists (P = .026) and when a specimen hold strategy was applied (P = .033). Most results (87%) did not alter antimicrobial therapy, with 2 negative results contributing to antimicrobial de-escalation. Conclusions CSF mNGS demonstrated higher diagnostic yield in patients whose specimens showed CSF pleocytosis and in those who had PID consultation. Diagnostic stewardship strategies were applied, including specimen hold approaches and PID consultation.

Abstract Background Central nervous system (CNS) infections are significant causes of morbidity and mortality, especially when diagnosis is delayed. Traditional cerebrospinal fluid diagnostic methods for CNS infections have limited sensitivity. Metagenomic next-generation sequencing of cerebrospinal fluid (MSCSF) is a newer diagnostic tool capable of detecting a broad range of pathogens in a single assay. Methods This retrospective cohort study evaluated the first-year experience with MSCSF at Mayo Clinic. A total of 422 tests performed during calendar year 2024 on adult patients at Mayo Clinic facilities and those tested through Mayo Clinic Laboratories were analyzed, with a subcohort analysis of the 210 Mayo Clinic patients performed to define patient characteristics associated with positive results and clinical outcomes. Results The sensitivity of MSCSF for detecting neuroinfection in the subcohort was 64%, with 43% of confirmed neuroinfections diagnosed by MSCSF only. Subjects who were immunosuppressed (odds ratio [OR]: 3.5, P = .008) and those with a high pretest probability of CNS infection (OR: 20.1, P < .001) were more likely to have positive results. Also, those who tested positive were more likely to die within the ensuing 30 days than those who tested negative (OR: 5.2, P = .001). Conclusions MSCSF can be a valuable tool for diagnosing CNS infection but should not be used indiscriminately. It appears to be most beneficial when there is a high clinical suspicion of CNS infection and conventional diagnostics, and available molecular tests, yield negative results. Its integration into clinical practice should be guided by diagnostic stewardship to maximize cost-effectiveness and clinical utility.

Abstract Background Intestinal colonization with carbapenem-resistant organisms (CRO) may be considered to inform empiric antibiotic selection for symptomatic infection. However, it is unclear how often gut colonizing CRE isolates are related to bloodstream isolates in hospitalized patients with bacteremia, especially in areas with high CRO prevalence. Minimum spanning tree of isolate relatedness Isolates and corresponding patient number (P1-P6) and rectal (R) or blood (B) specimen are noted within circles and allelic differences are numerated on linking lines between the circles. Group 1 and 3 isolates are possibly related to each other. Created with BioRender.com. Methods Patients with paired K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates cultured from surveillance rectal specimens and clinically collected blood specimens collected within 1-8 days of each other between 2019-2023 were identified at 3 Greek hospitals. Isolates were frozen and shipped to a reference laboratory in the U.S for whole genome sequencing (WGS) using Illumina MiSeq. Assembly and core genome multilocus sequence typing (cgMLST) analysis were performed with Ridom SeqSphere+ software. Isolate relatedness was based on the total number of allelic differences between each isolate: < 15, related; 16-50, possibly related; ≥ 51, unrelated. Results One hundred eight paired isolates were identified from 54 unique patients; 6 pairs and 12 total isolates underwent cgMLST. In 5 of 6 pairs (83%), the rectal isolate was collected prior to the bloodstream isolate. Rectal and bloodstream isolates were related to one another in 5 of 6 (83%) pairs and were unrelated in 1 pair. Two pairs collected from 2 distinct patients admitted to the same intensive care unit (patients 2 and 3, Figure) were possibly related (16-50 allelic differences) to each other. Conclusion In this pilot evaluation conducted in a setting of high CRO prevalence, KPC-producing K. pneumoniae isolated from paired rectal and blood specimens from individual patients were often related to each other, suggesting that intestinal colonization by this organism could inform empiric antibiotic treatment for bacteremia. Two patients from the same hospital unit had isolates that were possibly related, suggesting within hospital transmission. WGS is a powerful tool that can inform clinical and infection control practices. WGS of the remaining K. pneumoniae pairs and additional Acinetobacter baumannii pairs is ongoing. Disclosures Robin Patel, MD, I already disclosed so please pull from disclosure for other abstracts: Advisor/Consultant|I already disclosed so please pull from disclosure for other abstracts: Grant/Research Support|I already disclosed so please pull from disclosure for other abstracts: Honoraria

ABSTRACT The value of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed.