Citations

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 New insights into community-acquired pleural infection microbiology may enable development of more rapid and accessible diagnostics that can streamline treatment. This study systematically compared molecular approaches for pathogen detection in pleural fluid from patients with such infections. Considering the need for rapid results with minimum hands-on time and test complexity, off-label use of the cassette-based, random-access BIOFIRE Joint Infection Panel (BF-JIP, bioMérieux) with and without supplementary in-house polymerase chain reaction (PCR) assays was evaluated. Methods Pleural fluids from 162 subjects from the Mayo Clinic (Rochester, Minnesota) and Haukeland University Hospital (Bergen, Norway), where molecular tests are routinely used for analyzing pleural fluid, were studied. The performance of the BF-JIP was compared to 16S rRNA gene PCR followed by Sanger and/or next-generation sequencing (16S rRNA gene PCR/sequencing), an in-house PCR panel specifically designed for pleural space infections, and culture. Results The BF-JIP supplemented with 3 in-house PCR assays targeting Fusobacterium nucleatum group, Streptococcus intermedius/constellatus, and Aggregatibacter aphrophilus detected 96.8% of pleural infections, providing complete microbial profiles in 70.1%. In comparison, the detection rate of 16S rRNA gene PCR/sequencing was 92.2%, providing complete microbial profiles in 87.0%. Culture detected bacteria in only 31.9% of pleural fluids, providing complete microbial profiles in 15.3%. Conclusions These results underscore the importance of molecular approaches in diagnosing pleural space infections and demonstrate the feasibility of panel-based PCR-based diagnostics for these sometimes-complex infections. The BF-JIP, designed for testing synovial fluid, could be modified to provide a useful diagnostic for testing pleural fluid.

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.

The placenta is susceptible to infection by a number of viral pathogens, including severe acute respiratory syndrome coronavirus 2, which is associated with poor fetal outcomes. The histologic pattern of injury, termed severe acute respiratory syndrome coronavirus 2 placentitis, is characterized by a triad of increased perivillous fibrin deposition, intervillous histiocytes, and trophoblast necrosis. While the etiology of massive perivillous fibrin deposition (MPVFD) is mostly unknown, previous case reports of MPVFD in association with maternal Enterovirus (ENT) suggest that a subset of these cases are a consequence of undiagnosed viral infection. We evaluated 46 placentas collected between 2011 and 2022 with a diagnosis of MPVFD (n = 41) or chronic histiocytic intervillositis (CHI; n = 4). Combining methods of pan-viral metagenomic sequencing and targeted viral PCR, we detected Enterovirus DNA in 8 of 45 (18%) MPVFD and/or CHI cases. Seven of these positive cases were from MPVFD, and 1 was associated with a CHI diagnosis. Enterovirus A species (n = 7) were commonly identified, whereas one case had Enterovirus B. Histologic evaluation of these cases, including immunohistochemical staining for CD68, demonstrated increased intervillous histiocytes in Enterovirus-positive MPVFD cases in comparison with Enterovirus -negative, as well as evidence of trophoblast necrosis. Thus, we favor the terminology Enterovirus placentitis to describe this pathology. Overall, these findings suggest that Enterovirus is an underrecognized etiology of histologic MPVFD and, possibly, CHI. Further study to evaluate the recurrence risk of Enterovirus placentitis in comparison to MPVFD may help inform future fertility planning in patients with these diagnoses.

Abstract Background 16S ribosomal RNA (rRNA) gene PCR and sequencing may serve as an adjunctive diagnostic tool in complex infections, identifying bacteria in patients on antibiotics whose cultures may be negative. Data on the optimal clinical settings for utilizing this type of testing are limited. This study reviews a single center's 16S rRNA PCR/sequencing experience in children to identify clinical syndromes and optimize specimen choice for higher yield, leading to testing of appropriate patients. Methods A retrospective study was performed at Mayo Clinic, Rochester involving children aged 0 to 18 years whose normally sterile tissue or fluid samples underwent 16S rRNA gene PCR/sequencing. The assay involved an up-front real-time PCR assay, reported as negative or submitted to Sanger or next-generation sequencing depending on Ct values. Data was collected on patient characteristics and results of 16S rRNA gene PCR/sequencing and conventional tests. Impact on clinical decision-making was assessed as to whether the advanced molecular diagnostic changed antimicrobial therapy. Results A total of 162 tests were performed on 124 patients, including 47% females (58/124) with a median age of 9.6 (range, 2.2-15.0) years. Overall, 33/162 (20%) of tests were positive in 24/124 (19%) patients. 58% of positive tests (19/33) were from samples with negative bacterial cultures. Cerebrospinal fluid was the most common specimen tested (n=38; 31%). Positive results were more likely from fluid compared to tissue samples (OR [95% CI]: 3.1 [1.3, 7.1], p=.007; OR [95% CI]: 0.3 [0.1, 0.8], p=.007, respectively). Testing was positive in 5/10 (50%) pleural fluids tested, a higher positivity rate than any other specimen type (OR [95% CI]: 5 [1.3, 19.0], p=.011). No significant changes in antibiotics were made within 24 hours of reporting positive 16S rRNA gene PCR/sequencing results. Conclusion This study highlights the importance of selecting appropriate sample types for 16S rRNA gene PCR/sequencing in pediatric patients. The assay is most likely to yield positive results in children with pleural effusion and in other fluid samples such as subdural and synovial fluid. Further implementation science research is needed to achieve impact on clinical practice. Disclosures Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria

Abstract Background Definition of the microbiology of pleural space infection has been challenging due to the poor yield of conventional culture, a challenge that has led to recommendations for broad spectrum antimicrobial therapy for these infections. Since August 2019, we have offered a 16S ribosomal RNA gene PCR assay followed by Sanger or next generation sequencing, or reported as negative, depending on the PCR signal strength (e.g., crossing point) in clinical practice at Mayo Clinic. Methods Here, we describe the microbiologic and clinical findings of 57 pleural fluids testing positive by 16S rRNA gene PCR/sequencing from August 2020 to January 2023. Demographic data and clinical features, and results of pleural fluid and blood cultures were recorded. Analysis was performed using SAS software version 9.4 (SAS Inc). Results Median patient age was 63 years (range 5-83 years) and 75% were men. Pleural space infection was community-acquired in 84%. The most frequent symptom was dyspnea (80%), followed by chest pain (52%). 89% of patients had received antibiotics in the two weeks preceding specimen collection. Radiological consolidation was observed in 63% and pleural fluid was loculated in 58%. Pleural fluid and blood cultures were positive in 32 and 11%, respectively. The microbiology found with the sequencing assay is shown in the table. 23/57 (40%) detections were polymicrobial. Fusobacterium nucleatum group and/or Streptococcus intermedius were detected in 31/57 (54%) cases [including 28/48 (58%) in the subset with community-acquired infection], with additional facultative and/or anaerobic species in various combinations also found in 17/31 (55%) of these cases. Conclusion Based on analysis of pleural fluid using a targeted metagenomic sequencing assay in clinical practice, the most frequent organism profile involved in community-acquired pleural space infection is a combination of Fusobacterium nucleatum group and/or Streptococcus intermedius, with or without other normal microbiota, a grouping for which we propose the term FuSion (Fusobacterium nucleatum group, Streptococcus intermedius and oral normal microbiota). Disclosures Eva Carmona, MD, boehringer ingelheim: Advisor/Consultant Øyvind Kommedal, n/a, Pathogenomix inc.: Advisor/Consultant|Pathogenomix inc.: Stocks/Bonds Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant

Abstract Background Shotgun and targeted metagenomic sequencing have been shown in separate studies to be potentially useful for culture-free pathogen identification in blood and/or plasma of patients with infective endocarditis (IE). However, the 2 approaches have not been directly compared. The aim of this study was to compare shotgun metagenomic sequencing with targeted metagenomic sequencing (tMGS) for organism identification in blood or plasma of patients with IE. Methods Patients with possible or definite IE were prospectively enrolled from October 2020 to July 2021. Shotgun metagenomic sequencing was performed with the Karius test, which uses microbial cell-free DNA (mcfDNA) sequencing to detect, identify, and quantitate DNA-based pathogens in plasma. tMGS was performed using a 16S ribosomal RNA (rRNA) polymerase chain reaction assay targeting the V1 to V3 regions of the 16S rRNA gene. Results were compared using the McNemar test of paired proportions. Results Samples from 34 patients were investigated. The Karius test was positive in 24/34 (71%), including 3/6 (50%) with blood culture–negative endocarditis (BCNE), which was not significantly different from the positivity rate of tMGS (P = .41). Results of the Karius test were concordant with tMGS in 75% of cases. The Karius test detected 2 cases of methicillin-resistant Staphylococcus aureus among the 7 S. aureus detections, in accordance with results of phenotypic susceptibility testing. The combination of blood cultures, the Karius test, and tMGS found a potential causative pathogen in 33/34 (97%), including 5/6 with BCNE. Conclusions The Karius test and tMGS yielded comparable detection rates; however, beyond organism identification, the Karius test generated potentially useful antibiotic resistance data.

Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics.

OBJECTIVE Infuse bone graft is a widely used osteoinductive adjuvant; however, the simple collagen sponge scaffold used in the implant has minimal inherent osteoinductive properties and poorly controls the delivery of the adsorbed recombinant human bone morphogenetic protein–2 (rhBMP-2). In this study, the authors sought to create a novel bone graft substitute material that overcomes the limitations of Infuse and compare the ability of this material with that of Infuse to facilitate union following spine surgery in a clinically translatable rat model of spinal fusion. METHODS The authors created a polydopamine (PDA)–infused, porous, homogeneously dispersed solid mixture of extracellular matrix and calcium phosphates (BioMim-PDA) and then compared the efficacy of this material directly with Infuse in the setting of different concentrations of rhBMP-2 using a rat model of spinal fusion. Sixty male Sprague Dawley rats were randomly assigned to each of six equal groups: 1) collagen + 0.2 µg rhBMP-2/side, 2) BioMim-PDA + 0.2 µg rhBMP-2/side, 3) collagen + 2.0 µg rhBMP-2/side, 4) BioMim-PDA + 2.0 μg rhBMP-2/side, 5) collagen + 20 µg rhBMP-2/side, and 6) BioMim-PDA + 20 µg rhBMP-2/side. All animals underwent posterolateral intertransverse process fusion at L4–5 using the assigned bone graft. Animals were euthanized 8 weeks postoperatively, and their lumbar spines were analyzed via microcomputed tomography (µCT) and histology. Spinal fusion was defined as continuous bridging bone bilaterally across the fusion site evaluated via µCT. RESULTS The fusion rate was 100% in all groups except group 1 (70%) and group 4 (90%). Use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater results for bone volume (BV), percentage BV, and trabecular number, as well as significantly smaller trabecular separation, compared with the use of the collagen sponge with 2.0 µg rhBMP-2. The same results were observed when the use of BioMim-PDA with 2.0 µg rhBMP-2 was compared with the use of the collagen sponge with 20 µg rhBMP-2. CONCLUSIONS Implantation of rhBMP-2–adsorbed BioMim-PDA scaffolds resulted in BV and bone quality superior to that afforded by treatment with rhBMP-2 concentrations 10-fold higher implanted on a conventional collagen sponge. Using BioMim-PDA (vs a collagen sponge) for rhBMP-2 delivery could significantly lower the amount of rhBMP-2 required for successful bone grafting clinically, improving device safety and decreasing costs.