Saleh, Omar Abu

Abstract Background Plasma microbial cell-free DNA (mcfDNA) sequencing, commercially available from Karius® (Redwood City, CA), has been increasingly utilized in diagnosing endovascular infections. The 2023 updated Duke criteria incorporated detection of pathogen via metagenomic sequencing in its microbiologic criteria. We analyzed the clinical impact of mcfDNA sequencing in the diagnosis and treatment of suspected infective endocarditis (IE) cases. Methods We performed retrospective review of patients with suspected IE who underwent Karius® test at Mayo Clinic, MN, between 2017 to 2023. Results We preformed preliminary analysis of 25 patients. Analysis of baseline characteristics (table 1) revealed that these patients had significant predisposing factors for IE. Before accounting for mcfDNA sequencing, 3 (12%) met criteria for definite IE, 10 (40%) met criteria for possible IE, and 12 (48%) were rejected per 2023 Duke criteria. After incorporating mcfDNA sequencing result, 1 patient who was classified as possible IE was escalated to definite IE. This patient had previous infection of implantable cardioverter-defibrillator (ICD) with Streptococcus dysgalactiae. ICD was removed. She had development of endophthalmitis and stroke, 1 week and 2 weeks after the completion of IV antibiotics, respectively, with echocardiogram showing new tricuspid valve vegetation. Routine work-up was negative despite that she was not on any systemic antibiotics at the time of blood culture collection. mcfDNA sequencing detected Streptococcus dysgalactiae, making definite diagnosis of IE, leading to the physician’s decision to extend the IV antibiotic treatment (treatment escalation). Overall, after mcfDNA sequencing result, treatment decision was not impacted in 14 (56%), ongoing treatment was escalated in 4 (16%), de-escalated in 5 (20%), and antimicrobials was newly initiated in 2 (8%) (figure 1). Positive mcfDNA sequencing result often led to treatment escalation or initiation; negative result often led to treatment de-escalation (figure 2). Conclusion In challenging cases of suspected IE, mcfDNA sequencing led to alteration of antimicrobial treatment in 44% of cases. Further data collection and final analysis will include larger number of patients. Disclosures All Authors: No reported disclosures

Abstract Background Vascular graft infection (VGI) is a rare but serious condition. Pathogen identification can be difficult given the deep-seated nature. Microbial cell-free DNA next-generation sequencing (mcfDNA NGS) is an emerging tool in non-invasive microbiologic workup. One commercially available mcfDNA is the Karius Test (KT). There is a paucity of data regarding utility and impact of mcfDNA NGS testing in the evaluation of VGI. Methods We retrospectively evaluated adult patients who underwent KT for evaluation of VGI at our institution. Only patients who met the Management of Aortic Graft Infection Collaboration (MAGIC) classification for suspected or confirmed VGI were included. We investigated the concordance between KT and conventional microbiologic workup. Conventional workup (CW) included pathogen identification through blood cultures, peri-graft/graft cultures, or histopathology. Concordance was defined by identical pathogen identification on KT and CW. We examined the influence of the KT result on clinical management.Figure 1.KT Results for Confirmed and Suspected VGI. Results We identified 25 patients who met criteria for either suspected or confirmed VGI per the MAGIC criteria. Microbiology was undefined in 8 patients who had negative KT and negative CW. A total of 17 patients had positive workup with 6 having positive KT and negative CW, 3 having negative KT and positive CW, and 8 having both positive KT and positive CW (Table 1). KT was less likely to be positive for patients who met criteria for suspected infection (4/10; 40%) as opposed to confirmed infection (10/15; 66.67%), albeit non-significant (OR 0.33, p=0.188) (Figure 1). The KT led to antimicrobial de-escalation in 27.3% of patients with positive CW and 35.7% of patients with negative CW (Figure 2). Full concordance of KT and CW was noted in 3 patients (Table 2).Figure 2.Influence of KT on VGI cases with positive and negative conventional work-up. Treatment escalation was defined as the broadening of antimicrobial coverage due to KT results. Treatment de-escalation was defined as narrowing/discontinuation of antimicrobial coverage due to KT results. Conclusion We found that the positivity rate for KT was 56%. KT impacted management in 40% of cases. Patients with confirmed VGI had a higher likelihood of having a positive KT. KT was the only positive microbiological definitive test in one case. Further studies are needed to explore the utility and impact of KT in the workup of VGI. Disclosures All Authors: No reported disclosures

Abstract Background Minocycline is a second-generation tetracycline with a broad antimicrobial spectrum. It is often used in chronic suppression of musculoskeletal infections, given activity against gram positive organisms. Efficacy of Minocycline against gram negative skin and soft tissue infection has not previously been defined. In this study, we review in vitro minocycline efficacy against isolates from skin, muscle, and bone sources.Fig 1.Organisms isolated Methods We conducted a retrospective review of all minocycline susceptibility testing performed on musculoskeletal GNB isolates performed at the Mayo Clinic reference lab between 2013-2022. We describe the isolates broken down by site of culture and organism isolated. Then, we calculated the overall susceptibility of these isolates to Minocycline and susceptibility of the most common 6 organisms isolated.Fig 2.Sites of gram negative isolation Results During the study timeframe, we identified 13,567 gram negative isolates from musculoskeletal sites. Of 330 species identified, 19 were identified in at least 100 isolates (fig 1). Of 36 unique sites, 19 sites were each identified in at least 100 isolates (fig 2). 1920 isolates were tested for susceptibility to minocycline. Of these, 1584 (82.5%) were susceptible to minocycline. Of the most common organisms identified and tested for minocycline susceptibility, 99% of Stenotrophomonas maltophilia, 83% of Enterobacter cloacae, 80% of Escherichia coli, 68% of Acinetobacter baumanii, 1% of Proteus mirabilis, 53% of Klebsiella pneumoniae, 65% of Serratia marcescens isolates were susceptible to minocycline (fig 3). Pseudomonas aeruginosa isolates were not tested, due to innate resistance.Fig 3.Minocycline susceptibility compared to key organisms Conclusion A majority of skin and soft tissue cultures positive for Gram negative isolates were below the waist line. Of these, we reported organisms most commonly found. Minocycline showed in vitro activity against some commonly isolated gram negative organisms, especially E.coli, Acinetobacter, and Stenotrophomonas. Further research is needed into the efficacy of minocycline in gram negative skin and soft tissue infections, but it may represent a viable oral option, especially in the end of life. Disclosures All Authors: No reported disclosures

Abstract Background The introduction of shotgun metagenomic sequencing (sMGS) methods that identify the presence of microbial cell-free DNA (mcfDNA) in peripheral blood have shown promising results in various populations of patients. There is a pressing need to evaluate its application for osteoarticular infections (OAIs) in real-world setting. Baseline characteristics of the patients with suspected osteo-articular infections, including demographics, clinical characteristics, diagnosis, and KT results. Methods We retrospectively collected data on 73 adult patients who presented to the Mayo Clinic with suspected OAIs between 2019 and 2023, and for whom mcfDNA sMGS (Karius®, KT) testing was part of their diagnostic workup. We collected data on comorbidities, diagnostics, microbiological workout and its concordance with the KT. The primary objective was to evaluate its clinical impact on OAI diagnoses and management distinguishing into four categories: (1) KT was able to confirm an established diagnosis, (2) KT supported non-ID diagnosis, (3) KT established an unsuspected diagnosis, (4) KT did not add relevant information. Relevant microbiologic studies of patients with unsuspected diagnoses established by the KT (N=5) Results In our cohort KT was performed in 73 patients (Table 1). Among the infected individuals, KT yielded positive results in 22/43 (51.2%) cases. Out of these 22 cases, 11 (50%) showed agreement with conventional diagnostic workup, while in five (22.7%) cases, the KT established an unsuspected diagnosis, improving the diagnostic power from 11/43 (25.6%) to 16/43 (37.2%) (Table 2). Moreover, yielded a negative result in 19/26 (73.1%) patients with non-infectious diagnosis (Figure 1). Native Vertebral Osteomyelitis (NVO) diagnosis (p < 0.001) or OAIs with concomitant presence of endocarditis or endovascular infection (p = 0.005) were statistically associated with a definite, probable, or possible diagnostic certainty of KT result (Table 3). Overall, KT influenced antibiotic therapy in six cases (8.2%). Table 3 Factors associated with diagnostic certainty categories Conclusion This is the first retrospective study aiming to assess the real-world usefulness of the KT on the clinical management of patients with OAIs, with a special focus on NVO. It underscores KT's real-world application in cases where traditional diagnostic methods fall short. In highly complex OAIs cases, KT added 11.6% to the diagnostic power by establishing unsuspected diagnosis in 5 of 43 infective patients. Patients with NVO or OAIs with concomitant endocarditis or endovascular infections particularly benefited from KT testing. Figure 1 Distribution of KT results and their respective outcome according to the final diagnosis of suspected OAIs cases. Disclosures All Authors: No reported disclosures

Abstract Background Gram-negative bacteria (GNB) account for 5-23% of all periprosthetic joint infections (PJI), following total knee or hip arthroplasties (TKA, THA). Literature on the management of Gram-negative PJI is limited. This study aimed to describe our experience with the presentation, management, and outcomes of Gram-negative PJI.Table 1:Baseline characteristics (N = 126 cases)Data are N (%) unless otherwise specified. Abbreviations: BMT; Bone-marrow transplant, CCI; Charlson comorbidity index, CPPD; Calcium pyrophosphate crystal deposition, CRP; C-reactive protein, DMARDs; Disease-modifying antirheumatic drugs, ESR; Erythrocyte sedimentation rate, GERD; Gastroesophageal reflux disease, IQR; Inter-quartile range, PJI; prosthetic joint infection, SD; Standard deviation, TNF; Tumor necrotic factor, WBC; white blood cell Methods A retrospective review of our infected institutional total joint registry from 2012-2023 identified 125 patients with 126 GNB PJI (60 THA and 66 TKA) according to MSIS criteria for PJI. Seventy-two cases (57%) of GNB PJI in this series were classified as acute based on the duration of symptoms of less than 4 weeks. Treatment failure was defined as reoperation for any reason, persistent infection, or reinfection post-surgery. Baseline demographics, presenting symptoms, microbiological data, and treatment outcomes were reviewed.Table 2:Therapies (N = 126 joints)Data are N (%) unless otherwise specified. Abbreviations: DAIR; Debridement, antibiotics, and implant retention, IQR; Inter-quartile range Results For baseline demographics, see Table 1. Notably, 10% of patients included in the study were immunosuppressed. Fifty-five cases (43.6%) had a history of revision prior to the diagnosis of PJI, including 44 cases with history of prior PJI. Pseudomonas aeruginosa was the most commonly isolated pathogen (31.7%). Wound drainage was associated with polymicrobial growth (40.8%). Debridement, antibiotics, and implant retention (DAIR) was utilized in 42 cases with 9.8% failure rate. History of revision arthroplasty, chronic PJI and the presence of sinus tract were associated with higher odds of reoperation within 1 year. Eighty-four cases underwent resection arthroplasty (RA), 73 had antimicrobial spacer, of those, 60 patients completed two-stage revision (TSR). At the time of reimplantation, 45/60 (75%) of cases had negative cultures (Table 2). The overall failure rate in resection group failure rate was 45.2%. Wound drainage was associated with higher odds of reoperation in this group (Table 3). The Failure rate in this cohort was 33%. Fluoroquinolones had a 90% susceptibility rate but were used infrequently for chronic suppression (25%).Table 3:Predictor factors with reoperation for any reason within 1 yearData are N (%) unless otherwise specified.Abbreviations: DAIR; Debridement, antibiotics, and implant retention, PJI; Prosthetic joint infection Conclusion Pseudomonas aeruginosa was the predominant pathogen in GNB PJI, with a high failure rate observed in the resection group. History of revision arthroplasty, chronic PJI, sinus tracts and wound drainage were associated with increased risk of reoperation within 1 year.Figure 1:Distribution of failure events across the study Disclosures Nicholas Bedard, MD, Stryker: Advisor/Consultant

Abstract Background The underlying cause of fever of unknown origin (FUO) remains unidentified in up to 51% of cases despite systematic evaluation. Microbial cell-free DNA next-generation sequencing (mcfDNA-NGS) offers an agnostic, noninvasive approach to pathogen identification, but the utility and clinical impact of this assay in FUO remain unknown. Methods This retrospective cohort study evaluated adult patients referred for FUO evaluation at a tertiary medical center between November 2019 and November 2023. Patients underwent both standard microbiologic testing (ST) and mcfDNA-NGS. Diagnostic impact was assessed in 4 domains: new diagnoses, earlier time to diagnosis, avoidance of invasive procedures, and non-hypothesis-driven diagnoses. Logistic regression was used to identify predictors of positive mcfDNA-NGS testing. Results Among 176 patients, mcfDNA-NGS was positive in 44.3%, with 49% of these cases considered clinically significant. Infectious cause of FUO was identified in 39% of patients, noninfectious in 35%, and unknown in 26%. mcfDNA-NGS contributed to a positive diagnostic impact in 30% of cases, mainly by earlier diagnosis (16%) and potential for avoidance of invasive procedures (10%). Positive mcfDNA-NGS was significantly associated with higher Charlson comorbidity index score (odds ratio [OR], 1.22; P < .001) and white blood cell (WBC) count ≤4.5 × 109 cells/L (OR, 8.61; P < .001). Conversely, FUO without localization was associated with a decreased likelihood of positive mcfNDA testing (OR, 0.18; P < .001). Conclusions mcfDNA-NGS effectively complements ST in diagnosing FUO, providing earlier detection and minimizing invasive testing. Clinical predictors such as high comorbidity and low WBC count may guide the optimal use of mcfDNA-NGS in FUO. Prospective evaluation of optimal timing and use of mcfDNA-NGS and cost-benefit analysis in FUO is needed.

ABSTRACT A total of 1,925 Corynebacterium isolates were tested for antimicrobial susceptibility at the Mayo Clinic Microbiology laboratory (Rochester, Minnesota) from January 2012 to March 2023, with C. striatum (35.6%) and C. amycolatum (24.4%) identified as the predominant species. Species known to potentially carry diphtheria toxin were excluded. Common sources of isolation included skin and soft tissue (56.8%), bone and/or native joint synovial fluid (14.2%), urine (13.1%), sputum (6.1%), and blood (5.9%). For penicillin, susceptibility decreased from 47.5% (58 of 122) in 2012 to 20.6% (14 of 68) in 2023. Isolates also showed a decrease in susceptibility to erythromycin from 22.4% (26 of 116) in 2012 to 13.2% (9 of 68) in 2023. Susceptibility to trimethoprim-sulfamethoxazole averaged around 50% throughout the period. Notably, linezolid and vancomycin were universally effective in vitro against all species. The highest susceptibility rates among tested oral agents were to linezolid and doxycycline for non- C . striatum species. Daptomycin minimal inhibitory concentrations (MICs) of >256 µg/mL were observed for one C. amycolatum isolate, one C. tuberculostearicum isolate, and for seven C. striatum isolates, all from patients with prior daptomycin exposure. Daptomycin MICs of 2 µg/mL (nonsusceptible) were observed in one C. striatum isolate recovered from a daptomycin-naïve patient and in six C. jeikeium isolates, from both daptomycin-exposed and non-exposed patients. Significant variation in susceptibility profiles across different Corynebacterium species underscores the importance of performing antimicrobial susceptibility testing to guide effective treatment. Moreover, multidrug resistance observed in C. striatum poses substantial therapeutic challenges especially in patients requiring prolonged or chronic antibiotic suppression.

Background: Isavuconazole (ISA) has a favorable side effect profile that makes it attractive for treatment of invasive fungal infections (IFI). It carries FDA approval for invasive aspergillosis and mucormycosis, but there are fewer data for other organisms and non-pulmonary infections. We conducted this review to investigate how ISA performed at treating IFI, with an especial interest in these non-approved indications. Methods: We retrospectively identified and reviewed 131 patients who received ISA as treatment for IFI at our institution, some of whom received ISA as their first anti-fungal therapy and others who received ISA as either step-down therapy or salvage therapy. We identified the microbiologic cause of infection as well as the anatomic site involved for each patient. We then classified patients according to their response to ISA: namely cured, partially responded, or stabilized. Results: The majority of patients were immunocompromised (n = 76, 58%). ISA was used primarily as a secondary therapy (n = 116, 89%); either as a step-down/switching from other agents, or as salvage therapy. The most common reasons for switching to ISA were toxicities with prior agents followed by QT prolongation. Although pulmonary aspergillosis and mucormycosis were represented in more than half of the cohort, ISA was also used off-label for treatment of other organisms such as endemic fungi (n = 19, 15%) as well as central nervous system (CNS) infections (n = 15, 11%). We have described the detailed clinical characteristics of these CNS infections cases. The overall clinical response rate varied by type of infection and site involved (57–73% response rate). Conclusions: We demonstrated encouraging clinical responses, particularly outside the FDA-approved indications, as well as good tolerability. This report highlights the critical need for expanded scope of prospective studies to delineate the efficacy of this better-tolerated agent, especially in central nervous system infections.