Stefanie Desmet

Background: Hybrid-capture viral metagenomics improves sensitivity over untargeted metagenomics, but its performance across sequencing platforms and the definition of data-driven analysis cut- offs using real clinical samples have not been systematically evaluated. This study benchmarks the hybrid-capture workflow on mock and clinical specimens across short- and long-read platforms while introducing a new, reproducible approach for setting pathogen-calling cut- offs. Methods: The Twist Comprehensive Viral Research Panel was used with both AVITI short-read and Oxford Nanopore Technologies (ONT) long-read sequencing on 12 mock cerebrospinal fluid (CSF) external quality assessment (EQA) panel samples and on 14 CSF and 21 bronchoalveolar lavage (BAL) clinical specimens. Data-driven cut-offs for pathogen calling were derived using Latin hypercube sampling across genome coverage, abundance, and bleed-ratio parameters, then validated on the independent EQA datasets. Results: After cut-off application, both platforms detected 22 of 23 expected viral targets in EQA samples (95.7% detection-level sensitivity) with 100% specificity in negative controls. In clinical specimens, AVITI corroborated 26 of 30 (86.7%) and ONT 25 of 30 (83.3%) routine viral detections while maintaining 100% diagnostic specificity. Sensitivity was 100% for CSF but lower in BAL, particularly for low viral load targets, and all detections below a Cq value of 32 were identified by both platforms. Metagenomics also identified a potentially clinically relevant pathogen not tested by routine testing in two CSF samples. Conclusion: Hybrid-capture viral metagenomics performed comparably on AVITI and ONT for routine viral targets, with AVITI showing slightly higher genome recovery. In addition, the newly developed data-driven cut-off selection strategy offers a practical, reproducible method other laboratories can apply to their own workflows.

Pneumococcal infections caused by Streptococcus pneumoniae remain a leading cause of vaccine-preventable morbidity and mortality worldwide, particularly among older adults and individuals with immu...

In 2019, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) redefined the “I” category from “intermediate susceptibi

This article has been retracted. Please see this notice for details: https://doi.org/10.1093/infdis/jiag347.

Streptococcus pneumoniae serotype 23B, a non-vaccine serotype, has shown an increasing prevalence and penicillin non-susceptibility among carriage and invasive pneumococcal disease (IPD) isolates. Recently, a novel penicillin non-susceptible genotype has emerged, named 23B1. In the framework of the Belgian pneumococcal carriage study, we studied the prevalence of 23B ₀ /23B1 among 586 23B strains (2016–2022) in 172 day care centers from 6- to 30-month-old children and among 130 pediatric 23B IPD isolates (2007–2021). Pneumococci were whole genome sequenced to determine the capsular polysaccharide genotype and sequence type (ST). Antimicrobial susceptibility testing determined penicillin and amoxicillin MICs, as well as resistance to co-trimoxazole and levofloxacin. 23B carriage was stable during 2016 ̶ 2022 except in the 2020–2021 winter season when it increased. The proportion of genotype 23B1 compared to 23B ₀ decreased from 2016 ̶ to 2022 but remained consistently higher than 23B ₀ . In 2020–2021, an increase in the proportion of 23B1 was reflected in an overall increase in 23B carriage. All increases in 23B IPD cases were almost entirely driven by 23B1. The median penicillin MICs were significantly different for 23B ₀ (0.03 mg/L) and 23B1 (0.25 mg/L). In 2021, increased intermediate levofloxacin susceptibility was noted in 23B. 23B1-associated ST2372 was the most prevalent ST in carriage and IPD during 2013–2022. We show that an increase in 23B carriage among children was paralleled in pediatric IPD in Belgium, reiterating the utility of pneumococcal surveillance in the day care population. Serotype 23B is reported worldwide as an important pediatric non-PCV13 serotype with reduced penicillin susceptibility, with 23B1 as the presumed driver for the increased prevalence. IMPORTANCE: During the COVID-19 pandemic, the 23B serotype of Streptococcus pneumoniae has increased in prevalence in healthy carriage isolates from Belgian day care centers and pediatric (younger than 18 years of age) invasive pneumococcal disease (IPD) isolates. Additionally, an increase in penicillin non-susceptibility was also observed within this serotype. Recently, a genetic variant of 23B, named 23B1, was discovered, which is known to be related to decreased penicillin susceptibility. We showed that increases in 23B prevalence in healthy carriage and IPD cases always coincided with 23B1 expansions, leading to higher penicillin non-susceptibility rates. Increases in 23B in the day care population paralleled pediatric 23B IPD increases, indicating the vital role of day care monitoring of pneumococcal carriage. Countries should stay vigilant for prevalence increases in S. pneumoniae serotype 23B, given the decreased susceptibility to penicillin and co-trimoxazole of the 23B1 variant.

Determination of antimicrobial resistance (AMR) in pneumococcal isolates is important for surveillance purposes and in a clinical context. Antimicrobial susceptibility testing (AST) of pneumococci is complicated by the need for exact minimal inhibitory concentrations (MICs) of beta-lactam antibiotics. Two next-generation sequencing (NGS) analysis tools have implemented the prediction of AMR in their analysis workflow, including the prediction of MICs: Pathogenwatch ( https://pathogen.watch/ ) and AREScloud (OpGen). The performance of these tools in comparison to phenotypic AST following EUCAST guidelines is unknown. A total of 538 Streptococcus pneumoniae isolates were used to compare both tools with phenotypic AST for penicillin, amoxicillin, cefotaxime/ceftriaxone, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline. Disk diffusion was performed for all isolates, and broth microdilution was performed for isolates with reduced beta-lactam susceptibility. Demultiplexed FASTQ files from Illumina sequencing, covering the whole genome of pneumococci, were used as input for the NGS tools. Categorical agreement (CA), major error (ME), and very major error (VME) rates were calculated. For beta-lactam antibiotics, CA was high (>94%) associated with none or only one ME and VME (93% for predictions by AREScloud, while for Pathogenwatch, this ranged around 88%. For trimethoprim-sulfamethoxazole, CA was for both tools <86%. High VME rates were observed for erythromycin and tetracycline, higher for Pathogenwatch (53.6% and 47.0%, respectively) compared to AREScloud (14.3% and 19.1%, respectively). Both tools performed excellently despite the complexity of predicting beta-lactam resistance in pneumococci. Further optimization and validation are needed for non-beta-lactams since high (very) major error rates were observed.

Purpose Bacterial isolation is associated with worse outcomes after lung transplantation (LTx), and successful bacterial eradication is shown to improve long-term survival and pulmonary function. Outpatient Parenteral Antibiotic Therapy (OPAT) may be an effective therapeutic modality for bacterial eradication post-LTx. Methods A single-center, retrospective analysis of OPAT characteristics, efficacy, safety, and costs in non-cystic fibrosis LTx recipients. Results A total of 156 OPAT courses (from June 2019 to December 2022) were evaluated in 108 distinct LTx recipients. OPAT mainly consisted of dual antibiotic therapy (69%) for pulmonary bacterial isolation (97%), mostly Pseudomonas aeruginosa (66%). Successful eradication at 3 months post-OPAT was achieved in 71%. Eradication rate was significantly higher in patients treated after the first post-operative year (79%), compared to patients within the first year (61%) (p = 0.017). Eradication rate was similar for multidrug resistance (eradication rate 61%) versus no multidrug resistance (74%) (p = 0.116). Spirometry remained stable at 90 days post-OPAT. A statistically significant, but clinically negligible, increase in serum creatinine at 90 days post-OPAT was observed (1.33 mg/dL vs. 1.39 mg/dL, p < 0.001), yet unrelated to the antibiotic regimen used. OPAT-related hospital admissions occurred in 13% and line-related adverse events in 6%. Median number of hospitalization days saved per OPAT-course was 10 days (range 2–92), accounting for a total of 1841 avoided admission days and an estimated net cost reduction of 47% per treatment course. Conclusion OPAT is an effective and safe therapeutic modality for bacterial eradication post-LTx, associated with a significant reduction in hospitalization days and treatment costs.

Introduction Older adults face a higher risk of vancomycin-related toxicity given their (patho)-physiological changes, making early management of supratherapeutic exposure crucial. Yet, data on vancomycin exposure in older adults is scarce. This study aims to compare vancomycin concentrations between older and younger patients, emphasizing supratherapeutic concentrations and the effect of patient characteristics. Methods This observational retrospective study was conducted in the University Hospital of Leuven (EC Research S65213). We analyzed early (first) vancomycin concentrations between older (≥ 75 years) and younger patients. Multivariable analyses were conducted to evaluate the association between baseline patient characteristics with supratherapeutic exposure (logistic regression), and dose-normalized concentrations (linear regression). Results We included 449 patients aged ≥ 75 years (median 80) and 1609 aged < 75 years (median 61). In univariable analysis, the first-measured vancomycin concentrations were significantly higher in older adults (p < 0.001), who more frequently reached supratherapeutic concentrations (30.7% versus 21%; p < 0.001). In multivariable analysis, factors associated with supratherapeutic concentrations were decreased the estimated glomerular filtration rate calculated by using the Chronic Kidney Disease Epidemiology Collaboration equation (eGFR_CKD-EPI) [odds ratio (OR) of 0.98, confidence interval (CI) 0.97–0.98]. Supratherapeutic concentrations had inverse association with giving lower loading dose (OR of 0.59, CI 0.39–0.90), and lower maintenance dose (OR of 0.45, CI 0.26–0.77). Factors that predicted increased dose-normalized concentrations included decreased eGFR_CKD-EPI (coefficient of −0.05, CI −0.06 to −0.04), lower body weight (coefficient of −0.04, CI −0.05 to −0.03), increased blood urea nitrogen (coefficient of 0.02, CI 0.01–0.03), and delayed time to therapeutic drug monitoring (TDM) sampling (coefficient of 0.08, CI 0.06–0.09). Conclusions The absence of age as a significant factor in the multivariable analysis suggests that eGFR_CKD-EPI mediated the relationship between age and vancomycin exposure. Older adults may benefit more from vancomycin TDM.