Diagnostic Microbiology and Infectious Disease

Background: Pharmacogenomic point-of-care testing (PGx POCT) enables genotype-informed prescribing at the bedside, but implementation is limited by the lack of suitable reference materials and standardised external quality assessment (EQA). Methods: Fixed lymphoblastoid cell lines were formulated as swab-based materials at defined cellular inputs for MTRNR1 POCT. Materials were evaluated using Genedrive MTRNR1 assays, quantitative PCR, acceptance quality limit batch release, workflow perturbation testing and stability studies, then deployed in an international EQA programme. Results: Materials were homogeneous, workflow-compatible and reproducible, with 100% correct genotype calls at the lowest intended manufacturing cell load. Seven EQA surveys yielded 253 participant results, with full-mark rates between 87.5 and 100%, and six test failures. Most failures occurred with low cell-input materials near the assay limit of detection. The materials supported genotype concordance, performance monitoring and identification of workflow and sample-related variability. Conclusions: Workflow-compatible cell-based reference materials combined with EQA provide a scalable framework for quality-assured PGx POCT beyond specialist laboratories. International MT-RNR1 PGx POCT EQA programme deployment demonstrated feasibility in decentralised settings. The framework may provide a model for the implementation, verification and ongoing quality assessment of PGx POCT. Further evaluation across platforms, analytes, settings and participant groups is required to establish broader applicability.

Sepsis is a substantial contributor to neonatal mortality in Africa, with Klebsiella pneumoniae as the leading causative pathogen. Incorporating molecular tools such as whole genome sequencing (WGS) into routine surveillance of important bacterial pathogens, including those causing neonatal sepsis, can support targeted deployment of infection prevention and control measures and treatment interventions. This study aimed to understand the current practices and capacity for culture, identification and genomic analysis of bacterial pathogens in Africa, with a particular focus on K. pneumoniae, to support standardised genomic surveillance. A cross-sectional online survey was distributed to various clinician and scientist networks across Africa. All respondents completed a demographics section, while sections on laboratory test capacity, blood culture practices, WGS, and bioinformatics analysis were completed based on experience in these aspects. Responses were received from 52 institutions in 25 African countries. Among 51 institutions completing the laboratory test capacity section, 61% reported performing blood cultures, with the majority using manual commercial biochemical systems for species identification (71%) and manual disk diffusion for antibiotic susceptibility testing (82%). Participating institutions reported near-universal access to a -80{degrees}C freezer, centrifuge and thermocycler, but shaking incubators were less commonly available (67%). More than half of respondents (54%) indicated that they perform the entire WGS workflow (including DNA extraction, library preparation and WGS) internally, with most performing DNA extraction internally (88%) using column-based kits (70%). Outsourcing was progressively more common at later workflow stages. Infrastructure challenges that limit bioinformatics analysis capabilities were reported by all 23 respondents who completed this question, including limited access to high-performance computing and data storage space. This survey found sufficient capacity to support genomic surveillance of neonatal sepsis pathogens across participating African institutions. However, investments in blood culture, supply chains, sequencing, bioinformatics, and data dissemination capacity are needed to realise its full potential. These findings will support the development of standardised genomic surveillance protocols tailored to the diverse capacities of African laboratories.

Objectives: Capturing adequate blood volume for blood cultures is critical for accurate detection of bloodstream infections. Pediatric volume targets vary by age and weight, whereas adult targets are standardized. The BD BACTEC FXI Culture System (FXI) contains an integrated calibrated load cell capable of automatically reporting blood volume measurements for each vial loaded onto the system. This study evaluated the accuracy of the FXI's blood volume measurements in simulated pediatric and adult patients. Methods: Mock pediatric and adult blood draws were performed, using bagged whole blood, to replicate real-world collection protocols. Syringe-collected blood volumes ranged from 2.0 to 15.0 mL for pediatric patients, depending on mock patient weight, and were fixed at 40.0 mL for adults. Samples were inoculated into BD BACTEC Peds Plus/F, Plus Aerobic/F, and Lytic/10 Anaerobic/F Culture Vials, with a target volume of 2.0 to 10.0 mL per bottle. Reference blood volumes were determined gravimetrically using manually obtained pre- and post-inoculation weights with a blood-specific gravity of 1.055 g/mL and were compared to the automatically measured, gravimetric-based blood volumes reported by the BACTEC FXI Culture System. Results: Automated volume estimates were accurate to a mean error of -0.03 mL per bottle (SD, 0.40 mL; n=168; 95% CI, -0.09 mL, 0.03 mL) and -0.08 mL (SD, 0.79 mL; n=72; 95% CI, -0.26 mL, 0.10 mL) when assessing total volume collected per patient. Conclusions: Our findings demonstrate that the automated system can quantify blood volumes in BACTEC culture vials and support blood volume monitoring for pediatric and adult collections. The gravimetric approach is also amenable to full automation for efficient and accurate blood volume determination.

Background: Rapid genome sequencing (rGS) is a first-tier test in the neonatal intensive care unit (NICU). Implementation strategies to scale rGS for clinical care may change clinician roles. We analyzed the NICU rGS workflow to understand challenges rGS poses for clinical roles and responsibilities. Methods: The study took a qualitative approach to understanding the rGS workflow in Level III and Level IV NICUs. The five workflow phases studied included: i) identification of a patient with a suspected genetic disorder, ii) subspecialist consultation, iii) test selection and ordering, iv) return of results, and v) disclosure. Data were collected via job task journals, interviews, and workflow diagram construction with clinicians involved in rGS. Analysis included participant-guided workflow diagram refinement, qualitative thematic analysis, and identification of rGS workflow challenges. Results: We documented issues related to decision-making authority and scope of responsibility for clinicians, with the majority relating to complexities of subspecialist consultations. Issues included differences in: criteria for triggering consultations, expectations of consulted subspecialists' roles, test selection preferences, and responsibility for documenting clinical interpretations and disclosure. Conclusion: Subspecialist consultations in clinical rGS involved significant role ambiguity and communication challenges, requiring intentional implementation efforts to ensure clear clinical roles and responsibilities.

Background Best practice alerts (BPAs) used in emergency departments (EDs) to identify patients with possible sepsis lack specificity and some ED patients with sepsis BPAs are discharged. We evaluated factors associated with ED discharge following a sepsis BPA and with subsequent 7-day return admissions with sepsis or infection. Methods We conducted a retrospective cohort study using an electronic health record data query from five hospitals (2022-2024). Using logistic regression, we evaluated the association of participant demographics and clinical characteristics with ED discharge and factors at the initial ED visit associated with return admission. Results Among 62,068 ED visits with a sepsis BPA, one third resulted in ED discharge. Discharged participants were younger and more often female, Black, multiracial, and/or Hispanic; they more often had an initially normal pulse, respiratory rate, blood pressure, and lactic acid than admitted participants. Discharged participants more often had a fever on arrival (adjusted odds ratio (aOR) 6.03; p<0.001) and a white blood cell (WBC) count 11,000-14,999 (aOR 4.57; p<0.001) or 15,000-19,999/uL (aOR 2.01; p<0.001). One percent of discharged participants experienced a 7-day return admission with a diagnosis of sepsis or infection, which was associated with older age, White or Asian race, diastolic hypotension, WBC count [&le;]15,000/uL or <4,000/uL, and an index visit BPA response indicating sepsis or non-response. Conclusions Return admissions among discharged ED patients with a sepsis BPA are rare. Several factors are associated both with hospital admission at the index visit and return admission, indicating a need for additional risk-stratification tools.

Candida auris is an emerging multidrug-resistant fungal pathogen associated with healthcare-associated outbreaks, persistent colonization, and invasive infections. Increasing demand for surveillance has created a need for high-throughput methods capable of supporting large-scale screening programs. We developed and validated an automated laboratory-developed real-time PCR assay for detection of C. auris colonization on the Hologic Panther Fusion(R) open-access platform and compared its performance with the existing BD MAX assay. Analytical performance was evaluated by assessing limit of detection, accuracy, precision, specificity, inclusivity, reproducibility, and reagent and specimen stability. The Panther Fusion(R) assay demonstrated a limit of detection of approximately 18 CFU/reaction and showed 97% overall agreement with the BD MAX assay. Positive and negative percent agreement were 94% and 100%, respectively, with excellent agreement between methods ({kappa} = 0.94). No cross-reactivity was observed with non-C. auris organisms, all five major C. auris clades were detected, and assay performance remained stable across operators, reagent and specimen storage conditions. Following implementation, 26,838 clinical specimens were tested on the Panther Fusion(R) platform. Retrospective analysis demonstrated lower equivocal (0.28%) and indeterminate (0.09%) rates than those observed on the ABI and BD MAX platforms. Among PCR-positive specimens that underwent culture, the Panther Fusion(R) assay demonstrated 87.24% culture agreement. Because retrospective data were collected during different testing periods and patient populations, comparisons between platforms were not used to assess relative assay sensitivity or specificity. Implementation of the Panther Fusion(R) assay increased surveillance testing capacity from approximately 88 to 500 specimens per shift while maintaining robust analytical performance.

GeneCapture is developing a novel, point-of-care, rapid AST technique that, when fully automated, can accomplish AST within 2 hours directly from a raw sample, representing a significant advancement in diagnostic capabilities. For this study, a panel of uropathogen isolates was collected from clinical specimens positive for urinary tract infection from various laboratories in the southern US. A selection of these isolates were then spiked into fresh clean-catch urine samples to produce mock samples covering a variety of antibiotic responses. Testing was performed on 20 strains of Escherichia coli, the cause of over 80% of UTIs, and 6 strains each of Klebsiella pneumoniae and Proteus mirabilis, the next most common causes. Each strain was tested against the susceptible, intermediate and resistant (S-I-R) breakpoints of up to 5 appropriate antibiotics including Amoxicillin-Clavulanate, Cefpodoxime, Ciprofloxacin, Nitrofurantoin and Trimethoprim-Sulfamethoxazole. The new method uses a phenotypic assessment of bacterial growth, comparing responses from bacteria treated with antibiotics at S-I-R breakpoint concentrations to untreated controls as the metric for determining susceptibility. Exposure to the drugs for only 90 minutes gave a Categorical Agreement of 98% to gold standard culture results across the resulting 247 bacteria/antibiotic pairs. Testing on separate days of nine of these strains in triplicate gave a reproducibility of 97%. The sequential steps of this novel AST - sample processing, growth, labeling, and counting - can readily be performed on an automated cartridge that is currently under development. The presented work, which took place in a laboratory setting, demonstrates the efficacy of the assay.

Background: During emergency tracheal intubation, clinicians use waveform capnography or colorimetric carbon dioxide (CO2) detection to confirm exhaled CO2 from the endotracheal tube to determine its location. Both devices reliably yield a negative result when the tube is in the esophagus. However, in most emergency intubations the tube is correctly located in the trachea, and in this circumstance the relative diagnostic accuracy of the two devices is uncertain. If one device more often fails to detect exhaled CO2 despite correct tube placement (a false negative result), this could lead to unnecessary tube removal, repeated intubation attempts, and an increased risk of serious complications. Research Question: Among critically ill adults undergoing emergency tracheal intubation, is waveform capnography or colorimetric CO2 detection more sensitive for identifying an endotracheal tube correctly placed in the trachea? Study Design and Methods: The WAVE study is a pragmatic, prospective, paired diagnostic accuracy study conducted in emergency departments and intensive care units in the United States. The study compares waveform capnography and colorimetric CO2 detection (index tests) against a non-CO2-based reference standard for endotracheal tube location among 2,092 critically ill adults undergoing tracheal intubation. The primary outcome is the sensitivity of each index test for identifying a tube located in the trachea. The secondary outcome is specificity for identifying a tube located outside the trachea. Results: Enrollment began on April 21, 2025 and is ongoing, with completion anticipated in 2026. Interpretation: The WAVE study will provide prospective evidence comparing waveform capnography with colorimetric CO2 detection for determination of endotracheal tube location in critically ill adults. These findings may inform clinical practice and guideline recommendations regarding the optimal test of exhaled CO2 for use in the ED and ICU. Prespecifying the protocol and statistical analysis plan before completion of enrollment enhances the rigor, reproducibility, and transparency of the study.

Accurate discrimination between viral, bacterial, and inflammatory diseases in febrile children remains a major clinical challenge that contributes to diagnostic uncertainty, inappropriate antimicrobial use, and suboptimal clinical management. Host blood transcriptomics offer a promising strategy to improve diagnostic precision. The present study represents the largest integrative multi-cohort pediatric study of transcriptomic biomarker discovery, validation, and confirmation reported to date, integrating harmonized public transcriptomic datasets with an independent confirmation cohort comprising well-phenotyped patients to identify parsimonious host-response signatures for differentiating viral, bacterial, and inflammatory diseases. Transcriptomic signatures were derived from an integrated retrospective microarray multi-cohort (n=1,683), independently validated in a retrospective RNA-seq cohort (n=767), and confirmed by digital PCR in an independent cohort (n=29), demonstrating reproducibility across patient populations, transcriptomic technologies, and analytical platforms. The analysis identified binary signatures and a unified multiclass classifier that consistently achieved high diagnostic accuracy across all three study phases and outperformed more than 30 published host transcriptomic signatures. Decision curve analysis showed substantially greater clinical net benefit than C-reactive protein across clinically relevant decision thresholds. These findings provide a strong foundation for clinically deployable molecular diagnostics to improve patient triage, antimicrobial stewardship, and precision medicine in childhood infections.

Background: Surveillance tells laboratories which antimicrobial resistance genes are circulating now, but not which will change next. We built a model that forecasts, for each resistance gene and year, whether the following year brings a previously unseen allele (sequence variant), called emergence, or a change in the most common allele, called turnover. We tested it separately in Klebsiella pneumoniae and Acinetobacter baumannii. Results: We used genomes annotated with AMRFinderPlus: 2,385 gene-years across 331 genes for K.pneumoniae and 1,059 across 216 for A. baumannii. To remove the effect of sequencing effort, each gene-year was sampled to 10 records and the analysis repeated 25 times. Models were trained on earlier years and tested on later ones, and on genes held out of training. On later years every model ranked genes by risk better than simple baselines in both organisms, and results for held-out genes were close. In K.pneumoniae, turnover was predicted better than emergence (best area under the ROC curve, AUROC, 0.909 versus 0.819). In A. baumannii the order reversed (emergence 0.789, turnover 0.722). The two events also relied on different features. Emergence depended mainly on how many alleles a gene had already accumulated, in both organisms. Turnover in K. pneumoniae depended mainly on how dominant the leading allele already was, and a stability index added a small gain. In A. baumannii it was spread across many weaker features. There, 56% of changes in the leading allele involved only alleles already seen, against 23% in K. pneumoniae, which may make them harder to predict. Conclusions: Changes in resistance genes can be forecast a year ahead from routine genomic surveillance data, including for genes the model has not seen. How well it works, and what it depends on, differs between organisms, so models should be built and validated for each organism, not assumed to transfer. Such models could help laboratories decide which resistance genes to monitor most closely. Keywords: antimicrobial resistance; genomic surveillance; forecasting; machine learning; allele dynamics; Klebsiella pneumoniae; Acinetobacter baumannii

The 2026 Bundibugyo ebolavirus (BDBV) outbreak underscores the need for rapidly deployable molecular diagnostics. We developed and analytically validated reverse-transcription quantitative PCR assays detecting BDBV, Zaire ebolavirus, and Sudan ebolavirus. The platform includes a BDBV singleplex assay, a duplex assay with a human internal control, a four-target multiplex assay for ebolavirus differentiation, and a probe-free SYBR Green assay. We adapted the assays to a portable qPCR instrument, reducing runtime from 65 to 35 minutes, and validated lyophilized reagents to reduce cold-chain requirements. All TaqMan formats achieved a 95% limit of detection of 5 copies per reaction across instruments and reagent types; the SYBR Green assay achieved 50 copies per reaction. The assays detected viral RNA in contrived clinical samples without cross-reactivity among ebolavirus species tested. We shared the protocols in real time through Ampliphi (https://www.ampliphi.bio), a new open-access platform for rapidly disseminating diagnostic assays, and through protocol.io.

BACKGROUND: Conventional bronchoalveolar lavage (BAL) culture has limited sensitivity in lung transplant recipients due to universal antimicrobial prophylaxis and inability to detect non-culturable organisms. Metagenomic sequencing of BAL offers culture-independent pathogen identification, but concordance with conventional culture and clinical utility in the immediate post-transplant period have not been evaluated. METHODS: We prospectively enrolled 19 adult lung transplant recipients undergoing serial bronchoscopies during the immediate post-transplant period. BAL samples (n=33) were tested in parallel with conventional culture, microbial cell-free DNA (mcfDNA) sequencing (Karius Focus BAL; with quantitation determined by research analysis), and Oxford Nanopore sequencing using a unified three-tier organism classification framework. BAL host-response biomarkers were profiled concurrently. RESULTS: Karius reported Tier 1 pathogens in 19/33 episodes (58%) versus 8/33 by conventional culture (24%); organism-level concordance was low (Cohen's kappa 0.18). In 16 episodes where Karius reported a pathogen not recovered by culture, 8 across 3 participants represented predictive reports, defined as the same organism subsequently confirmed as causing invasive infection 2-46 days later, including fatal Pseudomonas pneumonia, Enterococcal surgical site infection, and Candidemia. Nanopore sequencing had low yield due to contaminating human DNA. Quantitative mcfDNA burden correlated positively with multiple alveolar inflammatory biomarkers including total protein, IL-6, and sST2. INTERPRETATION: BAL mcfDNA metagenomics identified clinically relevant pathogens not recovered by conventional culture, including predictive reports and non-culturable organisms. Quantitative mcfDNA signal correlated with alveolar inflammatory mediators, supporting the biological relevance of mcfDNA detection beyond conventional diagnostic classification and a role for metagenomic surveillance in the early post-transplant period.

Context: Cytological specimens allow minimally invasive, repeated sampling for diagnosis and molecular testing. However, cytology biobanking protocols have primarily been optimized for morphology, and the effects of preanalytical handling and storage on intracellular proteins remain unclear. Objective: To assess how fixation, staining, storage temperature, and duration affect intracellular protein preservation in cytological specimens and to identify conditions suitable for in situ proximity ligation assay (isPLA). Design: Human cancer cell lines were processed under different preanalytical conditions. Intracellular proteins and protein protein interactions were assessed by isPLA, fluorescence microscopy, and quantitative image analysis. Slides were stored at room temperature or minus 20 Celsius, with or without MayGrunwaldGiemsa (MGG) staining. Results: Storage temperature had the greatest effect on protein signal. Unstained slides stored at minus 20 Celsius retained strong signals for up to 24 months, whereas room temperature storage reduced detectable protein interactions by approximately 95%. MGG staining further reduced signal intensity during prolonged storage. Nevertheless, stained slides remained suitable for isPLA when staining, microscopic evaluation, destaining, and antibody labeling were completed on the same day. Prolonged room temperature storage of stained slides caused loss of antigenicity, increased autofluorescence, and false negative findings. Conclusions: Low temperature storage and standardized preanalytical handling preserve intracellular protein signals in cytological specimens. These conditions support reliable single cell protein profiling of archived samples and their use in biomarker research and translational pathology.

BackgroundFebrile neutropenia requires rapid empirical antibiotic therapy, with regimen selection shaped by local prescribing practice and pathogen patterns. We described starting treatment, later drug changes, microbiological findings, and inpatient outcomes. MethodsThis ambispective cohort comprised 96 episodes from 90 patients admitted to a tertiary-level teaching hospital. Treatment and outcome analyses used each episode as the unit of observation. Recorded variables included the starting antibiotics, later regimen modification, microbiology, hospitalization length, invasive fungal infection (IFI) as captured in study records, and disposition. Comparisons based on escalation status were exploratory. ResultCefoperazone-sulbactam appeared in 62/96 initial regimens (64.6%), amikacin in 42 (43.8%), teicoplanin in 28 (29.2%), and meropenem in 14 (14.6%). Eighteen episodes (18.8%) required a change that broadened or substituted antibacterial therapy. Among 31 microbiologically documented infections, 22 (71.0%) yielded Gram-negative organisms and 9 (29.0%) were polymicrobial. Median length of admission was 18.5 days in the escalation group and 9 days in the remainder (p<0.001). Mortality was 27.8% (5/18) with escalation and 5.1% (4/78) without it (p=0.011), with nine inhospital deaths overall (9.4%). ConclusionAntibacterial management differed within this cohort, with treatment escalation required in 18 of 96 episodes. Gram-negative organisms comprised most microbiologically confirmed infections. Escalation marked a clinically complex course with prolonged hospitalization and greater observed mortality; the observational design does not establish comparative efficacy or causation.

BackgroundAztreonam-avibactam (AZA) is a recently approved agent active against metallo-{beta}-lactamase-producing Enterobacterales. However, independent evaluations of commercially available antimicrobial susceptibility testing (AST) products remain limited. MethodsWe evaluated 56 carbapenem-resistant Enterobacterales isolates using four newly available AZA AST products, including two gradient diffusion (bioMerieux Etest; Liofilchem MIC Test Strip) and two disk diffusion (Hardy, Liofilchem) tests across three Mueller-Hinton agar manufacturers at two independent clinical laboratories. A diverse isolate collection enriched for nonsusceptible and breakpoint-adjacent MICs was used to rigorously assess categorical agreement. Broth microdilution (BMD) was used as the reference method. Essential agreement (EA), categorical agreement (CA), error rates, and reproducibility were analyzed. ResultsAll four commercial AST products demonstrated acceptable analytical performance compared with reference BMD. Gradient diffusion demonstrated high EA (bioMerieux 94.3%, Liofilchem 90.5%), whereas CA ranged from 83.3% to 89.0% across all four products. Minor errors accounted for all categorical discrepancies, and occurred almost exclusively among breakpoint-adjacent isolates. Media-related variability was modest, and quality control performance was generally high, with strong within-site precision and consistent performance across laboratories. ConclusionsThis study provides the first comprehensive head-to-head comparison of commercial AZA susceptibility testing products and supports their implementation in clinical microbiology laboratories.

BackgroundDespite the importance of oropharyngeal gonorrhoea in transmission, suboptimal antimicrobial responses and propensity for horizontal transfer of antimicrobial resistance at this site, it remains understudied. An oropharyngeal N. gonorrhoeae controlled human infection model (CHIM) represents a promising tool to study infection and undertake translational research. MethodsA panel of five contemporary N. gonorrhoeae isolates were subject to detailed characterisation to assess antimicrobial susceptibility, in vitro infectivity, cytotoxicity and serum sensitivity to inform challenge agent selection. A method for challenge agent manufacture, including release testing, was developed and validated. FindingsAll candidate isolates were able to infect the surface of pharyngeal and cervical cells in vitro. One isolate displayed an invasive phenotype, induced higher inflammatory cytokine production and displayed elevated serum resistance and was excluded. The remaining four isolates were minimally inflammatory, did not induce cytotoxicity and were susceptible to serum killing. Three of the four isolates grew in a defined liquid medium. Together these results led to the selection of a contemporary N. gonorrhoeae isolate suitable for use in CHIM. A challenge agent manufacture workflow was established and shown to reliably and reproducibly generate doses suitable for direct inoculation in an oropharyngeal CHIM. ConclusionPhenotypic characterization of candidate N. gonorrhoeae challenge agents led to the successful identification of a contemporary isolate suitable for implementation in a novel oropharyngeal gonorrhoea CHIM. We demonstrate the feasibility of a challenge inoculum manufacturing process that aligns with international best practice guidelines.

Hybrid capture approaches can detect a broad range of pathogens directly from clinical samples with greater sensitivity than untargeted approaches. Clinical adoption has been limited by long turnaround times, complex workflows, and perceived high costs. We iteratively developed and validated a rapid hybrid capture workflow based on Illumina's RPIP protocol (standard workflow). Rapid-RPIP v1 reduced wet-lab time from ~11.4 to ~7.3 h, for a turnaround time (TAT) of <15 h. Rapid-RPIP v2 further reduced wet-lab time by ~0.7 h and used Illumina Turbo Sequencing shortening the sequencing time from ~5.5 to ~2.5 h thus achieving a TAT of <10 h without compromising sensitivity and yielding higher RPKMs for RNA viruses than the standard workflow. This approach could be readily adapted to a broad range of clinical and public health applications.

Background Antimicrobial-resistant Gram-negative bacteria pose a major threat to global health. In the WHO Bacterial Priority Pathogen List (BPPL) 2024, several species were classified as critical or high priority for research and development of new antimicrobial therapeutics and diagnostics. However, access to well-characterised and clinically relevant bacterial isolate panels remains an unmet need for development and validation. Methods We constructed five panels of Gram-negative bacterial isolates, cultured from samples originally referred to the UK Health Security Agency Antimicrobial Resistance and Healthcare-Associated Infections Reference Unit for analysis between 2014 and 2025. For each isolate, antimicrobial susceptibilities were determined against a range of clinically relevant antibiotics, including third-generation cephalosporins, carbapenems, colistin and other last-line antibiotics. WGS was performed on all isolates, and previously described resistance determinants were characterised. Results The five panels consist of 145 isolates and represent 81 sequence types across Escherichia coli (30 isolates), Klebsiella pneumoniae (30 isolates), carbapenem-resistant Acinetobacter baumannii (30 isolates), carbapenem-resistant Pseudomonas aeruginosa (30 isolates), and a mixed panel of other healthcare-associated Enterobacterales species (25 isolates). Panels are highly representative of globally relevant strains, including those currently in circulation and on the WHO BPPL 2024, and of resistance mechanisms of public health importance. Conclusion The panels provide a diverse, comprehensive set of multidrug-resistant Gram-negative bacterial isolates, representative of strains currently in circulation globally. Selected isolates carry a breadth of important resistance mechanisms and are highly relevant to the current epidemiological landscape. The isolates and associated metadata are available to industry, academia, and other laboratories for use in developing novel antimicrobial compounds and diagnostic assays.

Nucleic acid extraction remains the principal infrastructure barrier to molecular influenza testing outside centralised laboratories, since bead-based purification is normally tied to mains-powered extractors and trained operators. We evaluated SmartLid, a centrifugation-free format in which a removable magnetic key shuttles paramagnetic beads through pre-aliquoted lysis/binding, wash, and elution buffers without pipetting or powered instrumentation, against an automated magnetic-bead extractor (Nextractor NX-48S) on 311 nasopharyngeal specimens from the 2024-2025 influenza season at a National Influenza Centre. Paired eluates were amplified under identical monoplex RT-qPCR conditions for influenza A(H1N1)pdm09, A(H3), and B/Victoria. Both methods gave 100% specificity (47/47 negatives; no false positives). Subtyping succeeded in 263/264 reference-positive specimens after SmartLid extraction versus 241/264 after automated extraction (99.62% versus 91.29%; difference 8.33 percentage points; discordant pairs 23 versus 1; McNemar P < 0.001). Across 240 complete pairs, cycle threshold (Ct) values were lower after SmartLid extraction (median paired difference -2.78 cycles; estimated location shift -2.60 cycles, 95% CI -2.82 to -2.37; P < 0.001) with rank-ordering of specimens conserved between methods (Spearman rho = 0.84). The advantage was preserved across all three subtypes and in both fresh and frozen specimens (adjusted P < 0.001). Specimens recovered only after SmartLid extraction had higher Ct values than dual-detected specimens (median 34.37 versus 28.54; P < 0.001), locating the gain near the assay detection limit. An instrument-free manual format can therefore exceed the extraction efficiency of an automated reference workflow, extending quality-assured influenza subtyping beyond centralised laboratories.

Objectives: To investigate whether ethambutol resistance in Mycobacterium avium complex is associated with the emergence of macrolide resistance. Methods: Patients who developed macrolide resistance during guideline-based treatment were included, and longitudinal analyses of minimum inhibitory concentrations and mutations in embB or the upstream region of embA were performed. Clinical, microbiological, and radiological characteristics were compared according to the mutation status of embB or embA upstream region, prior to the emergence of macrolide resistance. We further evaluated the impact of embB mutation on the development of macrolide resistance using in vitro time-kill assays. Results: Sixteen patients developed macrolide resistance during guideline-based treatment. None of these patients had an ethambutol minimum inhibitory concentration >=16 ug/mL or embB or embA upstream mutations at treatment initiation; however, 8/16 patients (50.0%) had an ethambutol minimum inhibitory concentration >=16 ug/mL at the time of macrolide resistance detection, and 7/16 (43.8%) had developed embB or embA upstream mutations prior to the emergence of macrolide resistance. Cavitary lesions were present in 1/7 (14.3%) patients with embB or embA upstream mutations. In strains with embB mutations, the minimum inhibitory concentration of ethambutol increased by 1-2 dilutions relative to that of pretreatment isolates, with a corresponding increase in the concentration required to suppress macrolide resistance. Conclusions: Ethambutol resistance may contribute to the development of macrolide resistance in patients with M. avium complex pulmonary disease, particularly in those without cavitary lesions.