European Journal of Clinical Microbiology & Infectious Diseases

BACKGROUNDAutomated antimicrobial susceptibility testing (AST) systems are crucial for accurate, timely detection of drug-resistant microbial isolates. This meta-analysis assessed the performance of the BD Phoenix ("Phoenix", BD Diagnostic Solutions), Vitek(R) 2 ("Vitek 2", bioMerieux), and DxM MicroScan WalkAway ("MicroScan", Beckman Coulter, Inc.) AST systems relative to common reference methodology. METHODSA systematic literature search in Ovid (MEDLINE and Embase) yielded 275 unique (not duplicated) records, with 44 additional records retrieved from handsearching; 39 studies met inclusion criteria. Categorical agreement (CA), essential agreement (EA), very major errors (VMEs), and major errors (MEs) for the three instruments were compared to a common reference method. Ratios of proportions were analyzed using random-effect meta-regression. RESULTSThe instruments did not differ significantly in CA, EA, or ME. Vitek 2 showed a higher overall VME rate than Phoenix ([~]44% higher; Vitek 2-to-Phoenix ratio = 1.44; p=0.062 [approaching significance]) and MicroScan (74% higher; ratio = 1.74; p=0.045). No appreciable difference was observed for VME between Phoenix and MicroScan. Subgroup analyses should be interpreted cautiously due to limited overall significance indicating varying performance across systems. Vitek 2 generally had higher relative VMEs for gram-negative organisms and lower relative VMEs for gram-positive organisms, whereas Phoenix showed the opposite pattern. MicroScan had relatively low VMEs when stratified by Clinical and Laboratory Standards Institute (CLSI) criteria; no differences in VMEs were observed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. CONCLUSIONAlthough some VME differences were noted, overall performance of the three systems was comparable. Organism- and drug-specific VME patterns--and updates to CLSI criteria over time--highlight the importance of continued monitoring of current breakpoints for all three instruments.

Nasopharyngeal (NP) swabs remain the dominant gold standard for respiratory infection diagnostics. While there has been increased use of alternative sample types since the COVID-19 pandemic, guidance on their use for detecting respiratory viruses is not yet definitive, especially for children. In this systematic review and meta-analysis, we aimed to compare the diagnostic accuracy and tolerability of multiple respiratory specimen types for detecting respiratory viruses in pediatric populations. Searches were conducted on July 17, 2025 in MEDLINE, Embase, Web of Science, and Scopus, with screening and data extraction performed in Covidence. English-language primary research articles published since 2000 comparing respiratory virus detection rates in children, using nucleic acid amplification tests between paired respiratory specimens, were included. Risk of bias was assessed using Quality Assessment of Diagnostic Accuracy Studies criteria. We calculated pooled sensitivities and specificities of index specimens: nasopharyngeal aspirates (NPA), mid-turbinate swabs (MT), anterior nasal swabs (ANS), oropharyngeal swabs (OP), and bronchoalveolar lavage fluid (BAL), as compared to the reference, NP swabs, using random-effects modeling, firstly without discrimination by virus. Index specimens were then grouped by sample collection site as nasal, oral, and lower respiratory tract (LRT) specimens for virus-specific analyses. Overall performance and statistical validity were evaluated by hierarchical summary receiver operating characteristic (HSROC) analysis. Data regarding sampling tolerability was also assessed. We screened 2,448 studies and identified 36 publications (total N participants = 10,687) that reported diagnostic test accuracy using paired index-reference data in children. Of these, 18 (total N participants = 4,310) used NP specimens as the reference and were included in the diagnostic test accuracy analysis. Virus-agnostic pooled sensitivity estimates indicated that MT (0.92%) performed most similarly to NP, though sensitivities of ANS (0.79%) and OP (0.70%) were also moderately high for detection of any respiratory virus. BAL sensitivity was the lowest (0.37%). All sample types demonstrated high specificity (0.98%-0.99%). Group estimates and HSROC statistics found that nasal specimens, when grouped, had the highest sensitivity and accuracy for all examined viruses, including for influenza (92%) and RSV (90%). By comparison, oral and LRT specimens performed less well, with more variability, though both showed moderately high sensitivities for RSV (78%, 76%, respectively) and influenza (82%, 80%, respectively), and LRT samples showed high sensitivity for HMPV (82%). Analysis of sample tolerability found that NP swabs consistently ranked as the least comfortable and least preferred, while nasal swabs and saliva both performed well. Datasets for LRT and oral specimens were sparser than for nasal, and this contributed to greater variability, underscoring the need for further diagnostic accuracy studies on alternatives to NP sampling. These data support the viability of nasal and oral alternatives to NP swabs and affirm their application in pediatric care, particularly in outpatient settings. Such alternatives could greatly improve sampling tolerability and increase global access, including in resource-limited settings, to accurate diagnostic methods for respiratory infections.

BackgroundAntimicrobial resistance (AMR) surveillance is essential for quantifying and monitoring the burden of AMR among World Health Organization (WHO) priority pathogens. We analysed Tunisian AMR surveillance system (TARSS) data across five sentinel hospitals from 2014 to 2022. MethodsWe conducted a retrospective isolate-level analysis for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp. Temporal, ward, and specimen associations were quantified using multivariable logistic regression models. Sex and age categories were explored in secondary models due to missingness. Temporal trends were assessed using Cochran-Armitage test, and co-resistance was summarised for third-generation cephalosporin and carbapenem phenotypes. We also evaluated temporal dynamics of 3GCR and CR profiles. ResultsA total of 35,525 E. coli, 14,325 K. pneumoniae, 9,679 P. aeruginosa, and 5,597 Acinetobacter spp. were reported to TARSS between 2014 and 2022. Mean annual MDR prevalence was high for Acinetobacter spp. (85.1%), moderate for K. pneumoniae (45.5%) and for P. aeruginosa (27.1%), and lower for E. coli (17.5%). Adjusted models indicated increased odds of resistance to several antibiotics, whereas E. coli showed decreased odds. Intensive care unit (ICU) and blood isolates were associated with higher odds of resistance in all pathogens. ConclusionThis nine-year multi-hospital analysis reveals a high prevalence of AMR across the four WHO priority pathogens, settings, and specimen types, with increasing resistance for some pathogen-antibiotic combinations. The higher odds of clinically important resistance amongst ICU and blood isolates support the use of ward-level antibiograms and stratified stewardship and infection prevention measures.

2.Streptococcus pneumoniae is the leading cause of empyema and pneumonia in children, and monitoring of effectiveness of polyvalent pneumococcal vaccines has been essential for controlling invasive pneumococcal disease (IPD) in children and elderly adults. Conventional serotyping of pneumococci has relied on Quellung reaction following laboratory culture, however more recently whole genome sequencing (WGS) has been implemented in many reference laboratories to enhance traditional typing. Pleural fluid samples from cases with empyema are often culture negative, limiting the utility of WGS and requiring polymerase chain reaction (PCR) or 16S rRNA sequencing to detect S. pneumoniae. These molecular methods have limited sensitivity and capacity to characterise pneumococcus in clinical samples, especially in specimens with a low pathogen abundance. This study applied capture-based enrichment (tNGS) to identify and characterise S. pneumoniae directly from pleural fluid samples. A total of 51 pleural fluid samples were subjected to tNGS with a custom probe panel, for 39 known positive fluids collected from IPD cases between 2018-2025 in New South Wales, Australia. tNGS results were benchmarked against molecular-based serotyping. Our tNGS achieved 100% sensitivity and specificity in detecting S. pneumoniae. Serotyping results were concordant with PCR and 95% (37/39) of S. pneumoniae PCR positive pleural fluid cases could be serotyped using tNGS. Standard molecular methods however could only determine serotype in 56% (22/39) of samples. This tNGS enabled 39% improvement in ability to directly identify and serotype IPD-associated serotypes of S. pneumoniae in difficult-to-culture pleural fluids can significantly enhance laboratory surveillance of IPD as well as our understanding of vaccine effectiveness. 3. Impact statementThere is currently a gap in understanding the pneumococcus serotype diversity causing infection within the pleural fluid space. The gold-standard Quellung method to determine serotype relies on culturing the pneumococci first. However, pleural fluids often remain culture-negative, and cases of pneumococcal empyema have been a historical blind spot in pneumococcal surveillance. This study offers a new methodology to close this gap and allow serotyping of previously untypable cases. The study demonstrated a targeted next generation sequencing (tNGS) approach to determine serotype without the need to first culture the bacteria. This novel use of tNGS targets part of the cps gene cluster, which determines serotype. To the best of our knowledge this is the first panel to do so. We have successfully serotyped 95% of pleural fluid S.pneumoniae PCR positive samples, where previously only 56% could be determined using conventional PCR typing methods. This demonstrates for the first time a novel tNGS method capable of determining the full serotype landscape causing pleural fluid infection. This development will enhance the understanding of vaccine effectiveness and contribute to the prevention of invasive pneumococcal disease. 4. Data summarySupplementary data containing reference cpsB genomes are available within this article. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. 1.5 RepositoriesENA project accession number PRJEB111154. All supporting data has been provided within the article or in supplementary data files. One supplementary data file is available with the online version of this article.

Abstract Introduction: Streptococcus is the second genus involved in bone and joint infections (BJIs) after Staphylococcus. Streptococcus agalactiae is the predominant Streptococcus species implicated in BJIs. However, unlike Staphylococcus-related BJIs, data on S. agalactiae infections remain scarce. Methods: We conducted a retrospective cohort study from the West Region cohort of the CRIOAc registry among six university hospitals including all microbiologically confirmed streptococcal BJI in adults between 2014 and 2023. Results: 1454 patients were included, with a median age of 67 years and 65% male. S. agalactiae was the predominant streptococcal species involved 423/1454(29%). The most prevalent comorbidities identified were obesity (378/1454;26%) and diabetes mellitus (343/1454;24%). Prosthetic joint infections (PJIs) were the most common (653/1454;45%), although diabetic foot osteitis was less prevalent overall, it was significantly more associated with S. agalactiae infections (48/423;11% versus 70/1031;7%, p=0.05). S. agalactiae BJIs were more frequently lower-limb infections and chronic infections (240/423;57% versus 502/1031;49%, p=0.04). Half of the cohort had a polymicrobial infection and were slightly more frequent with S. agalactiae BJIs (235/423;56% versus 498/1031;48%, p=0.1). These results were consistent with a sensitivity analysis excluding diabetic foot related osteitis. Logistic regression analysis identified arteriopathy (OR: 4.16; IC95:1.64-11.24, p=0.003), and obesity (OR: 2.57; IC95: 1.41-4.78, p=0.002) as specific risk factors for S. agalactiae BJIs. Conclusion: S. agalactiae emerges as a prominent and distinct pathogen in complex streptococcal BJIs, with specific risk factors such as arteriopathy, obesity and diabetes mellitus, and more chronic infections.

Urinary tract infection (UTI) is one of the most common community-acquired bacterial infections mainly caused by extraintestinal pathogenic Escherichia coli (ExPEC) strains. The high-risk Escherichia coli ST131 clone is a major global cause of this disease. The lineage rapid dissemination is associated to multidrug resistance (MDR), production of extended-spectrum beta-lactamase (ESBL), and multiple virulence-associated genes. Although we lack information about ExPEC high-risk clones in Latin America, we recently reported an increase in ST131 dissemination in Rio de Janeiro from 2015 to 2019. The present study aims to characterize virulence and resistance molecular and phenotypic features that may contribute to dissemination of E. coli ST131 in Rio de Janeiro, Brazil. We assessed a 133 E. coli ST131 strains collection obtained from urine of outpatients with suspected UTI, in 2019. We determined antimicrobial susceptibility, fluoroquinolones resistance genes, virulence factors associated genes and biofilm production of all strains and analyzed the frequencies by each clade or subclade. A higher incidence of women (92%) and elderly (65%) subjects was observed. Overall resistance to first- and second-line treatment for UTI antimicrobials ampicillin, ciprofloxacin and sulfamethoxazole-trimethoprim was detected in high rates (40%), with a major impact of subclade C2 strains that were resistant to almost all antimicrobials tested, 52% carry ESBL and 66% of strains harbor the aac(6)-Ib-cr ciprpofloxacin resistance gene. Clade B and subclade C2 showed higher virulence scores among the other clades. They present unique virulence profiles characterized by the presence of papGIII, sfa/focDE, and especially ibeA genes in clade B, and the afa/DrBC, papGII, hlyA, cnf1 genes in subclade C2. Over 50% of our strains are biofilm producers, characterized by weak (24%) and strong producers (32%). ESBL and MDR strains harbor mainly papA, papGII, hlyA, cnf1 and kpsMTII genes that plays a key role in ST131 colonization. Subclade C1 is the major biofilm producer (78%), despite its lower virulence score. We also detected higher incidence of papA (27%), hlyA (19%) genes and the RPAI(malX) marker (84%) in biofilm producer strains with a statistical association of sfa/focDE gene (9%). We can infer that Clade C strains might be responsible for ST131 dissemination and persistence in Rio de Janeiro.

1.Etiological diagnosis of lower respiratory tract infections (LRTIs) relies on sputum or bronchoalveolar lavage (BAL), which may be difficult to obtain or invasive. Exhaled breath aerosol (XBA) sampling offers a non-invasive alternative for pathogen detection. We evaluated the performance of the AveloMask, a face mask-based device designed to capture XBAs for molecular testing. In this prospective paired-sample study, hospitalized adults with pneumonia at three hospitals in Switzerland and Georgia provided an XBA sample using the AveloMask and a lower respiratory tract (LRT) specimen (sputum or BAL). XBA samples were analyzed by multiplex PCR using the Roche LightMix(R) panel and LRT samples were tested using the BioFire(R) FilmArray(R) Pneumonia Panel. Concordance between XBA and LRT samples was assessed using positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA). Ninety-three participants were enrolled and 63 participants provided paired samples. AveloMask sampling identified the dominant pathogen (lowest Ct value in the LRT sample) in 40/47 LRT-positive cases (85.1%). Across all targets, PPA was 61% (95%CI, 50-72%), NPA was 100% (95%CI, 99-100%), and OPA was 95% (95% CI, 92-96%). PPA was higher for bacteria than for viruses and lower PPA was largely driven by reduced detection of low-abundance or co-infecting pathogens. In a subset analysis, AveloMask results showed substantial overlap with standard-of-care testing and could have supported antimicrobial de-escalation. Breath aerosol sampling using the AveloMask enabled non-invasive molecular detection of LRT pathogens in pneumonia cases and may complement conventional standard-of-care testing, particularly when sputum is unavailable.

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

BackgroundPseudomonas aeruginosa is a major cause of healthcare-associated infections in paediatric settings, where its persistence in moist environments such as hospital water and wastewater systems poses a particular risk to neonates and immunocompromised children. AimThe aim of this study was to showcase the long-term survival and transmission of P. aeruginosa in a large tertiary childrens hospital in England which is crucial to develop strategies for water-safe care. MethodsEnvironmental P. aeruginosa isolates were collected from taps, sinks, showers, and baths in augmented care areas of a 330-bed tertiary childrens hospital built to NHS water-safety standards. Clinical isolates were classified as invasive (blood, cerebrospinal fluid, and bronchoalveolar lavage) or non-invasive (respiratory, urine, ear, abdominal, and rectal surveillance). Variable number tandem repeat (VNTR) profiles and metadata were extracted from PDF reports, de-identified, deduplicated, and curated using Python and R. FindingsThis retrospective study analysed nine-locus VNTR profiles of 457 P. aeruginosa isolates submitted to the UK Health Security Agency from a large tertiary childrens hospital, identifying 56 isolate clusters (each with [≥]2 isolates), of which 19 (34%) contained at least one invasive isolate. The most persistent cluster (Cluster 1, n=20) spanned from July 2016 to September 2024, containing environmental and clinical (invasive and non-invasive) isolates. ConclusionThese findings demonstrate long-term persistence of certain genotypes and temporal overlap between environmental and clinical isolates, highlighting the difficulty in detecting and eradicating P. aeruginosa in hospital water and wastewater systems and reinforcing the need for continuous rigorous water system controls.

ObjectivesListeria monocytogenes is an opportunistic pathogen, associated with foodborne infections that disproportionately affect newborns, elderly and immunocompromised patients. L. monocytogenes can be classified on the antigenic and related structural variation of cell-associated wall teichoic acid (WTA) molecules through conventional serotyping techniques. The WTA structure of serovars (SV) 1/2, 1/2*, 3 and 7 consists of a linear poly-ribitolphosphate (RboP) polymer either with or without decoration with rhamnose (Rha) and/or N-acetylglucosamine (GlcNAc). Of these four SVs, SV1/2 (WTA with GlcNAc and Rha) causes [~] 99% of all listeriosis cases. However, conventional serotyping cannot accurately discriminate between these four SVs, particularly SVs1/2* (WTA with Rha). MethodsHere we applied two identified monoclonal antibodies (mAb), with specificity for the RboP backbone or GlcNAc modification to develop a discriminatory serotyping scheme for SV1/2, 1/2*, 3 and 7. Isogenic mutants for the different SVs were created in L. monocytogenes SV1/2 strain EGD-e. The typing scheme was then adapted to an immnoblot assay and applied to a collection of 317 previously classified listeriosis isolates from the Netherlands Reference Laboratory for Bacterial Meningitis. ResultsBinding of the RboP-specific mAb was limited to EGD-e wild type (SV1/2), but increased significantly for isogenic EGD-e mutants representing SV1/2*, 3 and 7. In contrast, the GlcNAc-specific mAb only recognized EGD-e mutants representing SVs 1/2 and 3. The combined staining profiles of the two mAbs allowed accurate discrimination of the four SVs as verified on clinical isolates. Applying this typing scheme to 317 listeriosis isolates previously typed as SV1/2, we confirmed SV designation in >90% of isolates, but also identified SV1/2* (5.4%), SV3 (0.6%) and SV7 (0.3%) isolates. SV1/2* isolates were also identified among meningitis patients. ConclusionThe increased discriminatory capacity of L. monocytogenes serotyping provides a more detailed insight of the epidemiological landscape and the critical factors for L. monocytogenes infections.

BackgroundTo date, accessible diagnostic tools to identify whether a patients pneumonia is a bacterial, or viral infection, are not accurate or timely enough to prevent preemptive antibiotic administration. Relying on single biomarkers or clinical presentations has been insufficient. We aimed to incorporate a wide range of novel biomarkers and clinical presentations in a multivariable model and validate its capacity to differentiate cases of bacterial and viral pneumonia. MethodsData from 457 children aged 2-59 months, admitted to Kilifi County Referral Hospital, Kenya, with bacterial (n = 229) and viral (n = 228) infections, were used to develop and validate a predictive multivariable Poisson regression model to differentiate pneumonia etiology. The Receiver Operating Characteristic curve was used to assess biomarker performance and validate the model internally. ResultsSixty-three percent (63%) of the children presented with severe pneumonia. 72% with viral pneumonia had severe pneumonia, compared to 54% with bacterial pneumonia who had severe pneumonia. In crude analyses, chest-wall indrawing, cough, convulsions, crackles, angiotensinogen, and Serpin Family A Member 1 were significantly associated with pneumonia etiology, controlling for age. However, only chest-wall indrawing remained significant in multivariable analyses after controlling for age. The model demonstrated fair, but inadequate, discrimination, with an Area Under the Curve of 0.61. ConclusionAmong the children admitted to hospital with WHO defined pneumonia, a wide range of biomarkers and clinical presentations still failed to distinguish bacterial from viral pneumonia.

BackgroundRapid identification of pathogens and antimicrobial resistance (AMR) in bloodstream infections (BSIs) is critical for timely clinical management. Although blood culture is the reference standard, it is limited by turnaround time and incomplete resolution of resistance mechanisms. We evaluated metagenomic next-generation sequencing (mNGS) applied to flagged positive blood culture bottles to enhance diagnostic resolution and inform targeted molecular approaches. MethodsFifty-five flagged positive blood culture bottles from a tertiary care hospital in Bengaluru, India, were analyzed. Shotgun mNGS was performed directly on blood culture broth and compared with routine phenotypic identification and antimicrobial susceptibility testing (AST) from corresponding isolates. Antimicrobial resistance genes (ARGs) and plasmid replicons were profiled. ResultsmNGS showed high concordance with routine culture for pathogen identification (54/55; 98.2%) and improved species-level resolution across bacterial and fungal pathogens. Genotypic resistance profiles were consistent with phenotypic AST, identifying {beta}-lactamases, efflux-associated determinants, and target modification mechanisms. Diverse ARGs and plasmid replicons (Inc-, Col-, and rep-family) were detected, providing genomic context for resistance. Sequencing predominantly reflected the cultured organism, supporting high specificity in flagged blood culture material. ConclusionsmNGS applied to flagged blood culture bottles enables high-resolution characterization of pathogens and resistance determinants at a clinically actionable stage. The genomic insights generated provide a framework for developing targeted multiplex PCR assays that can reduce turnaround time and improve affordability compared with sequencing-based approaches. This strategy supports the use of mNGS as an adjunct to conventional diagnostics and as a bridge toward scalable, rapid, and cost-effective solutions for BSI diagnosis and AMR surveillance.

Background: The choice of hand-drying method affects microbial contamination levels but its economic consequences have not been systematically quantified. Methods: By applying a quantitative microbial risk assessment framework, we translated the documented contamination differential between jet air dryers and paper towels into infection risk estimates, and embedded these into an established health economic model of healthcare-associated infections in NHS hospitals and an illustrative productivity analysis for the EU workforce. Results: The median estimated avoidable HCAI cost attributable to jet air dryer presence in UK NHS clinical areas was 58 million pounds per year, representing 2.1% of total HCAI expenditure for the affected hospital population, with a 50% certainty interval of 33-84 million pounds. Extended to the EU workforce, the same contamination differential implied a median of 1.7 billion euros in annual productivity gains, due to reduced absenteeism, for a shift to use of paper towels in public restrooms. Conclusions: These findings suggest that hand-drying method selection carries measurable economic implications that are not currently reflected in facility management practice. The evidence supports the prioritisation of paper towels in clinical and public settings as a cost-effective infection control measure

BackgroundMucormycosis is a rapidly progressive and often fatal invasive fungal infection caused by moulds in the order, Mucorales. Early diagnosis is essential for effective clinical management; however, conventional diagnostic approaches such as culture and histopathology are slow, insensitive, and require specialist mycological expertise. Although molecular methods are available for disease detection, they are not widely accessible. At present, no enzyme immunoassay (EIA) exists for the detection of mucormycosis. MethodsA murine IgG1 monoclonal antibody (mAb), FH12, was generated against extracellular polysaccharides (EPSs) produced by Mucorales pathogens during active growth. The antibody was characterised for specificity, epitope stability, and antigen localisation using ELISA, immunoblotting, and immunofluorescence techniques. The mAb was incorporated into a Sandwich-ELISA and evaluated using culture filtrates, purified EPSs spiked into human serum, and tissue homogenates from a patient with cutaneous mucormycosis caused by Lichtheimia ramosa. ResultsmAb FH12 demonstrated pan-Mucorales specificity and no cross-reactivity with other clinically relevant yeasts and moulds. The epitope recognised by FH12 is periodate-insensitive and moderately heat-stable. The Sandwich-ELISA detected EPS antigens in human serum with limits of detection ranging from pg/mL to low ng/mL levels, and successfully identified the EPS biomarker in patient tissue homogenates. ConclusionThe FH12-based Sandwich-ELISA shows high sensitivity and specificity, and has the potential to be used as a laboratory-based adjunct diagnostic test for the detection of mucormycosis in humans.

Phage therapeutics are re-emerging as adjuncts or alternatives to antibiotics and their clinical translation will be enhanced with production methods that minimise downstream processing. We evaluated whether an endotoxin-reduced E. coli strain developed for production of recombinant proteins, ClearColi(R), can serve as a useful, safe phage production host without compromising yield and whether targeted receptor complementation can expand its utility. The parent strain BL21(DE3), and its lipid A modified derivative, ClearColi(R), were compared with respect to infection and generation of phage. Across a panel of 31 phage, a similar host range was observed between BL21(DE3) and ClearColi(R). To expand host range ompC was genetically engineered into the chromosome of ClearColi(R), thereby adding OmpC-dependent phage to its production capacity. Production metrics were broadly comparable between the hosts; efficiency of plating and final titres for representative phage were not significantly different; burst size varied by phage but without consistent host bias. Endotoxin activity in ClearColi(R)-propagated lysates was reduced by over 1000-fold relative to BL21(DE3), reaching the low hundreds of endotoxin units (EU) versus hundreds of thousands for BL21(DE3). Intravesical administration of ClearColi(R)-derived phage (LUC4) into pigs elicited no clinical abnormalities and no significant increases in circulating cytokines up to 48 hours after administration. ClearColi(R) allows efficient production of diverse phage with low endotoxin, reducing the requirement for downstream processing. Although its minimal LPS reduces its capacity for producing some LPS-dependent phage and its growth is slower than BL21(DE3), requiring optimisation for maximal phage titre, the safety and simplified manufacturing process support further development of endotoxin modified strains for phage production. Impact statementAntibiotic resistance is a current global problem and treatments based on phage and phage products already have a proven track record with particular bacterial infections, especially in the urinary tract. While progress is being made on in vitro phage synthesis, large scale bacteriophage preparations require a bacterial host for production, consequently toxic components in the initial lysate need to be removed or significantly diluted for safe clinical use. This is a study of the potential to utilise an endotoxin-reduced E. coli strain, ClearColi(R), to produce safer phage therapeutics. Such endotoxin modified strains should minimise the processing steps required and reduce overall production costs of a phage preparation. The research demonstrates that the endotoxin-reduced strain was able to produce a wide range of phage and for studied examples at phage titres equivalent to the more toxic parent strain. We also show that the strain can be modified to increase its host range and confirm the very low endotoxicity of basic phage lysates produced by the strain. Replicating this process to engineer additional low-toxicity bacterial production strains will accelerate the development of safer, more cost-effective phage therapeutics.

Urinary tract infections (UTIs) are among the most common infectious diseases worldwide, with Escherichia coli being the predominant uropathogen. The increasing prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains and their association with fluoroquinolone resistance pose a significant challenge to empirical therapy, particularly in community settings. The aim of this study was to determine the epidemiology and predictive factors associated with ESBL-producing E. coli and its concomitant fluoroquinolone resistance in community-acquired clinical isolates. A retrospective cross-sectional study was conducted analyzing 244 clinical E. coli isolates. Demographic and microbiological data were collected, including age, sex, sample type, and antibiotic susceptibility. Associations between variables and ESBL production were assessed using Pearsons chi-squared test, and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Of the isolates, 165 (68%) were ESBL-producing. A significant association was observed between age group and ESBL production (p < 0.001), with the highest frequency in the 20-39 age group. Most ESBL-positive isolates were obtained from women (73%), although odds ratio (OR) analysis suggested a non-significant trend toward a higher probability in men (OR = 1.29; 95% CI: 0.72-2.31). High rates of fluoroquinolone resistance were identified among the ESBL-producing isolates, with 30% resistance to levofloxacin and 35% to ciprofloxacin (p < 0.001). Urine samples showed the highest concentration of ESBL-positive isolates, with a significant association between sample type and resistance (p < 0.001). The high prevalence of ESBL-producing E. coli and its concomitant resistance to fluoroquinolones highlight a critical challenge for the empirical treatment of urinary tract infections in Mexico, underscoring the need to strengthen antimicrobial use management and local surveillance strategies.

Early detection of ovulation and pregnancy in the common marmoset is crucial for reproductive studies, yet hCG kits lack cross-reactivity with marmoset CG, and current methods remain labor-intensive. Here, we developed monoclonal antibodies against marmoset CG and CG{beta}, and established a non-invasive immunochromatographic CG assay. By eliminating invasive blood sampling, this assay supports 3Rs principles and enables practical endocrine monitoring. The assay detected urinary CG surges preceding ovulation, enabling efficient embryo recovery through artificial insemination (75%). Early pregnancy was detected at approximately 17 days post-ovulation. In addition, pregnancy detection in squirrel monkeys suggests conservation of CG features among certain New World primates. Overall, this simple, non-invasive assay provides a practical tool for marmoset research and establishes a foundation for future conservation-oriented reproductive monitoring following appropriate species-specific validation.

Wastewater surveillance is increasingly used for antimicrobial resistance (AMR) monitoring in urban environments, but low-resource settings often lack a piped sewerage system. Instead, coprophagous flies--flies that ingest feces--may serve as composite samplers for monitoring fecal wastes present in terrestrial environments. We evaluated whether the class 1 integron-integrase gene intI1 was associated with genetic markers of AMR and fecal source tracking markers (FST) in coprophagous flies collected from latrine entrances and food preparation areas in low-income urban Maputo, Mozambique. We quantified intI1, an enteric 16S rRNA target (for normalization), three FST markers, and 30 ARG targets using qPCR. We normalized concentrations of intI1 and each target to enteric 16S rRNA. We fit linear mixed models with a random intercept for housing compound to estimate within-fly associations between log10 relative abundance of intI1 and log10 relative abundance of each target with and without adjustment for fly taxonomic group, capture location, and standardized fly mass. We also modeled per-fly unique ARG count (i.e., number of ARG targets detected) using Poisson regression. Of 188 flies assayed, 176 passed internal controls; intI1 and enteric 16S rRNA were detected in 95% and 96% of flies, respectively. Higher relative abundance of intI1 was positively associated with ARG and FST targets, with the strongest associations observed for sulfonamide-(sul1: {beta} = 0.87; 95% CI: 0.81, 0.94; sul2: {beta} = 0.81; 95% CI: 0.73, 0.89), tetracycline- (tetA: {beta} = 0.78; 95% CI: 0.70, 0.85; tetB: {beta} = 0.69; 95% CI: 0.60, 0.79), and trimethoprim-related (dfrA17: {beta} = 0.78; 95% CI: 0.70, 0.86) genes. Associations with FST markers were weaker (i.e., human mtDNA: {beta} = 0.46; 95% CI: 0.37, 0.55; human-associated Bacteroides: {beta} = 0.34; 95% CI: 0.25, 0.43). Higher relative abundance of intI1 was also associated with a greater number of ARGs detected: each 10-fold increase in intI1 was associated with an 8% higher expected unique ARG count (aRR=1.08, 95% CI: 1.04-1.12). These findings support the need for further research across different settings exploring intI1 carried by coprophagous flies as a potential standardized screening target for AMR surveillance in unsewered terrestrial environments.

BackgroundPhage therapy is increasingly considered a promising alternative for treating multidrug-resistant (MDR) infections. However, its clinical application remains limited by challenges in isolating effective phages against resistant clinical strains and by the limited ability of in vitro assays to predict performance in real biological environments. While biological matrices are known to influence phage activity, these effects are not well characterised. MethodsA phage-resistant Pseudomonas aeruginosa isolate from a patient with recurrent MDR urinary tract infection was used as the model organism. Conventional isolation methods failed to recover effective phages, leading to the development of TEASER-i (Transient EDTA- and Ion-Assisted Sequential Enrichment & Recovery). Recovered phages were characterised using adsorption assays, one-step growth kinetics, and time-kill experiments. Their antibacterial activity was evaluated both in vitro and in ex vivo human matrices (whole blood, serum, plasma, and urine). Phage efficacy was quantified using maximum log reduction (Emax), area under the curve (AUC), and phage-to-bacteria ratio (PBR). ResultsA novel TEASER-i method optimised for difficult-to-treat Gram-negative infections, enabled recovery of a functionally effective Osewage-derived P. aeruginosa phage, which outperformed a Ourine-derived P. aeruginosa phage that showed slower replication and lower burst size. Phage activity varied significantly in blood, serum, and plasma. Urine supported the most sustained antibacterial effect. In many cases, early bacterial reduction was followed by regrowth. Sustained activity was associated with maintenance of favourable PBR values, while negative PBR corresponded to treatment failure. At 96 h, only two conditions maintained favourable phage load (log 10 PBR > 0): the S. aureus phage in urine (+1.66) and the sewage-derived P. aeruginosa phage in serum (+1.32). ConclusionsPhage efficacy depends not only on intrinsic lytic capacity but also on the ability to persist and amplify within specific biological environments. Conventional isolation and in vitro screening may therefore overestimate therapeutic potential. Combining optimised isolation strategies with ex vivo evaluation provides a more realistic framework for phage selection and clinical translation.

Objectives: To better define the clinical features of Acinetobacter spp. infection in Northern Thailand, including a comparison of hospital- and community-acquired infections (HAIs and CAIs). Methods: A prospective clinical study of Acinetobacter spp. infections at two Northern Thailand hospitals from 2019 to 2022, collecting data on sample sources, patient demographics, comorbidities, antimicrobial resistance profiles, and outcomes. Results: Of 129 enrolled patients, 81.4% had Acinetobacter spp. isolated from a respiratory sample. A significant minority (25.6%) of infections were CAIs, 33.3% of which were admitted to ITU within 24 hours of admission. Compared to HAIs, CAIs were significantly more likely to be caused by blood (15.2%, p=0.0258), wound (21.2%, p=0.0120), or urine infections (12.1%, p=0.0370). Acinetobacter spp. HAIs mainly occurred after admission to ITU (87.7%, p<0.0001) and were more likely to be multidrug-resistant than CAIs (76.3% vs. 34.4%, p<0.0001). Overall, the median length of hospital stay was 27 days and there was a 27.1% in-hospital mortality, which was increased in patients with CVA/brain (p=0.005), and multidrug-resistant (p=0.010) or carbapenem-resistant infections (p=0.003). Conclusions: These data define the clinical profile of Acinetobacter spp. infections in Northern Thailand, confirming their high mortality and demonstrating CAIs are a significant proportion of all cases.