Diagnostic Microbiology and Infectious Disease

BackgroundSickle cell disease (SCD) is the most common recessive genetic disorder, caused by pathogenic variants of the HBB gene. SCD is associated with a range of clinical manifestations, including vaso-occlusive crises, infections, and severe anaemia, which contribute to increased morbidity and mortality. The frequency of pathogenic alleles is high in Sub-Saharan African countries, with heterozygous carriers reaching up to 25% of the population. Several methods can be employed for molecular diagnostics, with HBB gene sequencing being the most precise. However, access to DNA analyses and sequencing in Low- and Middle-Income Countries (LMICs), where SCD prevalence is high, is limited. Understanding genetic profiles is crucial at both individual and population levels, as it can guide public health strategies and facilitate accurate genetic counselling. AimThis feasibility study aimed to demonstrate that a portable medical genetic laboratory (in suitcases) can be used to genotype individuals for the HBB A, S, and C alleles and their combinations within a few hours outside of a laboratory setting. Methods and resultsWe established a portable medical genetics laboratory capable of DNA extraction and isothermal DNA amplification using a commercially available kit for the A, S, and C alleles of the HBB gene. During one single study day, this portable lab was set up in a room where the Swiss Association of Patients with SCD was holding its annual meeting. We analysed the samples of 27 participants who were aware of their A, S, or C status. We collected buccal swabs and dried blood samples for genotyping. Genotype results for all participants were obtained within five hours after sample collection. In four cases, we observed discrepancies between the buccal swab and blood genotypes; three were resolved upon repeat testing, and one reflected donor chimerism following hematopoietic stem-cell transplantation. ConclusionsThis study demonstrates the feasibility and efficiency of using a portable medical genetics laboratory for rapid genotyping of HBB SCD alleles in community settings.This approach can improve access to molecular diagnostics in resource-limited environments. Such tools have the potential to significantly enhance local capabilities for genetic screening, counselling, and public health planning in regions heavily affected by SCD.

The incidence of vancomycin-resistant Enterococcus (VRE) is rising in hospitals in Canada, and resistance to last-resort antimicrobials including linezolid complicates treatment options for multidrug-resistant isolates. Recent reports from around the globe indicate that both linezolid and vancomycin resistance genes can be co-carried and mobilized by linear plasmids (named pELF) in Enterococcus species, often on the same backbone. We aimed to investigate linezolid resistance and linear plasmid prevalence in VRE bloodstream infection isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2009 to 2024. We found that screening for pELF linear plasmid ends in short reads was a reliable way to predict linear plasmid presence in large-scale surveillance data (100 % accuracy on 85 reference samples). Almost half of the isolates in our collection were predicted to carry pELF plasmids (45.4 %, 941/2071) and we found that this proportion has increased from 2018 (32.2 %, 59/183) to 72 % of isolates between 2021 and 2024 (2021: 68.5 % (115/168); 2022: 71.6 % (146/204); 2023: 72.8 % (166/228); 2024: 71.6 % (235/328)). This trend of increasing linear plasmid carriage is evident from 2018 to 2024 across the dominant emerging sequence types (ST80, ST17, ST117). Linezolid resistance based on phenotypic antimicrobial susceptibility testing was low (1.0 %, 21/2071). Using long read sequencing, we characterized the linezolid resistant isolates and confirmed pELF plasmid presence in 13/21 (61.9 %) isolates. Six isolates harboured pELF plasmids encoding linezolid resistance genes (optrA, cfr(D), poxtA) and five of these also encoded vancomycin resistance genes (vanA). We compared these six plasmids to 39 public plasmid sequences and clustered them using MOB-suite and pling. Overall, this study provides further examples of the co-carriage of vancomycin and linezolid resistance genes on mobile linear plasmids and shows that linear plasmid prevalence is detectable and increasing across VRE in Canada. IMPACT STATEMENTGiven the increasing prevalence of multidrug-resistant hospital-acquired pathogens, resistance to last-resort antibiotics is a global public health threat. Linezolid is a last-resort antibiotic used to treat vancomycin-resistant Enterococcus isolates, and the dissemination of linezolid resistance genes is significantly facilitated by mobile elements that can transfer between unrelated strains and species. Linezolid resistance genes have recently been described on linear plasmids and are often co-localized with other resistance genes on the same plasmid backbone. Consequently, understanding the features and distribution of linear plasmids and those harbouring linezolid resistance genes is crucial for pathogen surveillance and mitigation of resistance. In this work, we used long-read and short-read sequencing to characterize genomic epidemiology of linear plasmids across 16 years of Enterococcus surveillance data in Canada. This study furthers knowledge of linear plasmids by demonstrating that they are relatively common across vancomycin-resistant Enterococcus blood isolates and by providing more examples of co-localized vancomycin and linezolid resistance genes on the same linear plasmid backbone. DATA SUMMARYSequencing data and genome sequences were deposited in National Centre for Biotechnology BioProject PRJNA1279082, and accessions are listed in Table S1. Supplementary materials for this study are available at the Figshare portal through DOI: XXX.

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy worldwide. Improving the quality of life of people with SCD requires prenatal and neonatal screening. Our primary objective was to demonstrate that prenatal diagnosis of SCD is possible even in situations of poverty. Secondarily, we described the socioeconomic profile of couples seeking molecular diagnosis of SCD in Kinshasa, Democratic Republic of Congo. Methods This was a cross-sectional study conducted in Kinshasa between January 2020 and December 2025. During this study period, 107 couples underwent prenatal diagnosis. Prenatal diagnosis was performed using amniocentesis with FTA Elute technology. This diagnosis was confirmed at birth using cord blood DNA extracted via the conventional salting-out technique. Results The mean age of the pregnant women was 28 {+/-} 4 years. Eighty-one couples (75.7%) were Christian, nine couples (8.4%) were Muslim, and seventeen couples (15.8%) were animist. Eighty-two couples (76.6%) were known heterozygous AS couples, eleven (10.2%) were heterozygous couples, and fourteen (13.0%) were couples composed of one homozygous SS and one heterozygous AS partner. All pregnancies were singleton. Socioeconomic status was upper middle class (39.2%). The AS genotype was found in 79% of the fetuses. One intrauterine fetal death was observed after amniocentesis. In terms of handling, the FTA Elute technology reduces DNA extraction time to 30 minutes. It is easy to use. Results are available in less than 24 hours. Conclusion The FTA Elute technology is a reliable, less expensive, and easy-to-use prenatal screening technique for sickle cell disease. Sample transport and storage conditions are better suited to resource-limited settings.

Celiac disease (CD) is strongly associated with specific HLA DQ heterodimers, formed by HLA DQA1 and HLA DQB1 proteins. In particular DQ2.5 (DQB1*02 associated to DQA1*05) and DQ8 (DQB1*03:02 with DQA1*03) are present in virtually all celiac patients. HLA DQB1*02 is considered the main single genetic susceptibility marker and has been reported in 90 to 95% of CD patients. However, the distribution of these alleles may vary across populations, potentially impacting the performance of genetic screening strategies. In this study, we evaluated the prevalence of HLA DQ2.5 and DQ8 genotypes in celiac patients (n = 41) and an unbiased general population cohort (n = 60) from San Luis, Argentina, using a PCR-based genotyping approach. In addition, we assessed the feasibility of a simplified saliva direct PCR protocol for large scale testing. Overall, 95.1% of CD patients carried DQ2.5 and/or DQ8. Notably, 41.5% of patients were DQ8(+)/DQ2.5(-), and 36.6% lacked the DQB1*02 allele, indicating that DQB1*02 based screening alone would have reduced sensitivity in this population. In the general population, 53.3% of individuals carried CD associated genotypes, with a markedly higher prevalence of DQ8 compared to European cohorts. Genotype distributions deviated from Hardy Weinberg equilibrium in CD patients but not in the general population. We show that DQB1*03:02 is a reliable proxy for DQ8, allowing simplification of genotyping strategies, whereas DQA1*05 typing remains essential to discriminate DQ2.5 from other lower risk DQB1*02 carrying heterodimers. We also describe a saliva direct PCR approach showing a performance comparable to purified DNA based assays. These findings highlight the importance of population specific genetic data for optimizing CD screening strategies and foster the development of simplified, cost effective genotyping approaches for large scale applications.

Sequencing the respiratory tract transcriptome has the potential to provide insights into infectious pathogens and the hosts immune response. While DNA-based sequencing is more standard in clinical laboratories due to its stability, RNA assays offer unique advantages. RNA reflects dynamic physiological changes, and for RNA viruses, viral RNA particles directly represent copies of the viral genome, enabling greater diagnostic sensitivity. However, RNAs susceptibility to degradation remains a significant challenge, particularly in RNase-rich specimens like saliva. To address this, we conducted a systematic, combinatorial evaluation of 24 distinct mNGS workflows, crossing eight nucleic acid extraction methods with three RNA-Seq library preparation protocols. Remnant saliva samples (n = 6) were pooled and spiked with MS2 phage as a control. The SARS-CoV-2 virus was spiked into half of the samples, which were extracted using the eight different extraction methods (n = 3) and compared using RNA Integrity Number equivalent (RINe) scores and RNA concentration. The extracted RNA was then processed across the three library construction methods and subjected to short-read sequencing to assess all 24 combinations head-to-head. We compared methods based on viral read recovery and found that RINe and concentration did not correlate with viral detection. The Zymo Quick-RNA Magbead kit and the Tecan Revelo RNA-Seq High-Sensitivity RNA library kit were the extraction and library-preparation kits that yielded the most SARS-CoV-2 reads, respectively. Importantly, our combinatorial analysis revealed that any small variability attributable to different nucleic acid extraction methods was heavily overshadowed by differences in quality attributable to the RNA-Seq library preparation methods. These findings challenge the reliance on conventional RNA quality metrics for clinical metagenomics and underscore the need to redefine extraction quality standards for mNGS applications. IMPORTANCEmNGS is a powerful and unbiased approach towards pathogen detection that has mostly been applied to blood and cerebrospinal fluid samples. However mNGS has recently been applied to more areas including the respiratory pathogen detection space, with potential applications in both in-patient diagnostics and public health surveillance. Saliva samples are an ideal sample type for these use cases since they can be collected non-invasively. However, saliva is also a challenging sample type due to its high RNase activity and often yields low-quality nucleic acid. This study explores the feasibility of using saliva specimens in mNGS with contrived SARS-CoV-2 samples to optimize the combination of two factors: nucleic acid extraction and RNA-seq library preparation. Exploration in this area could enhance the sensitivity of saliva-based mNGS assays, with the goal of future expansion of this specimen type in clinical diagnostics and public health surveillance. Key PointsO_LIThe choice of RNA-Seq library preparation kit has a greater impact on pathogen detection than the nucleic acid extraction method. C_LIO_LIThe combination of Zymo Quick-RNA Magbead extraction kit and TECAN Revelo RNA-Seq High Sensitivity RNA library kit recovered the highest percentage of total SARS-CoV-2 reads. C_LIO_LIRNA quantity and RINe score do not correlate with viral read capture, indicating a need for an alternative metric to assess RNA quality for downstream mNGS clinical diagnostics. C_LI

iii.Background and ObjectivesThe "Pan-European Transfusion Research InfrAstructure" (PETRA) project was established to advance the use of donor, blood product, and patient datasets in Europe, aiming to benefit both patient and donor health. Here, the initial PETRA objective was to describe the landscape of existing donor and blood establishment (BE) databases. Materials and MethodsAn online survey was circulated to the European Blood Alliances BE members. The survey collected information on the feasibility of accessing donor data, and challenges and possibilities for linking these datasets with information on the associated blood products and transfusion recipients, and donors own health records. ResultsSeventeen BEs across 16 countries completed the survey. The majority could, in principle, link their donor data to product data (13 BEs (76%)) and recipient data (10 BEs (59%)), for research purposes. However, capabilities were limited and in only 29% of the BEs was the donor to recipients linkage an automated process. BEs reported significant challenges to achieve full vein-to-vein linkage, including legal constraints and lack of consent (11 BEs) and resources (10-14 BEs). IT and data issues as well as lack of knowledge and training were cited as obstacles by a minority of BEs. ConclusionWhilst the survey results suggest considerable interest in developing linkages between blood donors, their products, and recipients, many challenges remain due to a variety of obstacles. First steps in working towards a PETRA may be assistance to navigate legal frameworks as well as investing in resources and quality and harmonisation of data collections. iv. HighlightsO_LI17 blood establishments (BEs) in 16 countries responded to a survey on obstacles and opportunities for achieving vein-to-vein datasets. C_LIO_LIIn 59% of the BEs donor-to-recipient links can be established for research improving transfusion outcomes, but only in 29% this is an automated process. C_LIO_LIIn order to work towards a "Pan-European Transfusion Research InfrAstructure" (PETRA), legal frameworks, adequate donor consent and (financial and human) resources are the most common obstacles that require addressing. C_LI

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.

Respiratory syncytial virus (RSV), an approximately 15.2 kb negative sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV/A and RSV/B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 deidentified remnant clinical specimens (102 RSV/A; 112 RSV/B) with RT PCR Ct values <31 were included. RSV genomes were amplified using ARTIC style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV/A (median 53,433x; mean 51,076x) than RSV/B (median 49,699x; mean 46,945x), whereas genomic coverage was slightly lower for RSV/A (median 97.5%; mean 96.6%) than RSV/B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV/A and B.D.E.1 for RSV/B. For RSV/A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra and inter run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0 to 5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. These results demonstrate that the ARTIC-based RSV WGS assay enables near real time surveillance and strengthens data driven public health responses to future outbreaks.

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.

IntroductionHerpes simplex virus type 1 (HSV-1) is highly prevalent worldwide, making accurate serological testing essential for both clinical diagnosis and epidemiological surveillance. Automated chemiluminescent immunoassays (CLIAs) offer operational advantages over enzyme-linked immunosorbent assays (ELISAs); however, their diagnostic performance relative to Western blot (WB) confirmation in high-prevalence settings remains insufficiently characterized. Hypothesis/Gap StatementThe comparative diagnostic accuracy of CLIA- and ELISA-based assays for HSV-1 IgG detection, when benchmarked against a WB reference standard in endemic populations, remains unclear. AimThis study aimed to evaluate HSV-1 IgG seroprevalence and diagnostic performance of one CLIA and two ELISA platforms using Western blot as the reference method. MethodologyFour hundred archived serum samples from adult male craft and manual workers in Qatar were tested using the Mindray CL-900i CLIA, HerpeSelect ELISA, NovaLisa ELISA, and Euroimmun Western blot. Seroprevalence, diagnostic accuracy, and interassay agreement were assessed using WB as the reference standard, with equivocal and indeterminate results excluded from analysis. ResultsHSV-1 IgG seroprevalence estimates were comparable across assays: HerpeSelect 72.5%, Mindray 70.5%, NovaLisa 66.3%, and Western blot 66.5%, with no statistically significant differences (all p > 0.05). The Mindray CLIA demonstrated the highest diagnostic performance (sensitivity 95.7%, specificity 88.9%, accuracy 93.4%) and strong agreement with Western blot ({kappa} = 0.85). HerpeSelect showed substantial agreement ({kappa} = 0.81), while NovaLisa exhibited lower specificity. ConclusionCLIA- and ELISA-based assays produced comparable HSV-1 seroprevalence estimates in this high-prevalence population; however, diagnostic accuracy varied across platforms. The CLIA platform demonstrated the strongest agreement with Western blot, supporting its use in high-throughput settings, while confirmatory testing remains important to minimize misclassification. Key PointsO_LIWhat is known: HSV-1 serological diagnosis relies mainly on ELISA assays, while automated CLIA platforms are increasingly used in high-throughput laboratories but remain insufficiently evaluated against Western blot confirmation. C_LIO_LIWhat is new: This study provides a large head-to-head comparison of CLIA and ELISA platforms for HSV-1 IgG detection using Western blot as the reference standard in a high-prevalence population. C_LIO_LIClinical implications: Automated CLIA systems demonstrated strong diagnostic accuracy and may represent reliable high-throughput alternatives for HSV-1 serological screening in clinical laboratories. C_LI Impact StatementAccurate serological diagnosis of herpes simplex virus type 1 (HSV-1) is essential for clinical management, epidemiological surveillance, and public health decision-making, particularly in populations where infection is highly prevalent. This study adds to the existing literature by providing a large, head-to-head comparison of automated chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) platforms for HSV-1 IgG detection, benchmarked against Western blot confirmation in a real-world, high-prevalence setting. By demonstrating that different serological platforms can yield similar population-level seroprevalence estimates yet differ in diagnostic accuracy and specificity, this work highlights the risk of misclassification when confirmatory testing is not considered. The findings are of broad relevance to clinical microbiology laboratories, diagnostic services, and public health surveillance programs that rely on serological assays for HSV-1 screening. The study represents an incremental but important step in refining assay selection and interpretation, supporting more reliable laboratory diagnostics and improved understanding of HSV-1 infection burden in endemic populations. Data Availability StatementThe data that support the findings of this study are available from the corresponding author upon reasonable request.

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.

Abstract Background: Mean platelet volume (MPV) is a simple, low-cost biomarker that reflects platelet activation. Its prognostic value in septic shock remains controversial. We aimed to determine whether MPV at intensive care unit (ICU) admission is associated with hospital mortality in patients with septic shock. Methods: Retrospective cohort study of consecutive adults with septic shock (Sepsis-3 criteria) admitted to a single ICU. MPV, severity scores (SOFA, APACHE II, SAPS II), procalcitonin, and clinical data were collected. The primary outcome was in-hospital mortality. Spearman correlation, univariate and multivariate logistic regression (with Firth's correction), ROC curves, and subgroup analyses were performed. Results: Fifty-eight patients were included; mortality was 58.6%. MPV did not differ between non-survivors and survivors (13.09 {+/-} 1.37 vs. 12.66 {+/-} 1.45 fL, p = 0.259). MPV showed a weak correlation with procalcitonin ({rho} = 0.394, p = 0.002) but not with severity scores. In multivariate analysis adjusting for age, sex, SOFA and comorbidity count, MPV was not an independent predictor of mortality (OR 1.075, 95% CI 0.682-1.755, p = 0.749). The area under the ROC curve for MPV was 0.598 (95% CI 0.444-0.752), significantly lower than that of SOFA (0.837) and procalcitonin (0.836). Subgroup analyses showed no significant association between MPV and mortality in any stratum. Conclusions: In this cohort of septic shock patients, MPV at ICU admission was not associated with hospital mortality and had poor discriminative ability. Widely used severity scores and procalcitonin remain superior prognostic markers. MPV should not be used as a prognostic tool in septic shock. Keywords: Septic shock, Mean platelet volume, Mortality, SOFA, Procalcitonin, Biomarker

PurposeTo develop a pan-fungal lateral flow device (LFD); evaluating device performance with samples obtained from ex vivo porcine cornea infection models. MethodsFungal {beta}-glucan (PF1), human CLEC7A/Dectin-1/CLECSF12 protein (Fc Tag; PF2), and fungal melanin (PF3) antibodies were evaluated for binding to clinically relevant fungal and bacterial species (Aspergillus flavus, Fusarium keratoplasticum, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus) by immunofluorescence staining. PF1 and PF2 were evaluated in proof-of-concept, in-house LFD strips using cultured pathogens and ex vivo porcine corneal infection samples. The lead antibody (PF1) was validated in a commercially-developed prototype LFD. ResultsPF1 and PF2 discriminated target fungi from bacteria by immunofluorescence microscopy and in-house LFD strips. The lead candidate PF1 demonstrated good sensitivity (0.75) and specificity (0.94) with cultured fungal hyphae. Samples obtained from infected ex vivo cornea by clinically relevant methods confirmed excellent sensitivity (scrapes: 1.00, swabs: 0.94) and specificity (scrapes: 1.00, swabs: 0.83). The commercially-developed PF1-LFD prototype achieved perfect sensitivity (1.00) and specificity (1.00) when detecting and discriminating fungi from non-fungal ex vivo corneal swab samples. ConclusionsFeasibility of pan-fungal LFD application in microbial keratitis diagnosis was demonstrated - using {beta}-glucan as a pan-fungal target and a clinically relevant microbial keratitis ex vivo model. LFDs were able to differentiate fungal from bacterial samples, detect antigen present in corneal swabs, and provide a read-out within 20 minutes. Sensitivity and specificity values are comparable to currently used diagnostic tests. Translational RelevanceEarly discrimination of fungal keratitis cases is important to adapt treatment, and improve patient outcome.

Background: Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome) is a fatal neurodegenerative lysosomal storage disorder caused by impaired degradation of heparan sulfate (HS). Despite rapid advances in gene and enzyme therapies, there remains a critical need for an analytically validated, quantitative biomarker that accurately reflects central nervous system (CNS) substrate burden. Such biomarker would be a valuable tool in assessing disease progression and monitoring therapeutic efficacy. Objective: This study describes the method development, fit for purpose validation, and preliminary clinical application of a quantitative liquid chromatography-mass spectrometry (LC-MS/MS) assay for the HS-derived disaccharide N-sulfoglucosamine-glucuronic acid (GlcNS-GlcUA) in human cerebrospinal fluid (CSF), a critical biomarker for diagnosis, disease monitoring, and regulatory evaluation of emerging MPS IIIA therapies. Methods: A structurally defined GlcNS-GlcUA reference standard and its [13C6]-labeled internal standard were used in a derivatization and detection workflow employing 1-phenyl-3-methyl-5-pyrazolone labeling, and LC-MS/MS. Results: The method exhibited acceptable linearity across 0.005-0.500 nmol/mL (r[&ge;]0.9976), with intra- and inter-assay imprecision [&le;]3.5%CV and accuracy within 95%-110% of nominal concentrations. No matrix or hemolysis interference or carryover was observed, and the analyte remained stable during freeze-thaw storage conditions. Application of the method to 12 CSF samples from patients with MPS IIIA demonstrated quantifiable GlcNS-GlcUA levels ranging from 0.0054 to 0.106 nmol/mL, confirming suitability for clinical and regulatory use. Comparison of the MPS IIIA sample results between the development laboratory and the contract research organization laboratory support robust inter-lab assay transfer. Conclusions: This validated LC-MS/MS method establishes a regulatory-grade quantitative assay for measurement of CSF HS in MPS IIIA. Its high analytical sensitivity and reproducibility enable reliable assessment of CNS substrate reduction and pharmacodynamic response, supporting biomarker-driven therapeutic development and accelerated approval pathways for neuronopathic mucopolysaccharidoses.

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.

The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 g/ml for CAZ, 8 g/ml for LVX, 16 g/ml for MEM, and 8 g/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 g/ml, 8 g/ml, and 16 g/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document. IMPORTANCEThe Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for four antimicrobials, we encountered significant challenges in our analyses, including limitations with data for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide clinicians on treatment and supported the eventual removal of ECVs from the CLSI M100 standards document.

Background Oropouche virus (OROV) is an emerging vector-borne virus with rapidly expanding geographic range, increasing case counts, and growing evidence of severe outcomes including neuroinvasive disease and vertical transmission. Because OROV infection presents with nonspecific febrile illness that overlaps clinically with other viruses including dengue, zika, and chikungunya, accurate molecular diagnostics are essential for patient care and surveillance. Yet existing assays rely on single genomic targets and are vulnerable to detection failure as the virus evolves and reassorts. Methodology/Principal Findings To support diagnostic capacity, we developed and clinically validated a multiplexed qPCR assay targeting three regions of the OROV S segment, incorporating redundancy to preserve sensitivity across viral diversity while enabling robust clinical interpretation. The multiplex also includes an assay targeting RNaseP as an internal sample control to ensure adequate sample processing. We evaluated assay performance using both historical and contemporary OROV strains and validated the assay on contrived serum, plasma, and cerebrospinal fluid samples, assessing linearity, limit of detection (LOD), accuracy, specificity, precision, and sample stability. The assay met or exceeded all predefined acceptance criteria for clinical testing and achieved an LOD as low as 6 copies per reaction for contemporary outbreak strains. We further implemented a logic-based interpretation matrix that reduced false-positive risk while maintaining sensitivity near the analytical LOD. Conclusions/Significance Our assay sensitively and specifically detects OROV RNA in serum, plasma, and cerebrospinal fluid while incorporating safeguards against viral evolution and reassortment. The assay has been approved for use by CLIA at Nexus Medical Labs in 49 U.S. states, expanding access to timely OROV diagnostics in the United States and providing a durable framework for molecular detection of reassorting, rapidly evolving viruses as OROV continues to spread into new regions.

As part of preparedness activities supporting pathogens classified under the UK High Consequence Infectious Diseases (HCID) framework, we previously evaluated both a whole-genome tiling amplicon sequencing scheme and a pan-viral hybridisation capture approach (TWIST-CVRP) for sequencing Andes virus (ANDV). In light of the recent outbreak, we make available viral sequencing datasets generated using a historical ANDV isolate (Chile, 1997). In addition, we provide an evaluation of tiling amplicon scheme performance and present recommended primer updates informed by in silico comparison with the recently released outbreak genome. These datasets are intended to support benchmarking, validation, and optimisation of bioinformatic pipelines across the community.

Ganciclovir (GCV), and its orally available pro-drug valganciclovir (VGCV), are preferred therapies for treating congenital cytomegalovirus (cCMV), however, their use carries a significant risk of neutropenia for the child. This risk limits dosing and effectiveness of VGCV, particularly in the treatment of infants with cCMV infection, who are at increased risk for sensorineural hearing loss (SNHL). We hypothesized that an improved understanding of the pharmacokinetics (PK) and pharmacodynamics (PD) of VGCV in cCMV-infected infants at risk for SNHL would inform strategies for optimizing safe and effective VGCV dosing. Participants were enrolled in one of two clinical studies interrogating the PK, safety, and efficacy of VGCV treatment in cCMV-infected infants at risk for SNHL. GCV exhibited a short median half-life of 2.02 h and the median (range) area under the 24 h concentration-time curve (AUC24) was 60.8 (26.8, 99.4) g*h/mL. An AUC24 > 70 g*h/mL was associated with an elevated risk of neutropenia (Fisher's Exact p = 0.029). No associations between GCV PK and hearing outcomes were observed. Taken together, these results indicate vast inter-individual variability in GCV PK that is associated with dose-related toxicity, supporting the need for individualized dosing in the cCMV-infected population.

Background Reactivation of human herpesviruses (HHVs), particularly EBV, is associated with more severe acute SARS-CoV-2 infections and the development of Long COVID (LC). Observations of higher anti-EBV antibody levels in individuals with LC support the idea that chronic reactivation of HHVs could contribute to LC pathology. HHV shedding in saliva has also been previously associated with saliva hormone levels. This study aims to examine the relationship between salivary shedding of HHV DNA and LC symptoms, as well as cortisol, testosterone, and estradiol levels. Methods We enrolled 45 participants with LC, and 45 age-sex-matched controls. Surveys and validated health questionnaires were used to collect demographics, medical history, and symptom profiles. Saliva was self-collected at waking, 15, 30, and 45 minutes, and 8 and 16 hours after waking, across two consecutive days. Salivary cortisol, testosterone and estradiol were measured, and extracted nucleic acid was tested for EBV, HSV 1/2, HCMV and HHV-6 A/B using multiplex qPCR, plus SARS-CoV-2 and RNaseP using RT-qPCR. Findings Detection of salivary EBV and HHV-6 DNA was highest early in the morning. There were no significant differences in salivary cortisol, testosterone, or estradiol, or in EBV or HHV-6 shedding between the LC and control groups. However, salivary HHV-6 DNA levels were positively associated with a greater aggregated LC propensity score, as well as anxiety and depression scores. Interpretation The observed correlation between salivary HHV-6 shedding and symptom severity suggests HHV-6 may contribute to post-acute disease, though mechanisms remain unclear. While our study did not identify a relationship between salivary EBV shedding and LC, EBV may still play a role at earlier time points in the disease course, or in compartments not sampled here. These findings highlight the potential importance of HHV-6 in LC pathophysiology and underscore the need for longitudinal, multi-compartment studies of herpesvirus reactivation in LC.