The Lancet Microbe

Shiga toxin-producing Escherichia coli (STEC) are an important public health concern due to their association with foodborne gastroenteritis and severe outcomes including haemolytic uraemic syndrome (HUS), particularly linked to the stx2a subtype of the Shiga toxin. We investigated the temporal dynamics and acquisition of stx2a among STEC isolates submitted to the United Kingdom Health Security Agency (UKHSA) between 2016 and 2024. 12,888 whole genome STEC sequences and associated metadata were analysed. 31.9% of STEC isolates harboured stx2a, spanning 78 O serogroups with a marked shift from STEC O157 to non-O157 serogroups over time. STEC O26:H11 and STEC O145:H28 were the primary drivers of observed increases, most commonly associated with stx2a alone or in combination with stx1a. The widespread and increasing presence of stx2a across the STEC population in England highlights an emerging public health risk and demonstrates the value of routine genomic surveillance in monitoring high-severity Shiga toxin subtypes.

Third-generation cephalosporin (3GC)-resistant Klebsiella pneumoniae are an increasing public health threat in Tunisia, yet there is limited data on the circulating lineages and antimicrobial resistance (AMR) determinants underlying this threat. Here, we employed whole-genome sequencing (WGS) in the Tunisian AMR surveillance system (TARSS) to characterize the 3GC resistance mechanisms, population structure, virulence, and transmission across three participating sentinel hospitals in Tunis and Ben Arous. We sequenced a balanced sample of stored 3GC-resistant (3GCR) isolates from blood and urine collected between 2018 and 2022. Of 322 sequenced isolates, 286 (89%) were confirmed as K. pneumoniae, representing 28.5% of all stored 3GC-resistant isolates. The population structure was diverse (68 sublineages) and distinct between hospitals, although several globally distributed sublineages were detected across sites (SL383, SL101, SL307, SL15). Extended-spectrum {beta}-lactamases (ESBL) genes were detected in 77% of genomes, with blaCTX-M-15 (65.4%) and blaCTX-M-14 (8%) dominant at all sites and across diverse sublineages. AmpC genes occurred in 9%, and carbapenemase in 19.6% (blaOXA-48, 14.7%; blaNDM-5, 4.5%; blaNDM-1, 3.8%), with carbapenemases mainly observed amongst SL147 and SL383 at Hospital B (41.7%). Despite sequencing less than a third of the unique 3GCR infections in each hospital, we identified 24 probable nosocomial transmission clusters involving 64 isolates. Each cluster was restricted to a single hospital, although many were detected across multiple wards in the same hospital. The acquired virulence-associated locus (ICEKp) encoding yersiniabactin was common (48.6%). Hypervirulence-associated markers (encoding aerobactin, salmochelin, and/or hypermucoidy) were rare (8.7%) but increasing over time. These were mostly found in sublineages in which convergence of ESBL and hypervirulence has been reported in other settings (including SL147, SL101 and SL383), suggesting international dissemination of convergent strains. These findings show sustained ward-level nosocomial transmission of 3GCR K. pneumoniae lineages and site-specific differences in ESBL and carbapenemase burdens, which call for targeted infection prevention and control and for future routine integration of WGS into TARSS.

Klebsiella pneumoniae (KP) isolates belonging to multi-locus sequence type 258 (ST258) are a frequent cause of hospital-associated outbreaks and display extensive multidrug resistance. The KP ST258 lineage consists of two genetically distinct clades, called Clade 1 and Clade 2. These two clades are genetically related to one another, but are historically distinguished by having different capsular polysaccharide types. While bacteria belonging to both clades are isolated from clinical infections, Clade 2 is isolated more frequently compared to Clade 1. To investigate drivers of this difference in clade prevalence, we collected 172 clinical KP ST258 isolates from patients at a single medical center. Clinical review showed that patients infected with Clade 2 isolates were more acutely ill than Clade 1-infected patients, despite having fewer comorbidities. We also found that Clade 2 isolates were more resistant to killing by human serum, despite binding more complement protein C3 than Clade 1 isolates. Additionally, mice infected with a Clade 2 isolate had increased bacterial dissemination from the lungs to the liver and spleen than mice infected with a Clade 1 isolate, and this dissemination required an intact capsule locus. Increased dissemination in mice was not due to differential serum killing, as mouse serum was unable to kill isolates of either clade, but dissemination was associated with decreased macrophage uptake of the Clade 2 isolate. Taken together, these data suggest that KP ST258 Clade 2 is more virulent than Clade 1, though the specific mechanisms at play appear to differ between mice and humans.

Aminoglycoside drugs, amikacin, streptomycin, and amikacin liposome inhalation suspension are crucial for treating refractory Mycobacterium avium-intracellulare complex pulmonary disease. In Mycobacterium tuberculosis, cross-resistance occurs between amikacin and kanamycin, but not between amikacin and streptomycin in genetic drug susceptibility testing. However, the occurrence of cross-resistance among aminoglycosides remains unclear in M. avium-intracellulare complex. We aimed to evaluate cross-resistance among aminoglycosides to determine whether streptomycin or kanamycin remains effective after the development of amikacin resistance. This single-center retrospective study included 20 patients with amikacin-resistant M. avium-intracellulare complex harboring rrs mutations. Paired analyses of streptomycin and kanamycin minimum inhibitory concentration values before and after amikacin resistance development were performed. In addition, streptomycin- and kanamycin-resistant strains were generated in vitro and resistance-associated mutations were identified using whole-genome sequencing. No significant increase was observed in streptomycin minimum inhibitory concentration values following amikacin resistance. In contrast, kanamycin values uniformly increased to >256 g/mL after the acquisition of amikacin resistance. Furthermore, amikacin- and kanamycin-resistant isolates shared mutations at position 1408 in the rrs gene, whereas streptomycin-resistant isolates exhibited mutations at position 20 in the rrs gene. These results suggest that amikacin and kanamycin exhibit cross-resistance in M. avium-intracellulare complex, whereas amikacin and streptomycin may not. Two cases in our cohort in which streptomycin treatment was effective after the acquisition of amikacin resistance further support these findings. In conclusion, streptomycin may be a potential therapeutic alternative for amikacin-resistant M. avium-intracellulare complex pulmonary disease. Future studies correlating streptomycin minimum inhibitory concentration values with clinical outcomes are required.

Rationale: Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives: To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods: We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results: Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of [≥]2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions: Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance.

Infections caused by carbapenem-producing Enterobacterales (CPEs) are a persistent and growing threat in healthcare settings. Yet, current infection prevention and control (IPC) surveillance methods, which largely rely on the spatial and temporal proximity of patients, often misattribute or miss infection transmission events. Here, we develop and retrospectively evaluate an integrated methodology that combines analyses of ward-level patient movement data and whole-genome sequencing (WGS) data analyses, providing measures of bacterial and plasmid similarity. Specifically, we evaluate this methodology across two datasets: a CPE outbreak of diverse carbapenem types (103 genomes, January 2021 to March 2021) and an Imipenem-Hydrolysing beta-lactamase-positive CPE outbreak (82 genomes, June 2016 to October 2019), using standard clinical criteria and conservative genomic thresholds to quantify how often current IPC surveillance methods correctly identify genomically confirmed transmission events. Findings show that, across 3,423 patient contact-genome pairs, current IPC surveillance methods detected only 20.5% of genomically confirmed transmission events whilst maintaining 98.5% specificity, with missed events arising from temporal, spatial, and cross-species, mechanistic blindspots. In contrast, WGS-enabled IPC surveillance methods provided a 25 to 47-day earlier detection window and, in a linked economic evaluation, delivered annualised savings of up to GBP 3.6 million, as well as a return on investment exceeding 2-fold in 7 of 8 cost scenarios. By operationalising high-throughput WGS data analysis with clinically relevant patient movement data, we evidence that it may be possible to disrupt and thereby mitigate the effects of AMR-driven CPE outbreaks, supporting investigations into the adoption of WGS-enabled IPC surveillance as a standard-of-care tool.

Numerous factors may influence the optimal rollout of new gonococcal antibiotics. We compared eight rollout strategies using a gonorrhea transmission model and ranked strategies by the number of gonococcal infections and clinically useful antibiotic lifespan. Rankings were most sensitive to the starting ceftriaxone resistance prevalence and screening frequency.

Extended-spectrum beta-lactamase-producing Enterobacterales such as Escherichia coli and Enterobacter hormaechei represent a growing public health challenge in clinical settings, particularly in low-and middle-income countries, due to the escalating threat of antimicrobial resistance (AMR). In this study, we aimed to identify the antibiotic resistance genes present in E. coli (n=4) and E. hormaechei (n=3) clinical isolates. Multidrug-resistant phenotypes were confirmed using disc diffusion assays against 20 antibiotics. Whole-genome sequencing of resistant isolates was performed using Oxford Nanopore Technologies. Genome assembly and analysis revealed high-risk clones, including sequence type (ST) 1193 in E. coli and ST78 in E. hormaechei. All E. coli isolates harbored the blaCTX-M gene in their chromosomes along with point mutations conferring resistance to fluoroquinolones, while E. hormaechei isolates encoded blaACT in their chromosomes. Additionally, both species carried plasmids with multiple antibiotic resistance genes, including blaOXA and blaTEM, co-located with metal resistance operons, indicating the potential for horizontal gene transfer. BLAST analysis revealed high sequence similarity between the plasmids identified in clinical isolates and those previously recovered from environmental sources, highlighting the role of environmental reservoirs in AMR dissemination. Notably, no carbapenem resistance genes were detected in any isolate. These findings underscore the growing threat posed by multidrug-resistant Enterobacterales in clinical settings and emphasize the urgent need for strengthened infection prevention and control measures to mitigate AMR spread.

Drug-resistant tuberculosis (TB), characterized by prolonged treatment regimens and suboptimal treatment outcomes, remains a major obstacle to global TB elimination. Advances in sequencing technologies have enabled the development of machine-learning (ML) approaches, including deep-learning (DL) methods, to predict drug resistance directly from genomic data. However, a significant gap remains in translating these advances into clinical practice. While current approaches reliably predict resistance to first-line drugs, they show consistently lower and more variable performance for second-line drugs compared with traditional drug-susceptibility testing. To characterize these limitations and assess practical utility, we conducted a comprehensive survey and standardized benchmarking of current approaches for predicting TB drug resistance using whole-genome sequencing (WGS) data. Using systematic selection criteria, we identified 20 traditional ML and DL models from 8 studies and evaluated drug-specific versions across 14 second-line drugs within a unified framework. To account for methodological heterogeneity, the models were evaluated using three distinct feature sets reflecting variability in input representations. We trained and evaluated the models on different subsets of the WHO dataset, comprising 50,801 samples, and assessed generalizability using an external validation dataset comprising 1,199 samples. In the internal evaluation on the held-out WHO test dataset, traditional ML models using binary features achieved higher predictive performance than DL models. For example, XGBoost achieved the highest area under the precision-recall curve (PRAUC) scores (46%-93%) for 10 of the 14 drugs. However, performance varied substantially across drugs. Notably, the superior performance of traditional ML models -- even with limited feature sets -- highlights their applicability in low-resource settings. When evaluated on the external validation dataset, the performance of traditional ML and DL models was comparable, and neither class of models demonstrated substantial improvement over catalogue-based approaches, underscoring challenges in cross-dataset generalization. Overall, this benchmarking study provides a comprehensive and systematic evaluation of current approaches, establishes a rigorous evaluation framework for future comparisons, and identifies key methodological considerations necessary to advance robust drug resistance prediction in clinical settings. To enhance reproducibility and facilitate the application of TB-Bench to additional datasets and models, we have made the source code publicly available at https://github.com/BIRDSgroup/TB-Bench.

Background: Partial resistance to artemisinins (ART-R) has emerged in East Africa, associated with mutations in the Plasmodium falciparum kelch13 gene. It is currently unclear whether ART-R has implications for gametocyte production or for onward transmission to mosquitoes. Methods: In a cohort of uncomplicated malaria patients attending Kalongo Hospital in northern Uganda, we quantified carriage of PfKelch13 mutant parasites by conventional sequencing and droplet digital PCR (ddPCR) for the C469Y and A675V mutations. Prevalence and density of gametocytes and ring-stage parasites were assessed by microscopy and quantitative reverse-transcriptase PCR (qRT-PCR). Lumefantrine concentrations, indicative of prior malaria treatment, were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. Transmission potential of wild-type and PfKelch13 mutant parasites was assessed by mosquito feeding assays and complemented with molecular characterization of parasites in wild-caught mosquitoes from household resting catches. Findings: We enrolled 235 patients with symptomatic P. falciparum infection; PfKelch13 C469Y or A675V mutations were detected in 35.8% (78/218) of infections by sequencing and 59.1% (136/230) by ddPCR. Gametocyte carriage was 24.0% (56/233) by microscopy and 56.6% (133/235) by qRT-PCR and not associated with the abundance of PfKelch13 mutant parasites by ddPCR (p=0.603). Among a total of 227 mosquito feeds with patient whole blood, 1.4% (120/8745) of mosquitoes became infected. Mosquito infection rates were positively associated with gametocyte density ({beta} = 0.39, 95% CI = 0.23-0.59, p < 0.001) without an observed interaction with the abundance of PfKelch13 mutant parasites (p = 0.452). PfKelch13 C469Y or A675V mutations were detected in 40.1% (21/52) of malaria-infected bloodmeals of field-caught mosquitoes and in 28.0% (7/25) of sporozoite-positive mosquitoes. Interpretation: We conclude that pfkelch13 mutations are very common in patients in northern Uganda with uncomplicated malaria, mostly in multiclonal infections. We observed no evidence that ART-R affected gametocyte production or transmission to mosquitoes. Funding: Dutch Research Council (NWO)

Background: Prolonged SARS-CoV-2 infection in immunocompromised individuals may accelerate virus evolution within the host, raising concerns about the virus evading immunity, developing resistance, and forming novel variants of concern. However, the determinants and public health implications of within-host viral evolution in this population remain incompletely understood. Methods: We performed longitudinal analyses of SARS-CoV-2 genomes from 91 patients with COVID-19 who were classified as being severely or moderately immunocompromised. Using serial measurements of viral RNA loads and infectious titers, we modeled the shedding dynamics of the virus and stratified the infected cases by upper respiratory virus shedding duration to assess associations with within-host evolutionary dynamics. Results: Shedding modeling identified two profiles of shedding duration: intermediate and long. The long shedding profile (shedding lasting >21 days) was found in 14.8% of moderately immunocompromised cases and 72.1% of severely immunocompromised cases. Frequent single-nucleotide variants accumulated specifically in severely immunocompromised individuals with the long shedding phenotype, correlating positively with shedding duration. By contrast, mutations remained limited in moderately immunocompromised individuals with the long shedding phenotype and in severely immunocompromised individuals with the intermediate shedding phenotype. We identified mutations in the spike receptor-binding domain associated with monoclonal antibody resistance; however, we found no fitness-enhancing mutations for inter-host transmission, and antiviral drug resistance mutations were rare. Instead, mutations were introduced frequently and randomly across the entire viral genome. Conclusions: Prolonged upper respiratory virus shedding exceeding 21 days combined with severe immunocompromise is a risk factor of the accumulation of within-host SARS-CoV-2 mutations. Although no variants of concern emerged, the introduction of genome-wide random mutations suggests that the risk for novel variant generation cannot be excluded. These findings highlight the need for intensive antiviral strategies to limit shedding duration to less than 21 days in severely immunocompromised patients, and for immunological investigations to elucidate the host factors underlying residual shedding control in those who achieve clearance within this threshold.

BackgroundThe Toto Bora trial tested whether a course of azithromycin reduced rates of re-hospitalization or death in the 6 months following hospitalization among Kenyan children. We hypothesized that azithromycin would reduce enteric bacteria and increase carriage of macrolide resistance in the subsequent 3 months. MethodsKenyan children (1-59 months) hospitalized and subsequently discharged for non-traumatic conditions provided fecal samples before and 3 months after randomization to a 5-day course of azithromycin or placebo. Quantitative PCR identified enteropathogens and AMR-conferring genes in fecal samples. Generalized estimating equations assessed the impact of the randomization arm on pathogen and resistance gene detection, accounting for baseline presence and site. ResultsAmong 1,393 baseline stools, 12.4% had at least one bacterial enteropathogen, 94.7% had at least one macrolide-resistance gene, and 92.6% had at least one beta-lactamase-resistance gene identified. At month 3, children randomized to azithromycin had a 6.1% higher likelihood of carrying a macrolide resistance gene compared to placebo (adjusted prevalence ratio [aPR], 1.06; 95% CI, 1.04-1.08; P<0.001). Specifically, azithromycin randomization was associated with a higher relative prevalence of erm(B) (aPR, 1.09 [95% CI, 1.04-1.15]; P=0.001), erm(C) (aPR, 1.23 [95% CI, 1.14-1.31]; P<0.001), msr(A) (aPR, 1.14 [95% CI, 1.04-1.25]; P=0.007), and msr(D) (aPR, 1.07 [95% CI, 1.03-1.11]; P=0.001). There was no difference in overall bacterial pathogen prevalence (18.9% vs 17.3%) between randomization arms, but a slightly lower proportion of children had Shigella after randomization in the azithromycin arm (3% vs. 5%, aPR, 0.79 [95% CI, 0.62, 1.01]; P=0.063). InterpretationAzithromycin at hospital discharge was associated with higher carriage of macrolide-resistance-conferring genes in the post-discharge period compared with placebo, without significant declines in enteric pathogen carriage other than modest changes to Shigella. The potential benefits and risks of empiric azithromycin need to be considered, as children are increasingly exposed to this broad-spectrum antibiotic.

Acinetobacter baumannii is a major cause of severe hospital-acquired infections, with a steadily increasing global prevalence driven by a few clinically adapted lineages. Animals and natural environments also harbor A. baumannii populations, but assessing their connections to clinical lineages is limited by sparse genomic data and a lack of integrated sampling. We conducted a local One Health genomic epidemiology study, sampling, isolating, sequencing, and characterizing several hundred A. baumannii isolates from clinical, animal, and environmental contexts. Within a geographically restricted area, we recovered several globally distributed clinical lineages (international clones, ICs), as well as livestock- and environment-associated lineages shared across Europe, highlighting widespread dissemination beyond clinical settings. Isolates closely related to the emerging clinical lineage IC11 were found in livestock, but no other clinically associated lineages were detected outside clinical contexts. Among these, the epidemic superlineage IC2 was identified in both human and veterinary clinical settings, indicating that similar practices in human and animal medicine select for closely related opportunistic pathogens. We found that hospitals host distinct, antibiotic-sensitive endemic populations capable of causing infection. These populations belong to a diversifying clade spanning clinical and environmental contexts and carry a high load of insertion sequences. Strong plasmid conservation further suggests frequent horizontal gene transfer across ecological compartments. Overall, A. baumannii comprises diverse, context-adapted lineages with a high potential for global spread. Although intercontext transmission appears limited, plasmids may overcome these ecological barriers. Our findings underscore the need for integrated One Health surveillance to better understand transmission pathways and limit the emergence of clinically adapted strains.

Background Pseudomonas 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. Aim The aim of this study was to showcase the long-term survival and transmission of P. aeruginosa in a large tertiary children's hospital in England which is crucial to develop strategies for water-safe care. Methods Environmental P. aeruginosa isolates were collected from taps, sinks, showers, and baths in augmented care areas of a 330-bed tertiary children's 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. Findings This retrospective study analysed nine-locus VNTR profiles of 457 P. aeruginosa isolates submitted to the UK Health Security Agency from a large tertiary children's hospital, identifying 56 isolate clusters (each with [&ge;]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. Conclusion These 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.

Background: Binary interpretation of Mycobacterium tuberculosis (Mtb) interferon gamma release assay (IGRA) results discards information about recency of exposure and disease risk. We analysed quantitative IGRA responses to Mtb in a community--based survey to investigate associations with response magnitude and inform understanding of transmission dynamics. Methods: We included QuantiFERON--TB Gold Plus (QFT--Plus) results from 2,895 participants (10--40 years old) in Blantyre, Malawi. Bayesian regression models assessed the probability of a positive response ([&ge;]0.35 IU/mL), response magnitude, and associated factors. We also investigated associations with a TB2-TB1 differential >0.6 IU/mL (proposed to reflect recent transmission), and how hypothetical alternative IGRA positivity thresholds affected inference about age-- and sex--specific transmission. Results: 17.4% (503/2,895) of participants had positive TB1 or TB2 responses at the QFT--Plus positivity threshold (0.35 IU/mL). The distributions of TB1 and TB2 responses, among participants with positive QFT--Plus, were similar across age and sex. A TB2-TB1 differential >0.6 IU/mL occurred in 3.8% (109/2,895) of participants and was not associated with age or sex. However, participants with HIV had reduced odds of TB2-TB1>0.6 IU/mL (adjusted odds ratio 0.37 [0.14--0.93]). At higher hypothetical positivity thresholds, the mean predicted Mtb immunoreactivity prevalence among males exceeded that in females at an earlier age: at 19 years, predicted immunoreactivity prevalence ratios were 0.90 (0.83--0.99) and 1.02 (0.89--1.15) at 0.1 IU/mL and 0.5 IU/mL thresholds, respectively. Conclusions: Quantitative IGRA responses offer information about age-- and sex--specific immunoreactivity and transmission risks that dichotomisation obscures. In high-burden settings, quantitative IGRA responses may clarify Mtb transmission patterns and guide targeted public health strategies.

Background Five-biomarker-defined hypervirulent Klebsiella pneumoniae (hvKp) causes invasive infections, but its burden in bloodstream infections versus classical K. pneumoniae (cKp) is unclear. Methods This retrospective cohort study at a tertiary hospital in Japan included K. pneumoniae bloodstream infection episodes from January 2022-December 2024. hvKp was defined by the presence of all 5 genotypic biomarkers (rmpA, rmpA2, iucA, iroB, and peg-344). The primary outcome was abscess complications, and secondary outcomes were length of stay and antibiotic duration. Whole-genome sequencing was performed for 164 isolates. Results Among the 207 episodes, 28 (14%) were of hvKp. Abscess complication occurred in 17 (61%) hvKp versus 23 (13%) cKp episodes (adjusted odds ratio 10.7; 95% CI, 4.36-26.2). Median length of stay in hvKp versus cKp was 28 versus 14 days (adjusted ratio 1.60; 95% CI, 1.18-2.16) and median antibiotic duration was 43 versus 14 days (adjusted ratio 2.13; 95% CI, 1.64-2.77). These associations were attenuated after adjusting for abscess-related complications. No significant difference in 30-day mortality was observed, although the study was underpowered. Multidrug resistance was less frequent in hvKp strains than in cKp strains (11% vs. 30%; P = .040). Among the sequenced hvKp episodes, abscess rates varied across lineages, from 9 of 10 in ST23 to 1 of 4 in ST412. Conclusions Five biomarker-defined hvKp strains delineated a bloodstream infection subgroup with frequent abscess complications and prolonged care. hvKp and cKp present distinct clinical challenges; diagnostic tools distinguishing these subgroups may aid abscess evaluation and source control.

Background: Mucormycosis 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. Methods: A 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. Results: mAb 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. Conclusion: The 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.

Background: In Rwanda, genomic surveillance identified a dominant multidrug-resistant tuberculosis (MDR-TB) strain, the R3clone, responsible for approximately 70% of rifampicin-resistant TB cases. Its presence beyond Rwanda remains unexplored. Methods: Unique genetic signatures of the R3clone were defined using whole-genome sequencing of MDR-TB isolates from Rwanda. We developed a targeted qPCR assay detecting a clone-specific single-nucleotide polymorphism. With these tools, we screened isolates from neighbouring countries and public genomic repositories. Results: We identified 375 R3clone isolates, including 264 from historical Rwandan collections (1991-2021), 49 from recent Rwandan diagnostic routine (2021-2024), 25 from historical Burundi isolates (2002-2013), and 37 among public repositories from several countries. The R3clone-specific qPCR showed 100% specificity in distinguishing the R3clone from other MTBC (sub-)lineages. Transmission analysis revealed cross-border transmission of the R3clone within the Great Lakes Region. Conclusion: This study comprehensively assesses cross-border transmission of a dominant MDR-TB strain, highlighting the need for coordinated international surveillance.

Mpox poses an ongoing global public health threat, with case numbers rising beyond traditionally endemic regions in Central and Western Africa. Rapid detection of the causative agent, the Monkeypox virus (MPXV), is critical for outbreak control, yet laboratory infrastructure and trained personnel remain scarce in many affected areas. Point-of-care molecular diagnostics offer a practical solution by enabling timely testing without specialized equipment or elaborate nucleic acid extraction. We evaluated the performance of an extraction-free RNase HII-assisted amplification (RHAM) assay for MPXV detection by Pluslife Biotech, a novel isothermal amplification technology providing results in under 30 minutes. The Pluslife RHAM test demonstrated pan-MPXV clade reactivity, detecting all four MPXV clades (Ia, Ib, IIa, IIb) with high analytical sensitivity and no cross-reactivity to other poxviruses or other clinically relevant pathogens. The assay proved compatible with diverse clinical specimen types, including lesion swabs, oropharyngeal swabs, rectal swabs, urine, semen, and wound exudate. As part of routine diagnostics at the German Consultant Laboratory for Poxviruses, in a comprehensive evaluation of 206 clinical specimens against diagnostic real-time PCR, the Pluslife RHAM test achieved a diagnostic sensitivity of 94.2% (95% CI: 85.8-98.4%) and a specificity of 100% (95% CI: 97.3-100%). Notably, samples with higher viral loads (Ct <30) showed 100% sensitivity. Time-to-result correlated significantly with viral load, enabling faster diagnosis in high-viral-load cases. The Pluslife RHAM test represents a practical, sensitive, and rapid point-of-care solution for MPXV detection in resource-limited settings, combining strong analytical performance with operational simplicity to support timely outbreak response and clinical decision-making.

Objectives: Scrub typhus, caused by the bacterium Orientia tsutsugamushi, is frequently underdiagnosed due to its non-specific clinical presentation and the frequent absence of eschar. Most molecular diagnostic assays target single-copy genes of O. tsutsugamushi, which can limit diagnostic sensitivity. We aimed to develop an ultra-sensitive quantitative PCR (qPCR) assay targeting a highly repetitive element in O. tsutsugamushi genome. Methodology: We developed a SYBR Green-based qPCR assay (TranScrub) targeting a multicopy transposase gene of O. tsutsugamushi and compared its performance with assays targeting the 56kDa (single-copy) and traD (multicopy) genes. Diagnostic performance was evaluated using clinical specimens and a panel of blood-borne pathogens. The limit of detection (LOD) was estimated using serial dilutions of quantified template. The assay was further applied to dried blood spot (DBS) samples from patients with acute febrile illness of unknown aetiology, with positives confirmed by Oxford Nanopore amplicon sequencing. Results: Targeting the multicopy transposase gene enabled highly sensitive detection of O. tsutsugamushi, outperforming the conventional 56-kDa assay and matching the traD assay. TranScrub achieved a 91% sensitivity (29/32) and 100% specificity (77/77) using blood-derived DNA, with no cross-reactivity. The LOD was 0.024 genome equivalents/L. Among 81 DBS samples from acute febrile patients of unknown aetiology, 6 (7.5%) tested positive, all confirmed by sequencing. Conclusions: The transposase gene represents a novel target that improves molecular detection of scrub typhus. TranScrub enables sensitive and specific detection from both blood and DBS, supporting its use in clinical diagnosis and field surveillance.