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.

BackgroundSalmonella Isangi is an under-characterised serovar repeatedly associated with antimicrobial resistant hospital infections. Outbreaks of extensively drug-resistant (XDR) Salmonella Isangi occurred in close succession within hospitals in Malawi and South Africa, prompting us to characterise the serovar using epidemiologic, phenotypic, and genomic perspectives. MethodsIn Malawi, we integrated hospital blood culture surveillance with environmental sampling from neonatal wards and urban waterways. In South Africa, we analysed isolates from five hospitals involved in a regional outbreak. We used whole genome sequencing (Illumina and MinION) to characterise AMR genes and plasmids, assessed biofilm formation, disinfectant susceptibility, in vivo virulence, and analysed all publicly available Salmonella Isangi genomes. Findings224 / 345 (65%) of genomes in the global collection belonged to Salmonella Isangi sequence type (ST) 335. Of these, 221 (99%) originated from Malawi and South Africa, including the isolates recovered from both outbreaks. 199 (89%) ST335 genomes carried determinants of resistance to fluoroquinolones and third-generation cephalosporins, consistent with an XDR profile. In Malawi, a single ST335 clade caused the outbreak and was simultaneously present in both the hospital environment and nearby rivers. Inter-hospital transmission of a separate ST335 clade sustained the outbreak in South Africa. Closely related Malawian and South African isolates carried distinct plasmids encoding similar resistance determinants; evidence from our study and public databases suggests gene transfer via a cointegrate intermediate. Five non-outbreak South African ST335 isolates harboured additional carbapenem and macrolide resistance genes. Phenotypically, Salmonella Isangi ST335 resembled Salmonella Typhimurium in biofilm formation and disinfectant tolerance but was less virulent in mice. InterpretationSalmonella Isangi ST335 combines a locally untreatable XDR profile with nosocomial transmission and environmental persistence, suggesting a high potential for future outbreaks. A distinct and potentially greater threat lies in the horizontal spread of its resistance determinants to Salmonella Typhimurium and Salmonella Enteritidis, the two dominant invasive serovars in the region. Strengthened surveillance, integrating phenotypic testing with targeted genomics, is urgently needed. Its absence in Malawi, in contrast to South Africa, underscores inequities in preparedness for emerging AMR threats. FundingThis work was supported by the Wellcome Trust through the Core Grant (206545/Z/17/Z) and the COVID-19 Sequencing Grant (220757/Z/20/Z) awarded to MLW. Additional support was provided by the Global Health Research Professorship to Melita Gordon from the UK National Institute for Health and Care Research (NIHR) (NIHR300039). Peter Johnston is funded by the Liverpool Clinical PhD Programme for Health Priorities in the Global South, supported by the Wellcome Trust (223502/Z/21/Z). For open access, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission. Whole-genome sequencing of Salmonella isolates from South Africa was made possible by support from the SEQAFRICA project which is funded by the Department of Health and Social Cares Fleming Fund using UK aid. The views expressed in this publication are those of the authors and not necessarily those of the UK Department of Health and Social Care or its Management Agent, Mott MacDonald. Analyses in this study were supported in part through use of software and workflows developed under National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) grant R21AI178369. The NIH had no role in study design, data collection, analysis/interpretation, or publication decisions. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSSalmonella Isangi is a recurrent cause of antimicrobial resistant hospital outbreaks. We searched PubMed for Salmonella Isangi and related synonyms (to February 23rd, 2026) and identified 39 articles. No prior studies have examined transmission routes or provided phenotypic characterisation beyond antimicrobial resistance testing. Outbreaks have been reported from five hospitals on three continents, as well as a foodborne outbreak in China. Two major sequence types (STs) consistently appeared: ST335 and ST216. ST216 was widely geographically distributed and recovered from a variety of animal, meat, and environmental sources. ST335 was primarily associated with human clinical cases. Added value of this studyThis investigation was motivated by an outbreak of extensively drug-resistant (XDR) Salmonella Isangi at a hospital in Malawi. The Malawian outbreak occurred shortly before a multi-centre nosocomial outbreak in South Africa, and we provide insights from both in our analysis. We combined local epidemiology, phenotypic analyses, and global genomic characterisation to deliver a comprehensive description of the serovar. Both outbreaks were caused by ST335, which is the dominant sequence type in South Africa and Malawi, but by distinguishable clades in each country. In Malawi, genetically indistinguishable isolates were simultaneously circulating among patients, the hospital environment, and rivers throughout Blantyre City. Transfer of patients between hospitals is likely to have sustained the outbreak in South Africa. Recombination through a cointegrate intermediate may explain why the same resistance determinants are carried on distinct plasmid backbones within the Malawian and South African ST335 clades. We identify five ST335 isolates in South Africa that were not related to either outbreak and which harbour carbapenem and macrolide resistance genes in addition to an XDR genotype. Implications of all available evidenceXDR Salmonella Isangi ST335 is a major threat in Malawi because effective therapy requires antibiotics that are seldom accessible in routine care. The ability of ST335 to transmit in hospitals and to persist in the environment may increase the risk of future outbreaks. Salmonella Isangi readily acquires and maintains antimicrobial resistance determinants through diverse plasmid backbones and recombination, raising concern for transfer to locally prevalent invasive Salmonella serovars. National genomic surveillance of the kind that exists in South Africa is essential to track and contain further resistance emergence, but such surveillance does not exist in Malawi. There is an urgent need to expand genomic surveillance in low-income countries if the threat posed by Salmonella Isangi and other pathogens that drive antimicrobial resistance is to be recognised early and effectively contained.

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.

BackgroundTreatment response in canine leishmaniosis is driven by the dog host, the Leishmania parasite, and pharmacological factors, with drug resistance increasingly undermining the effectiveness of therapy. A direct quantitative PCR test (LeishGenR) was applied to 104 clinical samples from 95 dogs in the Mediterranean area diagnosed with leishmaniosis in veterinary clinical settings and testing positive for Leishmania infantum by PCR. The assay enabled rapid detection of genetic drug-resistance biomarkers for allopurinol (metk), meglumine antimoniate (mrpa), and miltefosine (LdMT), providing a clinically relevant, timely alternative to culture-based approaches by directly analyzing circulating Leishmania infantum amastigotes. ResultsThe assay (LeishGenR) showed high specificity (100%) and sensitivity (>87.5%) for genetic drug-resistance profile assignment and a strong correlation with whole-genome sequencing for gene copy number assessment (metk: r = 0.878; mrpa: r = 0.943 and LdMT = 0.691). Genetic drug-resistance biomarkers were detected in 24.3% of L. infantum DNA from clinical samples analyzed (20/82; 95% CI 16.3-34.6)), most commonly for allopurinol (13.4%; 95% CI 7.6-22.4), then meglumine antimoniate (9.4%; 95% CI 4.6-18.2), and for miltefosine (5.4%; 95% CI 1.8-14.8). Prevalence was higher in dogs previously treated for leishmaniosis. ConclusionThis study demonstrates the ability to detect genetic biomarkers of drug resistance in L. infantum directly from clinical samples of dogs with leishmaniosis. This method enables rapid, precise detection of genomic biomarkers, circumventing delays associated with culture-based methods and supporting more effective clinical management and surveillance. Among dogs with high parasitemia referred to clinics in Mediterranean regions sampled in this study, the findings reveal a significant prevalence of circulating L. infantum strains carrying genomic drug resistance biomarkers to standard treatments for canine leishmaniosis.

BackgroundReliable detection of latent Mycobacterium tuberculosis (Mtb) infection (LTBI) remains challenging, particularly in TB contacts and immunocompromised individuals, where interferon-{gamma} release assays (IGRAs) demonstrate variable sensitivity. IP-10, a chemokine produced at substantially higher concentrations than IFN-{gamma}, represents a promising immune marker. This study aimed to evaluate the diagnostic performance of two IP-10 based assays RIDA(R)QUICK TB (lateral flow) and RIDASCREEN(R) TB (ELISA), by comparison with QuantiFERON-TB Gold Plus (QFT-Plus) assay or a composite reference standard. MethodsA cross-sectional diagnostic accuracy study enrolled 99 adults: 49 with culture-confirmed active pulmonary TB, 30 close TB contacts and 20 individuals with autoimmune disease, in Bucharest, Romania. All participants underwent RIDA Quick, RIDA Screen and QFT-Plus testing. Indeterminate results for all assays were reclassified using a composite reference standard. ResultsAgainst culture in active TB cases, RIDA(R)QUICK TB demonstrated a sensitivity of 85.7% (95% CI: 72.8-94.1) and PPV of 97.7%, while RIDA(R)SCREEN TB achieved 91.8% sensitivity (95% CI: 80.4-97.7) and 97.8% PPV. Specificity and NPV could not be reliably estimated due to the near-absence of true-negative individuals. Agreement with QFT-Plus was moderate to good ({kappa}=0.47-0.93).ROC analysis performed against QFT-Plus as a comparator demonstrated good immunological discrimination for RIDA(R)QUICK TB (AUC = 0.828) and RIDA(R)SCREEN TB (AUC = 0.767), reflecting concordance with QFT-Plus rather than diagnostic accuracy against confirmed infection. ConclusionIP-10 based assays demonstrated higher sensitivity than QFT-Plus and excellent PPV across bacteriological standard, supporting their use as complementary tools for LTBI detection. Larger, more heterogeneous cohorts are needed to accurately define specificity and operational integration.

BackgroundAchromobacter spp. are emerging opportunistic pathogens, associated with chronic infections, antimicrobial resistance, and poor clinical outcomes. The Danish epidemic strain (DES) of A. ruhlandii is highly drug-resistant and adapted to the cystic fibrosis (CF) airway, yet its evolutionary history and defining genomic features remain poorly understood. MethodsWe analysed genome and antibiotic susceptibility testing data for 58 longitudinally collected DES isolates sampled over 21 years at Rigshospitalet, Denmark. We combined these with 79 publicly available A. ruhlandii genomes and applied phylogenomics to infer DES emergence and transmission, and genome-wide association studies (GWAS) to identify lineage-specific and adaptive genomic features. ResultsDES forms a distinct monophyletic clade within A. ruhlandii, estimated to have emerged around 1990, with no evidence of dissemination beyond Denmark. GWAS identified key lineage-defining traits, including acquisition of large mobile genetic elements, plasmid integration events, and enrichment of resistance and iron acquisition genes. In addition, we detected other epidemic A. ruhlandii lineages with evidence of long-term persistence and inter-country spread, sharing similar genetic signatures of adaptation. ConclusionsThis study elucidates the genomic features associated with chronic infection and epidemic potential in A. ruhlandii. The DES lineage illustrates how extensive horizontal gene transfer, high intrinsic resistance potential, and enhanced host-adaptation traits, such as increased iron acquisition, can facilitate the emergence and persistence of successful epidemic lineages. These findings highlight shared evolutionary signatures of epidemic A. ruhlandii and underscore the need for continued genomic surveillance to detect and monitor emerging high-risk lineages in chronic infections.

Conventional diagnostic methods (CDM) for Legionella preferentially detect L. pneumophila and frequently fail to identify non-pneumophila species (NPLS), obscuring the full clinical spectrum of infection and limiting surveillance accuracy. We analyzed plasma microbial cell-free DNA (mcfDNA) sequencing detections of Legionella spp. from a large clinical cohort tested between 2018 and 2024 and compared species distributions with culture and PCR confirmed cases reported in the most recent national surveillance datasets (2018-2021). To contextualize the clinical impact, we reviewed published reports in which mcfDNA sequencing was used to diagnose legionellosis (2021-2025) and evaluated real-world performance data from a hospital contributing 8.9% of detections within the cohort (Hospital A). mcfDNA sequencing identified proportionally fewer L. pneumophila, more NPLS, and fewer unresolved species than the CDC reports (all p<0.001). Among 15 publications describing 19 U.S. patients, 74% were immunocompromised and 79% had NPLS infections. Concordance between mcfDNA and CDM occurred in 31.6% of cases. At Hospital A with 36 detections, diagnosis was established by CDM alone in none, by both CDM and mcfDNA in 23.5%, and by mcfDNA alone in 76.5%, yielding an additive diagnostic value of 56.8% These findings suggest that plasma mcfDNA sequencing may improve detection of NPLS particularly in high-risk or diagnostically challenging patients and provide complementary data for both clinical diagnosis and epidemiologic surveillance.

BackgroundHousehold contact investigation for tuberculosis (TB) is limited by referral for clinic-based testing services. We evaluated the performance of in-home tongue swab (TS) testing among symptom-agnostic household contacts (HHC) to inform HCI screening strategies. MethodsWe conducted a prospective cohort study among HHC of TB patients in Eastern Cape, South Africa. In-home testing of sputum and TSs, with TSs pooled from up to three HHCs, was performed using Xpert Ultra on portable GeneExpert devices. Outcomes included diagnostic performance of TS testing relative to sputum and linkage-to-care outcomes. FindingsBetween June 2021 and October 2024, 909 HHC were enrolled; 99{middle dot}1% provided s TS, 31{middle dot}6% provided sputum. Overall sensitivity and specificity of TS testing was 61{middle dot}9% (95% CI: 38{middle dot}4%-81{middle dot}9%) and 100% (98{middle dot}9%-100%), respectively; sensitivity was 100% (47{middle dot}8%-100%) for individually tested swabs. Among two-swab and three-swab pools where 21 individual was sputum positive, 55{middle dot}6% (21{middle dot}2%-86{middle dot}3%) and 42{middle dot}9% (9{middle dot}9%-81{middle dot}6%) tested positive, respectively; TS sensitivity declined with decreasing sputum Ultra semi-quantitative category. 27 of 439 (6{middle dot}2%) households had an indictation of secondary TB; 13 (3{middle dot}0%) by sputum and TS, 11 (2{middle dot}5%) by sputum only, 3 (0{middle dot}7%) by TS only. Sputum testing identified 29 HHC with TB (yield=3{middle dot}2%); 25/29 (86{middle dot}2%) linked to care (median 1 day [IQR 1-2]). InterpretationWhile in-home molecular testing of sputum supported rapid linkage-to-care, and TSs enabled near-universal testing of symptom-agnostic HHCs, efficiency gains through pooled TS testing must be balance against sensitivity trade-offs. FundingU.S. NIH; Australian Department of Foreign Affairs and Trade; UK Foreign, Commonwealth and Development Office RESEARCH IN CONTEXTO_ST_ABSEvidence Before This StudyC_ST_ABSHousehold contacts (HHCs) of people with TB are prioritized for active case-finding (ACF) strategies due to their increased risk of developing TB disease. Household contact investigation (HCI), a widely recommended ACF strategy, is constrained by attrition from referral-based cascades and sputum-based testing. We searched PubMed and Embase for studies published in English from January 1, 2010 to January 31, 2026, using combinations of the terms "tuberculosis" or "TB" with "household contact," "contact tracing," "contact investigation," "screening," "triage," "in-home testing," "molecular testing," and "tongue swab." We also reviewed references listed in relevant articles. There are limited data describing microbiological testing strategies targeting HHCs conducted outside clinic settings, and fewer still that explore the integration of HCI and in-home molecular TB testing. Tongue swabs have emerged as a promising non-invasive, non-sputum specimen type for molecular TB diagnosis. However, most tongue swab performance data have been generated in clinic-based or symptom-prompted populations, with a marked paucity of data generated in populations at high risk for asymptomatic or paucibacillary TB, including HHC. Before this study, published work exploring the use of tongue swabs within in-home TB testing strategies was limited to two papers, both from our group, which focused on acceptability, feasibility, and preliminary costing and modeling analyses. To date, no published studies have assessed the diagnostic performance of tongue swab-based molecular testing relative to sputum-based testing among HHC, the use of tongue swab specimens as part of in-home testing strategies, nor the implication of pooled tongue swab testing to inform household-level screening and diagnostic escalation strategies. In addition, evidence describing verified linkage to TB treatment services following in-home sputum molecular testing was limited to one pilot study paper. Added Value of This StudyThis study is the first to evaluate in-home molecular TB testing using tongue swab specimens, and to incorporate household-level pooling of tongue swabs from multiple household members as a primary screening strategy. Near-universal swab collection substantially expanded access to microbiological testing in a population with limited sputum production. Although pooled swab testing exhibited reduced sensitivity compared with individual-level sputum testing, stratified analyses of tongue swab tests by sputum Xpert Ultra semi-quantitative categories demonstrate that this reduction reflects a biological gradient associated with low mycobacterial burden. Importantly, pooled swab testing identified TB among contacts unable to produce sputum, increasing diagnostic yield beyond sputum-dependent approaches. The study also documents the increase in diagnostic yield when implementing a symptom-agnostic testing strategy among HHC, and rapid, verified linkage to clinic-based TB treatment services following in-home sputum testing. Implications of All the Available EvidenceCollectively, the available evidence supports reframing TB household contact investigation from individual-level referral for clinic-based testing toward in-home testing models, including the use of household-level screening with diagnostic escalation. Near-universal, in-home collection of tongue swab specimens enables substantially broader microbiological assessment than sputum-dependent strategies and facilitates detection of TB among asymptomatic and sputum-scarce HHCs, individuals frequently missed by referral-based approaches for clinic-based sputum collection and testing. Our findings show that the reduced sensitivity associated with pooled tongue swab testing follows a predictable biological gradient related to mycobacterial burden rather than a technical failure of pooling. Pooled swab testing should therefore be understood as a household-level screening strategy within a sequential diagnostic algorithm, not a replacement for individual diagnosis. For TB programs, efficiency gains and expanded coverage achieved through pooling must be balance against sensitivity trade-offs. Household-level screening using pooled specimens can focus downstream referrals and may improve programmatic efficiency without requiring universal individual testing. Future research should evaluate optimized diagnostic algorithms that integrate pooled, non-sputum testing with diagnostic escalation, assess impact on linkage-to-care and prevention outcomes, and define the role of pooled testing within scalable, community-based TB case-finding strategies.

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 [&ge;]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.

Background: Global syphilis rates have risen dramatically since the early 2000s. Genomes can be used to inform rational control and intervention strategies by enhancing surveillance and ensuring vaccines have broad global utility. However, although >1000 Treponema pallidum genomes are now available from high-income countries, genomic data from Africa remain limited. Methods: We combined samples from 1198 participants recruited into a genital ulcer aetiology study in Botswana, Ghana, Uganda and Zimbabwe (collected 2022-2023) with 276 samples from national syphilis surveillance in South Africa to generate 147 novel African T. pallidum genomes (collected 2006-2023). Combining these with 167 publicly available African genomes and 1062 genomes from 24 non-African countries, we performed contextual population genomic analyses to understand the T. pallidum genomic diversity and transmission within and between African countries and the rest of the world. Findings: Contrasting with previous studies showing global circulation of highly similar T. pallidum, we found remarkable diversity amongst African T. pallidum. Of 56 sublineages, 20 were exclusively found amongst 6 African countries, 31 were found amongst 24 non-African countries, and 5 were found in both. Sublineage sharing between Africa and the rest of the world was rare, with 83.8% of African syphilis caused by locally circulating sublineages. Only 20.1% of African syphilis was resistant to macrolides (global average = 68.6%); where resistance occurred, this was strongly linked to introduction of global sublineages into Africa. Interpretation: African T. pallidum is characterised by locally circulating strains not found globally. Since sublineage sharing between countries is low, cataloguing African T. pallidum diversity will require intense local sampling in many countries. These findings will inform ongoing strategies for genomic surveillance and vaccine design, whilst contributing to our understanding of the spread of antimicrobial resistance in Africa, enabling refined treatment guidelines based on local data. Funding: Wellcome and the Gates Foundation.

BackgroundNeutralising antibody titres are widely used as key immunogenicity endpoints in Ebola virus (EBOV) vaccine and monoclonal antibody clinical trials. However, direct comparison of results across studies remains challenging due to the use of heterogeneous neutralisation platforms, ranging from pseudotyped viruses to live EBOV assays. These limitations restrict assay standardisation, validation, scalability, and compliance with good clinical laboratory practice (GCLP), particularly in outbreak-prone and resource-limited settings. There is an unmet need for neutralisation assays that combine biological authenticity with clinical-trial compatibility. MethodsWe developed and optimised a fluorescence-based microneutralisation assay using a biologically contained EBOV lacking the essential VP30 gene (EBOV{Delta}VP30), enabling multi-cycle viral replication under containment level 2 conditions. Using a defined panel of serum samples from Ebola virus disease survivors and EBOV-negative controls, we benchmarked EBOV{Delta}VP30 neutralisation titres against previously generated data obtained with wild-type EBOV and pseudotyped virus platforms. Assay performance was evaluated in terms of sensitivity, reproducibility, discrimination between positive and negative samples, and correlation with live virus neutralisation. Calibration was performed using the WHO International Standard for anti-EBOV immunoglobulin. FindingsThe EBOV{Delta}VP30 microneutralisation assay robustly distinguished EBOV survivor sera from negative controls (p < 0{middle dot}0001) and demonstrated a strong correlation with live EBOV neutralisation titres (Spearman {rho} = 0{middle dot}8725). This correlation exceeded that observed for HIV-1-based pseudotyped assays and for the vesicular stomatitis virus-based platforms. The fluorescence-based read-out showed comparable sensitivity to conventional immunostaining, supporting its suitability for high-throughput and standardised implementation. Importantly, assay conditions were compatible with BSL-2 laboratories and GCLP-aligned workflows. InterpretationBiologically contained EBOV{Delta}VP30 provides a clinically relevant and scalable alternative to existing neutralisation platforms, bridging the gap between pseudotyped assays and wild-type virus testing. By improving biological relevance while maintaining accessibility and standardisation, this assay has the potential to enhance comparability of immunogenicity data across EBOV vaccine and therapeutic antibody (pre-)clinical trials, aligning with global outbreak preparedness and trial harmonisation objectives. FundingStated in acknowledgement section of manuscript. Research in contextO_ST_ABSEvidence before the studyC_ST_ABSBefore starting this study, we reviewed published work on how neutralising antibodies against Ebola virus are measured in vaccine and monoclonal antibody research. We searched PubMed, Web of Science, and reference lists of key review papers for studies published up to mid-2025, without restricting by language. Search terms included "Ebola virus", "neutralising antibodies", "neutralisation assay", "pseudovirus", "live virus", and "clinical trials". We focused on studies describing neutralisation tests using wild-type Ebola virus as well as commonly used pseudotyped virus systems. From this body of evidence, neutralisation assays using wild-type Ebola virus are considered the most biologically relevant but can only be performed in biosafety level 4 laboratories. This limits their availability, scalability, and use in clinical trials. Pseudotyped virus assays can be performed under lower biosafety conditions and are widely used, but multiple studies have reported variable performance and inconsistent agreement with live virus results. Although biologically contained Ebola viruses have been developed and used in laboratory research, their application as neutralisation assays and their direct comparison with both live virus and pseudotyped systems using the same human serum samples had not been systematically studied. As a result, it remained unclear whether such systems could support reliable immunogenicity assessment in clinical trials. Added value of this studyThis study shows that a biologically contained Ebola virus lacking the VP30 gene can be used to measure neutralising antibodies in a robust and scalable way under biosafety level 2 conditions. By directly comparing this system with wild-type Ebola virus and widely used pseudotyped assays using the same set of human serum samples, we demonstrate that neutralisation results obtained with the biologically contained virus closely align with those of the wild-type virus reference assay. The assay reliably distinguishes samples from Ebola survivors and uninfected individuals and can be read using different detection methods, making it compatible with GCLP-aligned workflows and suitable for further qualification and validation in support of clinical development. This work provides clear evidence that biologically contained Ebola virus can combine biological relevance with practical usability. Implications of all the available evidenceTogether with existing evidence, our findings indicate that biologically contained Ebola virus offers a valuable new option for measuring neutralising antibodies in vaccine and monoclonal antibody clinical trials. By reducing reliance on high-containment laboratories while preserving key features of authentic virus infection, this approach can improve the consistency and comparability of immunogenicity data across studies and sites. Broader use of such assays could support better decision-making during clinical development and strengthen outbreak preparedness. More generally, this work highlights how biologically contained viruses can help advance research licensure of medical countermeasures for high-consequence pathogens in ways that are directly relevant to human health.

ObjectivesTo quantify how urine sample type and polymicrobial context impact antimicrobial resistance (AMR) in urinary tract infections (UTIs), using routine diagnostics at scale. MethodsIn this retrospective, single-centre study, we analysed 188,687 urine cultures from the Institute of Medical Microbiology, University of Zurich, Switzerland (January 2015 to May 2023). We compared midstream urine (MU), indwelling catheter (IDC), and intermittent catheter (IMC) samples. Samples were classified as negative, bacteriuria, or UTI, by meeting a microbiological UTI threshold ([&ge;]105 CFU/mL). We compared sample types using covariate-adjusted regression and constrained ordination for community composition. In bimicrobial cultures, we assessed co-occurrence using adjusted pairwise odds ratios and degree-preserving permutation null models, supported by partner-choice analyses. AMR was modelled as acquired resistance (AR) and total resistance (TR: acquired + intrinsic) probabilities, with predictor contributions quantified using mutual information. ResultsAmong 186,819 MU, IMC, IDC samples, 56,867 met the UTI threshold. Catheter-associated UTIs (IDC and IMC) were ~60% more likely to be polymicrobial than MU samples. Community composition differed by sample type (p<0{middle dot}001). In IDC, Escherichia coli was less prevalent than in MU, but device-associated pathogens like Pseudomonas aeruginosa and Candida albicans were enriched. Most species-pairs showed no increased co-occurrence after adjusting for covariates, but a subset showed reproducible enrichment across methods (e.g., C. albicans-C. glabrata). Organism identity was the dominant determinant of AMR, with the highest mutual information across AR and TR. AR was higher in IDC for common uropathogens (e.g., E. coli). Co-isolation with hospital-associated partners (e.g., Enterococcus faecium) was associated with further AR increase. From 2015 to 2023, AR increased from ~48% to ~60%, with rising {beta}-lactam (+{beta}-lactamase inhibitor) resistance and declining fluoroquinolone resistance in Enterobacterales. ConclusionsSample type and co-isolated partners provide clinically actionable information beyond pathogen identity and could support more context-aware reporting and empiric prescribing.

Colonization of the gastrointestinal (GI) tract by vancomycin-resistant Enterococcus faecium (VREfm) often precedes bloodstream infection and serves as a reservoir for onward patient transmission in healthcare settings. Routine clonal isolate-based sequencing often underestimates within-patient diversity, and can miss transmission involving low-abundance and co-colonizing strains. Here we applied culture-enriched metagenomic sequencing to matched GI tract and blood VREfm populations collected [&le;]14 days apart from 35 patients with positive VREfm blood cultures collected between 2020 and 2025 at a single hospital. GI populations exhibited greater within-patient diversity than bloodstream populations, including multi-strain colonization in five patients. Among single-strain populations, variant analysis suggested distinct environment-specific pressures between the GI tract and bloodstream environments. To assess transmission using culture-enriched metagenomic sequencing, we compared all 70 VREfm populations against 470 contemporary clinical VREfm isolate genomes collected from the same hospital and identified 19 putative transmission clusters, including six clusters involving multi-strain populations. Together, these results demonstrate how culture-enriched metagenomic sequencing improves resolution for assessing within-patient VREfm diversity and enhances the detection of transmission events that could be missed by clonal isolate-based surveillance. Impact StatementVREfm bloodstream infection is often seeded from bacteria colonizing the gut. The genetic diversity within gut and blood VREfm populations, and the role of this diversity in bacterial transmission, has been difficult to resolve as genomic surveillance typically relies on sequencing a clonal clinical isolate from each patient. Using culture-enriched metagenomic sequencing of matched GI tract and bloodstream VREfm populations from 35 patients at a single hospital, we found that the GI tract reservoir contained VREfm populations with greater strain and variant diversity than populations collected from the bloodstream. By integrating population sequencing with a large collection of VREfm clinical isolate genomes, we further demonstrate that different strains co-colonizing the GI tract of the same patient can reside in multiple putative transmission clusters, revealing potential transmission links that clone-based approaches are likely to miss. These findings demonstrate the potential utility of culture-enriched metagenomic sequencing for higher-resolution hospital surveillance of bacterial transmission. Applying this approach to other bacterial pathogens could improve our ability to detect and interpret transmission involving heterogeneous microbial populations that colonize and infect hospitalized patients. Data SummaryPatient demographic data and clinical characteristics can be found in Table S1 (online Supplementary Material). All sequencing data generated in this study has been deposited in the National Center for Biotechnology Information (NCBI) under BioProject PRJNA901969, with sample accession numbers listed in Table S2. Sequences used to construct the local reference strain database are available at NCBI BioProject PRJNA475751, with accession numbers listed in Table S3. Variants identified in single-strain blood and GI populations are listed in Table S4. Accession numbers for clinical isolate genomes included in transmission analyses are listed in Table S5. RepositoriesSequencing data generated in this study is deposited in the National Center for Biotechnology Information (NCBI) under BioProject PRJNA901969.

BackgroundClostridium perfringens can cause life-threatening extraintestinal infections in immunocompromised patients, an area in which we have little information regarding strain factors that impact patient risks and outcomes. MethodsWe conducted genomic-epidemiologic analyses on C. perfringens isolates from 70 patients seen at Brigham and Womens Hospital over 2021-2024. Genomic analyses evaluated strain profiles within a broader context of 2,321 C. perfringens genomes from foodborne, veterinary, clinical, and environmental sources to identify factors associated with invasive infections. ResultsOf 70 patients with C. perfringens infections (mean age 67.6 years), 32 had invasive infections, of which two-thirds had active malignancies, and more than half were immunocompromised. Patients with invasive infections had a significantly higher 90-day mortality of 43.8% vs. 18.4% (p=0.035) and a higher median Charlson Comorbidity Index (6 vs. 3; p=0.003). Notably, no patient isolates were clonal, verifying the absence of hospital-based transmission. Patient isolates showed increased carriage of hyaluronidases (nagHIJKL), sialidases (nanIJ), and perfringolysin O (pfoA). Genomic-epidemiologic analyses identified a new independent association between the NagL hyaluronidase (OR 3.90, 95% CI 1.14 - 16.24) in highly morbid invasive infections. ConclusionWe present a comprehensive genomic analysis of C. perfringens and of strains infecting immunocompromised patients, including epidemiologic associations of the hyluronidase NagL, NanIJ sialidase, and perfringolysin O in highly morbid invasive infections. These genes provide potential markers to identify high morbidity strains that can infect these populations and to further elucidate their role in invasive infections.

BackgroundMalaria transmission in southwestern Uganda is low, but persists despite ongoing control efforts. Identifying whether infections are locally sustained or imported by travelers is critical for guiding interventions. We integrated epidemiologic surveillance with parasite genomics to characterize imported malaria episodes at three health facilities in southwestern Uganda. MethodsBetween January 2023 and June 2024, we enrolled microscopy-confirmed malaria cases at three health facilities, Maziba and Muko (very low transmission) and Kamwezi (low-to-moderate transmission), administered travel history questionnaires, and collected dried blood spots for genotyping. Plasmodium falciparum infections were genotyped using MAD4HatTeR, a highly sensitive multiplex amplicon sequencing panel targeting 165 diversity markers and 38 drug resistance loci. Complexity of infection and pairwise relatedness were estimated using MOIRE and Dcifer, respectively. Plasmotrack, a Bayesian transmission network framework, was used to infer network structure, transmission directionality, reproduction numbers, and importation rates. ResultsAmongst malaria cases, recent overnight travel was common in Maziba (87%) and Muko (96%) but infrequent in Kamwezi (12%). Most travel in cases from Maziba and Muko was from high-transmission regions in northern and eastern Uganda. Parasites in Maziba and Muko cases exhibited higher within-host diversity and lower within-site relatedness compared to those in Kamwezi cases. Transmission network inference identified most infections in Maziba and Muko as imported, with the majority of inferred secondary transmission linked to recent travelers. In contrast, Kamwezi showed multiple highly related clusters, indicating sustained local transmission. Validated and candidate markers of artemisinin partial resistance (K13 P441L and R561H) were more prevalent in Kamwezi. ConclusionMalaria in Maziba and Muko was driven largely by importation from other parts of Uganda, while local transmission played a larger role in Kamwezi . Tailored interventions addressing travel-associated risks and local transmission, supported by travel histories and parasite genetic data will be valuable to advance malaria elimination in this region.

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.

ObjectiveThe rapid availability of phenotypic antimicrobial susceptibility results is crucial for the timely detection of multidrug-resistant Gram-negative organisms and for guiding optimized treatment strategies. Recently, novel methods have been introduced that enable direct antimicrobial susceptibility testing (AST) from positive blood cultures. However, their performance has not yet been systematically compared in head-to-head evaluations. This study aimed to assess the analytical performance of two rapid AST approaches--the agar diffusion-based EUCAST rapid AST (RAST) method and the automated QuickMIC system--using a challenging collection of highly resistant Gram-negative organisms. MethodsA total of 101 Gram-negative bacteria (Escherichia coli, n = 24; Klebsiella pneumoniae, n = 22; Acinetobacter baumannii, n = 30; Pseudomonas aeruginosa, n = 25) were spiked into blood cultures and processed according to the respective AST workflows. Broth microdilution (BMD) was performed from pure cultures as the reference method. Time to result (TTR), categorical agreement (CA), and essential agreement (EA) with BMD were evaluated. Boruta analysis was applied to identify genetic determinants associated with AST errors. ResultsOverall TTR for QuickMIC was 3 h 44 min with a CA of 86.2%, an EA of 92.3 % for Enterobacteriaceae and 97.0 % for non-fermenters. Overall CA of RAST ranged from 90.7%-93.7% across reading time points. Overall, very major discrepancy rates were low (QuickMIC n=0.7%, RAST n=0.1%). Presence of NDM-5 and KPC was most frequently associated with errors for QuickMIC and EUCAST RAST, respectively. ConclusionsBoth rapid AST approaches yielded robust results in this diverse and highly resistant bacterial study population, directly from positive blood cultures, with a short turnaround time. These findings underscore the potential of rapid AST methods to facilitate timely optimization of antimicrobial therapy in bloodstream infections, even in the context of extensively drug-resistant pathogens. ImportanceAccurate antimicrobial susceptibility testing (AST) is essential for stewardship and effective therapy, especially as rising antimicrobial resistance increases the risk of empiric treatment failure. Traditional AST methods are limited by slow turnaround times, creating a need for rapid alternatives. This study evaluated the diagnostic accuracy of two rapid AST methods--EUCAST RAST and QuickMIC--using 101 genetically characterized, carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii tested directly from positive blood cultures. Broth microdilution served as the reference. Both rapid assays provided results within 3.5-6 hours and demonstrated high categorical and essential agreement with few very major discrepancies. Incorrect results were more common in isolates harboring NDM-5 and KPC carbapenemases. Overall, the findings support EUCAST RAST and QuickMIC as reliable tools for challenging resistant pathogens and highlight their potential to enable earlier detection of carbapenem-resistant phenotypes and more timely initiation of appropriate, last-resort antimicrobial therapy.