The Lancet Microbe

Background: Antimicrobial resistance (AMR) is a global public health problem. Infections caused by extended-spectrum beta-lactamase (ESBL) and carbapenemase (CP) -producing Enterobacterales (E) threaten individuals and healthcare systems worldwide. Symptomatic infection caused by Enterobacterales is typically preceded by asymptomatic colonisation and often occurs in the most vulnerable individuals, thus interrupting asymptomatic transmission is desirable. The dominant transmission routes across the healthcare continuum including hospitals, intermediate care, and long-term care facilities are not well understood. Methods: Here we present a protocol describing a genomic surveillance framework developed for the Tracking Antimicrobial Resistance Across Care Settings (TRACS) Liverpool programme, which aims to identify critical ESBL-E transmission points in hospitals and care homes in Liverpool, UK. Our study integrates individual participant and healthcare facility data, validated standard operating procedures for taking and culturing stool, rectal, environmental, and staff samples, and genomic sequencing of ESBL-E, and statistical modelling approaches into a research framework for ESBL-E genomic surveillance. Discussion: There is a need for improved epidemiological and laboratory approaches to studying bacterial transmission. Drug-resistant enteric bacteria are a highly tractable marker of the movement of all enteric bacteria, and interventions designed to interrupt transmission of drug-resistant bacteria are expected to have a broader healthcare impact. This protocol provides a standardised, reproducible approach for identifying ESBL-E, tracking acquisition events, and linking clinical and environmental isolates through whole-genome sequencing.

Background: People living with HIV (PLHIV) in sub-Saharan Africa exhibit high rates of pneumococcal carriage compared to HIV-uninfected adults, despite antiretroviral therapy. We established a novel controlled human infection model of experimental pneumococcal carriage in people living with HIV to understand carriage dynamics in this at-risk population. Methods: Seventy-five virally suppressed and clinically stable PLHIV and 75 HIV-uninfected controls were inoculated with escalating doses of pneumococcus serotype 6B. Carriage acquisition and density were determined by microbiological culture of nasal wash samples collected before and up to 14 days after inoculation. Adverse events were identified by active and passive surveillance. Participant-reported acceptability was established using a Likert scale. Findings: No serious adverse events occurred. Mild adverse events were similar between groups (19% [14/75] in PLHIV, 13% [10/75] in HIV-uninfected; p=0.505). More than 90% of participants reported acceptability with all study procedures. Experimental carriage occurred in 21% (16/75) of PLHIV compared with 36% (27/75) of HIV-uninfected participants (adjusted odds ratio 0.39 [95% CI 0.16-0.91]). Among PLHIV without detectable cotrimoxazole, 28% (8/29) acquired experimental carriage. Carriage clearance rates were lower in PLHIV (hazard ratio 0.44 [95% CI 0.14-1.42]). Interpretation: In carefully selected PLHIV with effective viral suppression and clinical stability experimental pneumococcal carriage acquisition did not exceed that in HIV-uninfected adults, even after accounting for antibiotic use, natural pneumococcal co-colonisation, and sociodemographic differences. These findings suggest that high carriage prevalence in PLHIV in sub-Saharan Africa may be driven more by prolonged carriage duration than increased susceptibility to acquisition. This model provides a platform to investigate mechanisms underlying carriage susceptibility and impaired clearance in PLHIV and to evaluate interventions aimed at reducing the carriage burden in sub-Saharan Africa. Funding: Wellcome Trust

Shigella sonnei is a human-adapted enteric pathogen with a very low infectious dose and increasing antimicrobial resistance. In high-income settings, transmission is multimodal including sporadic cases/outbreaks associated with food and travel, as well as sustained transmission among sexual networks of men who have sex with men (MSM). Whole-genome sequencing (WGS) now underpins national shigellosis surveillance in the United Kingdom. Hence, consistent, communicable genotyping is essential for case linkage and trend detection across heterogeneous transmission modes. Here, we evaluate the performance of WGS genotyping approaches for granulating outbreaks of S. sonnei shigellosis, particularly considering differential performance in dense sexual transmission where highly clonal MSM-associated sublineages pose distinct clustering challenges. Specifically, we compare performance of the current practice approach (10 SNP-distance clustering based on SNP address [t10]), allele-based methods (EnteroBase cgMLST/HierCC [HC5]), a pathogen-specific genotyping scheme (sonneityper), and two k-mer based approaches (PopPUNK and KPop), on a bona fide UK surveillance dataset (n = 3,639 isolates from between 2016 and 2022), and stratify analyses by demographics (i.e. presumptive MSM [pMSM] versus non-pMSM). Comparison metrics indicate that t10 clustering method groups data more broadly than HC5, and k-mer-based methods may capture genetic variation independent from SNP or allele-based approaches. Clusters derived from k-mer-based methods offer similar resolution to HC5 and reflect different demographics, but had unconvincing utility for this pathogen. These findings suggest a transmission context-aware surveillance workflow for shigellosis in high income settings: anchor routine communication on a portable allele-based backbone and augment with more granular, complementary methods (e.g., k-mer-based micro-partitioning or phylogenetic analysis) in comparatively low genomic-density regions of population structure (e.g., pMSM transmission lineages) to stabilise clusters and reduce artefactual chaining.

Background: Shigella is a leading cause of childhood diarrhea in low- and middle-income countries and is increasingly resistant to first-line antibiotics. We conducted a surveillance study to determine the incidence, genomic characteristics, and AMR profiles of Shigella infections in children under five with moderate to severe diarrhea (MSD) in Lusaka, Zambia. Methods: Between 15 September 2020 and 30 November 2021, a prospective cohort study of 1,400 children under five was enrolled during a community census in a peri-urban setting and passively followed for 9.5 months for MSD. During enrollment, socio-demographic data were collected using electronic questionnaires, while clinical data were collected through the DHIS platform. The main outcome, Shigella in diarrheal stool in under 5 children, was detected using culture and Loop-mediated Isothermal Amplification (LAMP) targeting the ipaH gene. Cox proportional hazards models were used to assess the incidence and risk factors of Shigella (ipaH) infections. Whole-genome sequencing (WGS) was used to characterize the genomic diversity and antimicrobial resistance genes, complemented by phenotypic antibiotic susceptibility testing. Results: There were 230 first episodes of Shigella over a follow-up time of 9,581.7 child-months, yielding an incidence of 24.0 (95% CI 21.1-27.3) cases per 1,000 child-months, with the highest incidence among 2 to 3-year-olds. The key risk factors identified were the water source (p=0.025) and age group (p=0.014). Genotypic characterization revealed 10 S. flexneri, 9 S. sonnei, and 3 S. boydii. The S. sonnei isolates formed two clusters, differing in virulence factors and plasmid profiles, indicating two possible circulating strains. Shigella isolates exhibited phenotypic and genotypic multidrug resistance, including against trimethoprim, aminoglycosides, and beta-lactams. Plasmid-mediated quinolone resistance (qnrS1) was identified in four S. flexneri isolates, with these genes located on the IncFIB(K) plasmid, highlighting the potential for horizontal transmission and spread of quinolone resistance in this region. No phenotypic and genotypic resistance to macrolides, the first-line treatment for Shigella in Zambia, was observed. Interpretation: We report a high burden of Shigella with multidrug resistance, including resistance to fluoroquinolones. These findings highlight the increasing resistance of Shigella to first-line antibiotics and underscore the importance of developing safe and effective vaccines, improving WASH conditions, and ongoing AMR surveillance. Funding: The EDCTP2 program, supported by the European Union, the Faculty for the Future Foundation (FFTF), the Netherlands Organization for Health Research and Development (ZonMw), and Health-Holland AMR-Global, Gloria, and Track-AMR.

Serological testing is important for assessing past exposure and immunity, but interpretation can be complicated by antibody cross-reactivity between closely related viruses. We assess this challenge for Usutu virus (USUV) and West Nile virus (WNV), flaviviruses that recently emerged in Europe. We analysed samples from wild blackbirds collected in the Netherlands between 2016-2022. Samples (N=1742) were screened using an NS1-based protein micro-array, with positives confirmed by Focus Reduction Neutralization Tests (FRNT). We jointly estimated seroprevalence and antibody responses by fitting a Bayesian latent-variable model to FRNT values. Estimates of homologous and cross-reactive antibody responses were used to improve interpretation of observed titres for serosurveillance. Estimated seroprevalence varied across time and regions between 4.9% (95%CrI 3.5-6.7) to 18.5% (95%CrI 14.9-22.7) for USUV and between 2.4% (95%CrI 1.3-3.8) to 6.4% (95%CrI 3.9-9.6) for WNV. These were 1.5 (USUV) to 2.4 times (WNV) higher than estimates based on the current threshold-based algorithm. USUV induced a higher antibody response and was more likely to induce a cross-reactive response than WNV. Our classification algorithm informed by these estimates showed high sensitivity (WNV: 0.88, USUV: 0.97) and specificity (both: >0.99). Our results illustrate how quantitative frameworks can improve serological interpretation in settings with co-circulating pathogens.

Background: Conjugative plasmids encoding New Delhi metallo-beta-lactamase (blaNDM) pose a threat for the spread of carbapenem resistance among healthcare acquired pathogens. Plasmid-associated outbreaks of blaNDM-producing bacteria can involve multiple bacterial species and persist over long time periods, making their detection and control difficult. We systematically studied the genomic epidemiology of blaNDM-encoding plasmids detected within a single hospital system over a five-year period. Methods: blaNDM-producing isolates were collected from clinical cultures as part of the Enhanced Detection System for Healthcare-Associated Transmission (EDS-HAT) genomic sequencing active surveillance program, or during infection prevention and control (IP&C) investigations. Isolates were identified as blaNDM producers by polymerase chain reaction (PCR); the presence of plasmid-encoded blaNDM genes was confirmed by sequencing on both Illumina and Oxford Nanopore platforms. Plasmids were clustered using Pling and bacterial relatedness of host isolates was evaluated with split kmer analysis. Electronic health record data were used to identify shared unit-level spatiotemporal exposures and epidemiologic links within both plasmid and host clusters. Results: We identified 61 blaNDM-producing isolates collected from 54 patients sampled between November 2020 and July 2025. Isolates belonged to 15 Enterobacterales species; Enterobacter hormaechei was the most frequently sampled species (n=23, 37%), and blaNDM-5 was the most frequently observed blaNDM allele (n=36, 59%). We observed six clusters of genetically similar blaNDM-encoding plasmids each containing 2-28 isolates, and eight singleton plasmids. The two largest plasmid clusters consisted of a highly conserved 46 kb IncX3 family blaNDM-5-encoding plasmid (n=28 plasmids, 9 species) and a more variable 98-201 kb IncC family blaNDM-1-encoding plasmid (n=12 plasmids, 6 species). Epidemiologic investigation paired with whole genome sequencing identified spatiotemporal associations between shared patient exposures and putative plasmid and bacterial transmission clusters, suggesting that unit-level exposures contribute to plasmid dissemination. Finally, analysis of publicly available sequences showed that the most prevalent plasmids detected, IncX3(blaNDM-5) and IncC(blaNDM-1), also demonstrated high global prevalence. Conclusions: This study demonstrates the diversity of blaNDM carrying plasmids within a single hospital system and their capacity to cause prolonged, multispecies outbreaks. Integrating whole genome sequencing with epidemiologic data identified unit-level spatiotemporal overlap as a likely contributor to plasmid dissemination in the hospital.

Ebola virus disease (EVD), caused by the Ebola virus (EBOV), is characterized by high morbidity and mortality, with 16 distinct EVD outbreaks reported in the Democratic Republic of the Congo (DRC), alone. As part of the formal response to the 2018 outbreaks in Equateur and North Kivu provinces, a recombinant vesicular stomatitis virus-Zaire Ebolavirus envelope glycoprotein vaccine (rVSV-ZEBOV-GP) vaccine was deployed under emergency use. While clinical trials have evaluated vaccine safety and efficacy, there is a paucity of real-world data documenting antibody durability for longer periods post-vaccination. Here, we present serologic data from 1081 individuals in Beni, North Kivu (n = 599) and Mbandaka, Equateur (n = 482) who were vaccinated during the outbreaks--with samples from baseline up to five-years following vaccination. Using a multiplexed immunoassay, we show sustained anti-EBOV GP reactivity: at year-5 collection, 72% of individuals naive at time of vaccination remained seroreactive to EBOV GP. Stratifying by site, antibody titers remained significantly elevated after baseline across all post-vaccination timepoints in both linear and logistic mixed-effects models. Pre-existing EBOV GP reactivity at baseline was the strongest independent predictor of antibody response in Mbandaka, associated with higher titers and greater odds of seropositivity (OR = 3.87, 95% CI: 2.50-6.01, p-value < 0.001), consistent with a boosting effect among previously exposed individuals. However, this was not replicated in Beni (OR: 0.66, 95% CI: 0.27-1.58, p-value = 0.348). In Mbandaka, among those recipients who reported receiving a booster dose, the odds of seroreactivity were 12.75-fold (p-value < 0.001) and 3.68-fold higher (p-value = 0.04) at 4.2 and 5-years post-vaccination, respectively, in comparison to odds of reactivity at three weeks following administration of the initial dose. Occupational groups with zoonotic or community-level exposure had trending lower odds of seroreactivity relative to healthcare workers, most consistently in Beni. Ultimately, these data indicate that five years following administration of the rVSV-ZEBOV-GP vaccine, most vaccinated individuals retain detectable anti-EBOV GP antibodies. While correlates of protection for EVD are not well established, sustained IgG seroreactivity to EBOV GP may serve as a marker for future understandings of the durability of and variation in immune responses to this high-consequence pathogen.

Global efforts to control and eliminate malaria are threatened by the emergence and spread of antimalarial drug resistance. The World Health Organization recommends surveillance of molecular markers of resistance as a complementary approach to therapeutic efficacy studies. Here, we report the first regional analysis of malaria drug resistance markers from genomic surveillance across six southern African countries spanning diverse transmission intensities and geographies. Dried blood spots, collected from rapid diagnostic test-positive individuals in Angola, Eswatini, Namibia, and Zambia in 2023, Mozambique in 2022, and South Africa between 2022 and 2024 using a standardized collection method, were analyzed using a Plasmodium falciparum targeted amplicon sequencing protocol. The distribution of resistance markers was spatially heterogeneous. Markers of artemisinin partial resistance (ART-R) were rare, never detected in >1.3% of samples from any country. However, over 30% of samples from eastern Namibia and Zambia's Western and Central Provinces carried the candidate kelch13 P441L ART-R marker. Other ART-R markers (kelch13 R515K, P553L, P574L, A675V) were detected at low frequencies in all countries except Mozambique. The wild type mdr1 N86 allele, potentially associated with reduced lumefantrine susceptibility, was near fixation across all countries. The sulfadoxine-pyrimethamine (SP) resistance dhps-dhfr quintuple mutation was approaching fixation in most districts, except northern Angola, where dhps K540E prevalence was lower and the crt K76T chloroquine resistance marker more frequently detected. This pronounced spatial heterogeneity underscores the need for timely high-resolution local resistance data generation and sharing to safeguard antimalarial drug efficacy and guide malaria control and elimination strategies across the region.

Summary Background Persistent transmission from relapsing Plasmodium vivax infections threatens malaria elimination programs in the Asia-Pacific and Americas. Tools to identify people at risk of relapse are urgently required. We aimed to validate a panel of eight P. vivax serological biomarkers for predicting future relapses. Methods In this observational study, soldiers returning from malaria-endemic Papua to non-endemic East Java, Indonesia, were screened at enrolment using antibody measurement (Luminex) and trained random forest classification algorithms, then followed for 6 months. Active case detection was performed fortnightly by microscopy. Algorithms classified soldiers as recently infected (last nine months) and thus at risk of relapse, based on anti-vivax antibody measurements at enrolment. Findings Between December 2018 and July 2022, 592 soldiers were enrolled, with 553 completing follow-up; 119 experienced a P. vivax relapse. Of these, 102 were correctly classified as at risk of relapse at enrolment, corresponding to 86% sensitivity and 86% specificity, with an AUC of 0.92. Interpretation P. vivax serological biomarkers can identify people at risk of relapse with high sensitivity and specificity and could be used as a novel public health intervention, P. vivax serological testing and treatment (PvSeroTAT), to reduce relapse-driven transmission.

Background Artemisinin derivatives are central to first-line treatment of both uncomplicated and severe Plasmodium falciparum malaria. Emerging artemisinin partial resistance in East Africa threatens to spread across the continent. Methods In two cross-sectional studies in Zambia in 2024, we genotyped the artemisinin resistance-associated gene Pfkelch13. In Kaoma, western Zambia, we evaluated the percentage of patients with day-3 parasite positivity following treatment with artemisinin-based combination therapy, and ex vivo parasite susceptibility to dihydroartemisinin (the active metabolite of artemisinin). We also assessed longitudinal changes in Pfkelch13 mutation prevalence in Kaoma using isolates collected from 2018 through 2026. Results We identified a novel mutation, Pfkelch13 A724E, in 52% (113 of 217) of isolates from Western Province, 51% (94 of 184) of isolates from North-Western Province, and 11.7% (229 of 1,949) of isolates country-wide. In Kaoma, 28% (21 of 75) of patients carrying Pfkelch13 A724E mutant parasites before treatment were parasite positive on day 3, compared with 0% (0 of 23) of patients with the wild-type allele (P=0.003). Within day-3 positive patients, the proportion of A724E mutant parasites increased significantly after treatment (P = 0.013). The prevalence of Pfkelch13 A724E in Kaoma increased steadily from 0% (95% confidence interval [CI], 0 to 22%) in 2018 to 79% (95% CI, 73 to 85%) in 2026. Conclusions A novel Pfkelch13 mutation conferring partial resistance to artemisinin is spreading in Zambia. Additional clinical evaluations are urgently needed in the region. (Funded by the Gates Foundation, INV-048316).

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

Background. The intrapulmonary pharmacokinetics of antimicrobial agents used to treat nontuberculous mycobacterial (NTM) pulmonary disease remain poorly characterized, limiting the optimization of dosing regimens. This study characterized the plasma and intrapulmonary pharmacokinetics of azithromycin, ethambutol, rifampicin, clofazimine, and amikacin, as well as their penetration into pulmonary lesion sites. Methods. We prospectively enrolled patients undergoing guideline-based treatment for NTM pulmonary disease who were indicated for surgical resection at a single center in Japan. Drug concentrations were measured in the plasma and lung samples, and analyzed using a population pharmacokinetic model. The lung lesion site, cavity, or nodule/bronchiectatic were evaluated as covariates of the plasma-to-lung partition ratios. Results. Twenty-four patients were enrolled in the study. Antimicrobial agents other than rifampicin and amikacin accumulate in the lungs at concentrations > 40-fold higher than those in the plasma. Notably, the intrapulmonary half-life of ethambutol, which has not been well-characterized to date, is estimated to be approximately 2 months, indicating prolonged retention within the lungs. Evaluation of drug penetration into cavities and nodular/bronchiectatic lesions showed no clearly reduced concentration compared to that of normal lung tissue. However, in the single case where the caseum was obtained, azithromycin, ethambutol, and rifampicin levels exhibited clearly lower concentrations. Conclusions. Ethambutol shows a prolonged intrapulmonary half-life, suggesting sustained lung exposure even with intermittent dosing. The absence of clearly reduced drug penetration into lesion sites suggests that lesion phenotype alone may have limited value in guiding drug selection.

Background: The ongoing Bundibugyo virus disease (BDBV) outbreak in Central Africa highlights the absence of approved vaccines specifically targeting BDBV. Whether licensed Zaire ebolavirus (EBOV) vaccines induce cross-reactive immunity against BDBV remains largely unknown. Methods: We performed an immunogenicity analysis using serum samples from participants enrolled in the PREVAC randomized clinical trial evaluating licensed Ebola vaccine strategies in West Africa. Samples collected at day 28 (D28) and month 3 (M3) following vaccination with rVSV{Delta}G-ZEBOV-GP or Ad26.ZEBOV/MVA-BN-Filo were assessed using a multiplex Luminex assay against glycoproteins from multiple filoviruses, including EBOV Kikwit, EBOV Mayinga, BDBV, Sudan virus, Reston virus, and Marburg virus. Results: A total of 179 samples were analysed. Detectable cross-reactive antibody responses against BDBV were observed across vaccine groups, timepoints, and age categories. However, BDBV responses remained substantially lower than homologous EBOV responses. In rVSV recipients, median BDBV responses (net MFI) reached 282 (IQR 164-644) at D28 compared with 1788 (832-3311) against the homologous Kikwit antigen. Similar patterns were observed following rVSV booster vaccination and Ad26.ZEBOV/MVA-BN-Filo vaccination. The heterologous Ad26/MVA regimen demonstrated increasing BDBV responses between D28 and M3. Conclusions: Licensed EBOV vaccines induced detectable but quantitatively reduced cross-reactive antibody responses against BDBV. Although no direct assessment of vaccine efficacy against BDBV disease was possible, these findings support the plausibility of partial heterologous immunity following EBOV vaccination. In the absence of approved BDBV-specific vaccines, these data support the urgent evaluation of currently available Ebola vaccines during BDBV outbreaks and reinforce the importance of developing broadly protective pan-filovirus vaccines.

Identifying and characterizing zoonotic pathogens in wildlife is essential for understanding disease risk to humans. In Sub-Saharan Africa, many people live with bats in their houses and are exposed to their pathogens, yet little is known about the bacterial pathogens in Afrotropical bat species. Globally, Bartonella spp. (bartonellae) and hemotropic Mycoplasma spp. (hemoplasmas) are common bacterial pathogens in bats, and some lineages are known to spill over and cause infections in humans. To evaluate this disease risk, we screened three common synanthropic bat species in Kenya, and their ectoparasites, for hemoplasmas and bartonellae and assessed their relatedness to known human pathogens. Of 767 bats across 21 sites, 17.9% of bats were Bartonella spp. positive and 19.3% were hemoplasma positive. Bat ectoparasites had similar Bartonella prevalence (13.5-25.0%) and, for most bat species, ectoparasite loads were not associated with increased likelihood of Bartonella infection. We found that Bartonella lineages displayed phylogenetic overlap between different bat species and ectoparasites, suggesting pathogen sharing between species, while hemoplasma lineages corresponded strictly to host taxonomy. Finally, we found that 16S rRNA sequences from one heart-nosed bat (Cardioderma cor) were 97.85% similar to a human-associated hemoplasma found previously in Schreibers bats (Miniopterus schreibersii) in Spain. We show that synanthropic bats host bacteria of potential public health concern, highlighting the need to investigate the emerging impacts of these pathogens on human health in Kenya and elsewhere in Sub-Saharan Africa.

Rationale: Efficacious dose selection for anti-tuberculosis drugs has traditionally relied on achieving plasma exposures above the minimum inhibitory concentration, but this approach has not consistently aligned with clinical outcomes. Objectives: We sought to identify early pharmacokinetic-pharmacodynamic targets most predictive of clinical efficacious dose. Methods: We conducted a back-translational, pharmacokinetic-pharmacodynamic simulation-based analysis of 15 anti-tuberculosis drugs. Using pharmacokinetic data from multiple biological matrices and a range of pharmacodynamic metrics, we established candidate exposure-response targets for attainment. We systematically evaluated the predictive accuracy of each target pair against established clinical doses to formulate a decision-making framework linking key drug properties to the most predictive targets. Measurements and Main Results: Depending on the target used, projected clinical doses varied widely - both within and across compounds - highlighting the importance of target selection for dose projection and go/no-go decisions. In general, targeting cellular lesion-level drug exposures relative to in vivo preclinical potency provided an effective approach for early dose selection. However, for highly penetrating drugs, targeting site-of-action therapeutic exposures in the caseum was more predictive of clinical dose. Based on these findings, we developed a preliminary dose prediction tool that enables drug developers to estimate clinically relevant dose ranges of compounds using in vitro and early in vivo data. Conclusions: This work establishes and validates a simple, evidence-based framework to standardize early translational decision-making on dose selection of anti-tuberculosis candidates in development.

Background: As tuberculosis (TB) incidence declines, transmission increasingly concentrates into vulnerable populations. There is an urgent need for affordable surveillance strategies to monitor trends, identify high-risk groups and target interventions. Mycobacterium tuberculosis (Mtb) immunoreactivity surveys indirectly detect transmission and therefore undiagnosed infectious disease. Methods: We conducted a cross-sectional community-based interferon-gamma release assay (IGRA) survey in children aged 1-4 years in Blantyre, Malawi. Community-representative participants were recruited using novel convenience sampling in health facilities alongside random household sampling, and tested for Mtb immunoreactivity using QFT-Plus IGRA. We constructed hierarchical Bayesian logistic regression models for IGRA positivity, with neighbourhood-level random effects. Findings: Of 1,545 participants, 102 (6.6%) had a positive IGRA: an annual risk of Mtb infection (ARTI) of 2.7% (95% CrI 2.2-3.2%). Immunoreactivity was higher in the poorest third of households (8.7% vs 4.9%; adjusted odds ratio: 1.88, 95% CrI 1.08-3.01) compared to the richest, but was not associated with HIV exposure, malnutrition or reported household TB exposure. There was substantial between-neighbourhood heterogeneity (ARTI range 1.1-4.1%). There was no association between neighbourhood-level TB case notifications and ARTI. Interpretation: An innovative convenience sampling approach identified a high burden and substantial spatial variation of recent TB transmission, which did not correspond to case notification rates. This strategy could support identification of high-risk populations, monitoring of trends and targeted public health interventions.

Background Healthcare-associated infections pose a major burden to neonatal health worldwide and remain difficult to track in low-resource hospitals because patient movement data and pathogen genomic data are rarely integrated into actionable transmission models. Existing approaches are often restricted to specific settings, highly structured electronic health records (EHRs), or analyses focused on either patient movements or pathogen characteristics alone. To address this gap, we developed PathoPath, an open-source integrative modelling platform, and evaluated its utility in a high burden paediatric hospital in Dhaka, Bangladesh. Methods PathoPath is an open-source R package that combines electronic health records with whole genome sequencing data to generate contact networks from direct and indirect contacts using minimal structured inputs. We retrospectively applied PathoPath to 373 cases of Klebsiella pneumoniae species complex (KpSC) infection identified in 2021 at the largest paediatric referral hospital in Dhaka, Bangladesh. Ward level patient movement trajectories were used to reconstruct contact networks, and genomic data from isolates from children <60 days were integrated to identify probable dissemination of bacterial clones and antimicrobial resistance plasmids. Findings PathoPath identified 750 direct contacts among 317 patients, forming 25 connected components, with the largest including 93 patients. KpSC infections were identified across 21 of 37 wards, with the neonatal intensive care unit accounting for 77.9% of all cases. Integration of genomic and network data distinguished sustained clustering of ST147 from multiple probable inter-clonal dissemination events involving IncFII plasmids carrying blaNDM-5 and/or blaOXA-181 within ST16. Four dominant sequence types accounted for 65.6% of sequenced isolates, and carbapenemase genes were detected in 95.8%. Interpretation PathoPath reconstructs hospital-wide contact networks and integrates them with pathogen genomics to map probable dissemination of pathogens and antimicrobial resistance using minimal structured clinical data. It could support more targeted infection prevention and control in hospitals where granular digital records are not available.

First documented detection of Vibrio paracholerae in a Costa Rican foodborne outbreak. Genomic analysis confirmed species identity, revealing limitations of conventional PCR and MALDI methods. Findings underscore the need for genomic surveillance to accurately characterize emerging enteropathogens and support public health systems.

Where malaria transmission declines, the remaining infections are increasingly low-density and asymptomatic, forming a persistent reservoir that is difficult to track using conventional epidemiological approaches. However, genomic data from such community-level infections remain scarce, limiting the ability to track parasite lineages, detect clonal expansions, and identify persistent chronic infections in pre-elimination settings. Here, 78 single-genotype P. falciparum genome sequences are analysed from community infections within a small area of The Gambia, where malaria transmission has substantially declined over recent decades. Pairwise identity-by-descent (IBD) analysis revealed generally low genetic relatedness among parasites, consistent with ongoing recombination and genetic mixing at the community scale. Nevertheless, eight clusters of near-identical genomes (IBD > 0.9) were identified, enabling the inference of recent de novo mutations that differentiate these genomes. Across these clusters, 43 de novo single-nucleotide polymorphisms and 19 short indels were identified using long-read-derived reference genomes. The observed pattern of mutation in natural infections broadly resembled that previously reported from laboratory mutation-accumulation experiments, including a strong transition bias and enrichment of G:C[-&gt;]A:T substitutions. These results demonstrate that combining IBD analysis with de novo mutation detection enables fine-scale resolution of parasite relatedness and recent transmission history. As malaria transmission continues to decline, such approaches may become increasingly valuable for tracking local transmission, identify parasite lineages, and potentially distinguish persistent infections from reintroduction events.

Effective filtration and concentration of stool specimens is an essential pre-analytical step for reducing fecal debris and improving organism recovery using microscopy-based ova and parasite (O&P) examination. This study evaluated three commercially available fecal sedimentation-based filtration/concentration systems, ParaPak SpinCon (Meridian Bioscience), Mini Parasep SF (Apacor), and the newly-available ParadeviceReingenuity), for qualitative parasite detection and workflow logistics using conventional and artificial intelligence (AI)-assisted microscopy. Forty clinical stool specimens (20 parasite-positive and 20 parasite-negative) were processed with the 3 devices, and the resultant 120 wet mount and 120 trichrome stained smear preparations were examined using conventional microscopy. Trichrome-stained slides were also scanned at 40x magnification using a Hamamatsu NanoZoomerS360 flatbed digital slide scanner and images were analyzed using the Techcyte Fusion Human Fecal Trichrome AI algorithm. Positive and indeterminate digital findings were confirmed by conventional glass slide microscopy. Slides and digital images were reviewed in a blinded manner. Concordance was assessed among the 360 initial evaluations (microscopy and AI-assisted), and discrepant parasitology results were resolved through re-review and specimen reprocessing as needed. Final qualitative agreement across slide/image evaluations using all three concentration systems was 100%. Minor discrepancies in protozoan and white/red blood cell detection/identification were noted in 5 and 7 cases, respectively, and likely reflected sampling and observer variability. While the three concentration systems produced equivalent qualitative results, the Paradevice and Mini Parasep SF offered the most streamlined workflows. These findings support the Paradevice and Mini Parasep SF as efficient, analytically equivalent systems that are compatible with traditional and AI-assisted O&P workflows.