The Lancet Microbe

Background: Invasive non-typhoidal Salmonella (iNTS) disease causes an estimated 605,000 cases and 76,000 deaths each year, concentrated in sub-Saharan Africa, where the African Salmonella Typhimurium sequence type 313 (ST313) lineage predominates. Vaccine development is hampered by an absence of efficacy data and undefined correlates of protection. Methods: We conducted a phase 1, randomised, double-blind, dose-escalation controlled human infection model (CHIM) in healthy UK-resident adults, who were randomly assigned 1:1 to oral challenge with S. Typhimurium 4/74 (ST19, associated with gastrointestinal disease) or D23580 (ST313, associated with invasive disease). Dose-escalation was guided by a Bayesian continual reassessment method (CRM). The primary endpoint was Salmonella diagnosis, defined as sustained fever [≥]38{degrees}C on [≥]2 occasions [≥]12 hours apart and/or bacteraemia. Trial registration ClinicalTrials.gov (NCT05870150). Findings: Between August 2023 and December 2024, 50 participants were enrolled (25 per strain). 105 CFU was the maximum feasible dose, with CRM-estimated attack rates of 57.9% (95% credible interval 37.3 - 73.8) for D23580 and 47.4% (26.7 - 65.7) for 4/74. There were no serious adverse events. We found no clinical, microbiological, or immunological difference between the two pathovariants. Higher baseline serum anti-O-antigen IgG was associated with reduced disease (adjusted OR 0.42, 95% CI 0.17 - 0.90) and higher baseline faecal anti-lipopolysaccharide IgA with reduced colonisation (OR 0.12, 95% CI 0.01 - 0.59). Interpretation: This S. Typhimurium CHIM is safe, reproducible, and provides a platform to generate early efficacy signals and candidate correlates of susceptibility, thereby de-risking future iNTS vaccine trials. The absence of a phenotypic difference between the invasive and gastrointestinal pathovariants in immunocompetent adults suggests that host factors, rather than pathogen adaptation alone, shape the invasive phenotype seen in endemic settings. Funding: Wellcome Trust.

Background and Aims The optimal treatment for first episodes and first recurrences of Clostridioides difficile infections (CDI) is unknown and there is emerging evidence for pulse and taper (P-T) regimens. Therefore, we sought to estimate the relative efficacy of treatment options. Methods MEDLINE and CENTRAL were searched from database inception to May 21, 2025 and unpublished conference abstracts were searched from recent infectious disease conferences. RCTs on the treatment of first episodes or first recurrences of CDI comparing fixed-dose or P-T regimens of fidaxomicin or vancomycin were included. The primary and secondary outcomes were 40- and 56-day CDI recurrence, respectively. A random-effects network meta-analysis on the risk ratio (RR) scale was conducted using a standard regimen (10-14 days) of vancomycin as the comparator. Treatments were ranked using the surface under the cumulative ranking curve (SUCRA). Results 8 RCTs were included comprising a total of 2181 patients. For 40-day recurrence, fidaxomicin P-T had the highest probability of ranking best (RR=0.10, 95%Confidence Interval [95%CI]=0.10-0.49, SUCRA=1.00), followed by vancomycin P-T (RR=0.49, 95%CI=0.32-0.76, SUCRA=0.61), fixed-dose fidaxomicin (RR=0.61, 95%CI=0.49-0.76, SUCRA=0.39), and, finally, fixed-dose of vancomycin (SUCRA=0.00). The treatments ranked in the same order for 56-day recurrence, though only 3 RCTs reported on this timepoint. Conclusion Vancomycin P-T, fidaxomicin P-T, and fixed-dose fidaxomicin were all superior to a fixed-dose vancomycin. Head-to-head comparative effectiveness RCTs are needed to quantify their relative effect sizes of and impact on long-term prevention of recurrent CDI.

Background: Vancomycin-resistant Enterococcus (VRE) species are common healthcare-associated pathogens that cause difficult-to-treat infections. Whole genome sequencing of patients has revealed a substantial burden of patient-to-patient VRE transmission in hospitals, with patients in intensive care units (ICUs) at particularly high risk of acquisition. However, few studies adequately characterize the pathways of VRE transmission between patients in acute care settings, a necessary step to identify current gaps in infection prevention practices. By harnessing genomic clustering analyses of whole genome sequences of VRE isolates from patients, environmental surfaces, and healthcare providers (HCP) in ICUs, we aim to reconstruct indirect pathways of pathogen movement to identify patterns of VRE spread and opportunities for transmission prevention. Methods and Findings: We collected daily samples (N = 6848) from ICUs in two hospitals over 13 weeks from four main sampling sources: patients, HCP hands, patient rooms, and shared surfaces. Samples were cultured on selective media and sent for whole genome sequencing (WGS). We used genomic thresholds to identify clusters of related VRE isolates and distinguish unrelated isolates. VRE was detected in samples from 20 out of 322 unique occupant-stays (6.22%). VRE isolates were detected from all sampling sources except for shared surfaces. A total of 44 unique VRE isolates were identified, 43 Enterococcus faecium (VREfm) and one Enterococcus faecalis (VREf). Two distinct patterns of VREfm spread were observed: 1) an outbreak setting with observed patient-to-patient transmission and low VRE diversity, and 2) high VRE diversity and pathogen movement between occupant-stays facilitated by persistent HCP and environmental contamination, but no observed transmission events. VRE detection probabilities were not significantly different between occupant-stays in outbreak and non-outbreak settings (OR = 0.63, 95% CI (0.23, 1.83), p = 0.32). However, inclusion of VRE isolated from non-patient samples increased the number of occupant-stays with VRE detection from 6 to 20, a 3.3-fold increase, as compared to patient samples alone. Inclusion of non-patient samples also increased the number of VRE multi-isolate genomic clusters detected by 7-fold. Our findings are limited because sampling was primarily conducted in ICUs. Due to the combination of short ICU stay durations and imperfect test sensitivity, VRE transmission events were probably underdetected. Conclusions: Our findings characterize the complex nature of VRE transmission pathways in ICU settings. Even without an ongoing outbreak, we found substantial evidence of VRE movement between occupant-stays, facilitated by a combination of HCP hands and environmental surfaces. This study highlights the importance of environmental sampling for understanding VRE transmission potential, which is likely to be underestimated using patient sampling alone. We recommend that future studies incorporate follow-up sampling after discharge to better understand the true burden of transmission.

Bacteriophages that infect Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever, are regarded as specialized, because all previously characterized phages depend on the Vi capsular polysaccharide for infection. Whether capsule-independent infection strategies exist has remained unclear. Here we identify environmental phages that infect S. Typhi both in the presence and absence of Vi. Screening 140 urban wastewater samples from Dhaka, Bangladesh, where typhoid is endemic, we recovered phages infecting a Vi-deficient S. Typhi strain from 41 samples (29%). All 41 phages also infected the isogenic Vi-expressing host, although 28 did so with 10- to 105-fold lower infection efficiency, and suppressing capsule expression increased susceptibility to 23, indicating an inhibitory effect of Vi on infection by many these phages. All 41 phages infected S. Paratyphi A and nine infected a monophasic S. Typhimurium, a broader host range than the Vi-dependent phages, which were restricted to Vi-expressing Typhi. Across 26 circulating genotypes, capsule suppression increased susceptible genotypes per phage by 1.51 on average (Wilcoxon p = 5.76 x 10-6), though four genotypes remained resistant to all phages tested, indicating additional determinants of susceptibility. Whole-genome sequencing of 27 phages identified three genera in two families, predominantly Teetrevirus (19/27); TerL phylogeny separated these from classical Vi-dependent phage lineages. Together, these findings reveal a broader-host-range component of Typhi phage ecology and show that Vi dependence is not a universal feature of phages capable of infecting S. Typhi.

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).

Introduction: Plasma metagenomic next-generation sequencing (mNGS) may detect pathogens in solid organ transplant (SOT) recipients, but optimal patient selection and result interpretation remain uncertain. Methods: Physicians reviewed SOT recipients with first-instance clinical plasma mNGS testing (Karius, Inc.) and determined consensus microbiological diagnoses, clinical impact of results, diagnostic yield, and clinical outcome. mNGS results were compared to microbiological diagnoses. A HIPAA-compliant large language model (GPT-4) was used to analyze electronic medical record (EMR) data and predict risk of infection with atypical bacteria, invasive fungi, mycobacteria, or parasites (collectively: pre-specified organisms of presumed significance, POPS) and identify patients who had positive-impact mNGS testing. Results: Of 145 SOT recipients, 119 (82.1%) had positive tests, 42 (29.0%) had [&ge;] 1 POPS organism, and 27 (19.1%) had [&ge;] 1 organism causing positive clinical impact. Positive impact was highly correlated with POPS status, with 24 (88.9%) of 27 positive-impact organisms categorized as POPS (P<0.001). GPT-4 scores accurately identified patients with POPS diagnoses (AUC 0.86), and assigned higher scores to patients with positive test impact (P=0.001). mNGS testing had highest sensitivity for atypical bacteria (82.4% sensitivity) and lower sensitivity for Aspergillus spp (53.3% sensitivity). Detection of greater numbers of organisms by mNGS was associated with increased mortality risk (odds ratio 1.32 per organism detected). Discussion: Plasma mNGS is a valuable clinical tool in SOT recipients. Positive clinical impact is associated with detection of atypical bacteria, fungi, mycobacteria, or parasites. GPT-4 analysis of EMR data identifies patients at risk of infection from these organisms and most likely to benefit from mNGS testing.

Background Despite widespread vaccination, pertussis remains a poorly controlled disease globally and results in substantial annual morbidity and mortality, particularly in young children. Controlled human infection models (CHIMs) using the causative agent Bordetella pertussis are promising systems to enable the study of pertussis disease pathogenesis and immunology and to rapidly assess vaccines and therapeutics. While a pertussis CHIM that produces asymptomatic infection has been established in Europe, the development of a CHIM that leads to symptomatic illness would be advantageous for evaluating vaccine efficacy against both infection and disease. Methods Healthy participants 18-40 years of age were inoculated intranasally with one of eight doses (ranging from 104 to 108 colony forming units (CFU)) of the pertactin-producing B. pertussis isolate D420 at the challenge facility within the Canadian Center for Vaccinology (Nova Scotia, Canada). The study occurred in two stages. In stage one, the B. pertussis dose was escalated in cohort groups of five to six participants until reaching an endpoint where 70-90% of participants exhibited mild (non-severe, Grade 1 or 2) symptomatic infection, defined as the Human Infectious Dose 70-90 (HID70-90). In stage two, additional challenges were conducted for doses below, at, and above the identified HID70-90 to characterize the emerging pertussis model. For all challenge doses, participants were closely monitored during an inpatient stay of up to 24 days and post-discharge for laboratory-confirmed infection, pertussis symptoms, safety, and IgG antibody responses to four B. pertussis antigens including pertussis toxin, filamentous hemagglutinin, fimbriae, and pertactin. All participants received a five-day course of azithromycin, where timing of initiation depended on B. pertussis testing and symptoms. The study was conducted between July 4, 2022 and March 19, 2025. Findings Seventy-five participants were inoculated with one of the eight B. pertussis D420 challenge doses and completed the inpatient stay. From the stage-one dose escalation, we found that 107 CFU of B. pertussis D420 was the lowest dose that achieved the HID70-90, where 9 of 12 participants (75.0%) exhibited mild symptomatic infection. Following stage-two challenges, 16 of 22 total participants at 107 CFU (72.7%) developed mild symptomatic infection, thus verifying the HID70-90. The symptomatic infection rate below the HID70-90 at 5x106 CFU of D420 was 20.0% and above the HID70-90 at 5x107 and 108 CFU were 58.3% and 55.6%, respectively. Symptoms with elevated frequency for symptomatic infection (relative to background symptoms in non-infected) included nasal congestion, runny nose, fatigue, malaise, and cough. At the HID70-90, 50% of symptomatic infections included cough. Serological analyses of the four highest (stage-two) challenge doses (5x106, 107, 5x107, 108 CFU) revealed that antibody titres increased over time post-challenge. Seroconversion for at least one of the four studied antibodies was nearly twice as common for symptomatic (70.0%) than asymptomatic (35.7%) infection and was absent (0%) for non-infected. All infections were cleared following azithromycin treatment (100%) and there were no study-related serious adverse events. Interpretation A safe and reproducible symptomatic pertussis CHIM was achieved, providing a model for research on pertussis disease pathogenesis and immunology and for assessing vaccines and therapeutics. (Clinicaltrials.gov, NCT05136599).

Abstract Introduction. Culture data guide orthopedic-infection management, yet the organism spectrum, resistance, and no-growth fraction are reported inconsistently and mostly within proprietary registries. We characterized these in a public, reproducible dataset. Methods. Retrospective cross-sectional study using MIMIC-IV version 3.1, a de-identified single-center US database. Episodes with an International Classification of Diseases diagnosis of prosthetic joint infection (PJI) or native osteomyelitis were identified; organism-spectrum and no-growth analyses were restricted to the 46% with at least one deep musculoskeletal culture (tissue or bone, synovial or joint fluid, implant sonication), so the benchmark describes culture-sampled, not all, coded episodes. Proportions carry exact 95% CIs; variation was tested by logistic regression with Benjamini-Hochberg control, and an out-of-fold logistic model quantified how well no-growth was anticipated by structured data. Results. Of 7697 episodes (median age, 60 years; 35.5% female), 1089 were PJI, 5715 native osteomyelitis, and 893 other device infection. Among 7700 deep specimens (3560 episodes; 2603 patients), 35.7% showed no growth (patient-clustered 95% CI, 34.0%-37.3%). The fraction was higher in PJI than osteomyelitis (48.6% vs 26.6%) but rose with sampling intensity (24.5% to 50.7%), indicating differential ascertainment. S. aureus led (32.5%; 43.3% methicillin-resistant), and PJI was less often polymicrobial than osteomyelitis (adjusted OR, 0.44). No-growth was weakly anticipated by structured data (out-of-fold AUROC, 0.63). Conclusions. About one-third of deep specimens from code-defined orthopedic infection showed no growth. This specimen-level fraction differs from a criterion-confirmed culture-negative-infection rate and depends on sampling intensity; it is released as a re-runnable benchmark on identical open data, not a transferable rate.

Background Mupirocin, a widely used topical agent for decolonization of Staphylococcus aureus, is increasingly compromised by resistance. Although plasmid-mediated mupirocin resistance is a recognized cause of decolonization failure, its role in facilitating hospital-wide transmission is unknown. Methods We conducted genomic surveillance of S. aureus at two interconnected urban hospitals where mupirocin decolonization is routine. Genome sequencing of >10,000 isolates was integrated with patient data to identify transmission and resistance determinants. Bacterial phenotypes and fitness were evaluated in vitro and in murine colonization models. Findings Genome sequencing identified 475 hospital transmission events; none were detected by conventional surveillance. The mupA (ileS2) resistance determinant, carried on conjugative plasmids, was enriched eightfold in methicillin-resistant S. aureus (MRSA) relative to methicillin-susceptible strains. mupA was associated with nearly a threefold greater chance of hospital transmission, especially within endemic healthcare-associated MRSA lineages, and was enriched twofold in hospital-onset infections compared with admission colonizing isolates. Multiple independently evolved inactivating mutations in the essential chromosomal gene ileS1 co-occurred with mupA, creating plasmid addiction in which mupA became indispensable for bacterial survival. Addiction arose most frequently within the dominant community-acquired MRSA lineage, where plasmid carriage reduced colonization fitness in mice. Plasmid-containing strains exhibited stringent-response activation, explaining the fitness costs and collateral tolerance to disinfectants, such as ethanol and peroxide. Although addiction reduced S. aureus fitness, it increased plasmid transfer, and addicted variants spread across hosts, demonstrating adaptation that mitigates these costs. Unexpectedly, we identified a mupirocin-dependent vulnerability to isoleucine limitation, revealing a potential strategy to target mupA-mediated resistance. Interpretation Plasmids promote hospital transmission of mupirocin-resistant S. aureus and create an evolutionary trap in which antibiotic use selects for bacterial dependence on otherwise costly resistance elements. This dependence revealed a collateral bacterial vulnerability that could be exploited to target resistant strains and preserve the effectiveness of mupirocin.

Nucleic acid extraction remains the principal infrastructure barrier to molecular influenza testing outside centralised laboratories, since bead-based purification is normally tied to mains-powered extractors and trained operators. We evaluated SmartLid, a centrifugation-free format in which a removable magnetic key shuttles paramagnetic beads through pre-aliquoted lysis/binding, wash, and elution buffers without pipetting or powered instrumentation, against an automated magnetic-bead extractor (Nextractor NX-48S) on 311 nasopharyngeal specimens from the 2024-2025 influenza season at a National Influenza Centre. Paired eluates were amplified under identical monoplex RT-qPCR conditions for influenza A(H1N1)pdm09, A(H3), and B/Victoria. Both methods gave 100% specificity (47/47 negatives; no false positives). Subtyping succeeded in 263/264 reference-positive specimens after SmartLid extraction versus 241/264 after automated extraction (99.62% versus 91.29%; difference 8.33 percentage points; discordant pairs 23 versus 1; McNemar P < 0.001). Across 240 complete pairs, cycle threshold (Ct) values were lower after SmartLid extraction (median paired difference -2.78 cycles; estimated location shift -2.60 cycles, 95% CI -2.82 to -2.37; P < 0.001) with rank-ordering of specimens conserved between methods (Spearman rho = 0.84). The advantage was preserved across all three subtypes and in both fresh and frozen specimens (adjusted P < 0.001). Specimens recovered only after SmartLid extraction had higher Ct values than dual-detected specimens (median 34.37 versus 28.54; P < 0.001), locating the gain near the assay detection limit. An instrument-free manual format can therefore exceed the extraction efficiency of an automated reference workflow, extending quality-assured influenza subtyping beyond centralised laboratories.

Background: Tenofovir-lamivudine-dolutegravir (TLD) is an effective, single-tablet ART regimen but the causes of virologic failure that can occur despite adherence to TLD are incompletely understood, especially when resistance mutations in integrase (IN) are absent. Noncanonical resistance mutations in nucleocapsid (NC) have been selected in cell culture and are associated with decreased dolutegravir (DTG) susceptibility in vitro. Samples from the ACTG A5381/Hakim study were examined to assess the potential contribution of NC mutations to virologic failure on TLD. Methods: A5381/Hakim was an observational cohort study that enrolled individuals with HIV-1 RNA >1000 copies/mL when initiating TLD as first line ART or switching from failing non-nucleoside reverse transcriptase inhibitor (NNRTI)-based or protease inhibitor (PI)-based ART. Whole HIV RNA genome next generation sequencing was performed on paired plasma samples from study entry and confirmed virologic failure in 56 participants receiving TLD for [&ge;] six months. Mutations relative to the HIV-1HXB2 reference genome were identified with DeepChek(R) software using a mutation reporting threshold of five percent mixed-base frequency. Canonical drug resistance mutations (DRMs) were identified within the DeepChek(R) software using the Stanford HIVdb v9.8 algorithm. Site-directed NC and IN mutants were tested for susceptibility to DTG, raltegravir and cabotegravir in the TZM-bl HIV-1 indicator cell line and evaluated for infectivity and replication in multi-round assays. Results: Mutations in NC that emerged between TLD initiation and virologic failure were identified in 11 of 56 (20%) participants. Of these 11, three participants also had IN mutations at virologic failure but not at study entry, suggesting dual selection. Selected mutations in the NC zinc-finger domain included V13I, K20R, E21V, N27I/S, A30T, K34R, K38R, K41G/R/N, Q45R and/or M46I, alone or in combination with other NC and/or IN mutations. Specific NC mutations (K20R, N27I, A30T, K41N, M46I) conferred a significant decrease in DTG susceptibility. The combination of certain NC with IN mutations (e.g. NC N27I and IN R263K) conferred greater reduction in susceptibility to DTG in vitro than either mutation alone. Conclusions: This work provides the first clinical evidence of NC mutation selection in individuals on failing TLD ART and shows that NC mutations, combined with IN mutations, can further decrease susceptibility to DTG. Our findings support additional investigations of the contributions of mutations outside of IN to virologic failure of integrase inhibitor-containing ART regimens.

Objective: To consolidate publicly available United States National Antimicrobial Resistance Monitoring System (NARMS) human isolate data spanning 2004-2021 and comprehensively quantify trends in fluoroquinolone resistance, tetracycline resistance, ceftriaxone resistance, and early-stage azithromycin resistance across four NARMS-monitored enteric bacterial pathogens: nontyphoidal Salmonella, Campylobacter jejuni, Campylobacter coli, and Shigella spp. Methods: Annual resistance percentages were compiled from publicly available CDC, FDA, and USDA NARMS Human Isolates Surveillance Reports and Integrated Reports. Multi-year reference-period averages were assigned to their period midpoint years. Ordinary least-squares linear regression was applied to each pathogen-drug series. Slope estimates, 95% confidence intervals, Pearson correlation coefficients, and p-values were computed. A sensitivity analysis was conducted excluding reference-period midpoint years. Results: Ciprofloxacin decreased susceptibility in Salmonella rose from 2.4% to 11% (slope 0.62 pp/year, 95% CI 0.39-0.85; p less than 0.001). Ciprofloxacin resistance in C. jejuni rose from 21% to 34% (slope 1.68 pp/year, 95% CI 1.10-2.25; p=0.001) and in C. coli from 28% to 45% (slope 2.08 pp/year, 95% CI 0.97-3.19; p=0.007). Shigella showed a 5.8-fold increase (underpowered, n=3). Tetracycline resistance in C. jejuni and C. coli rose significantly. Salmonella multidrug resistance declined (slope -0.34 pp/year; p=0.001) while ceftriaxone resistance rose (slope 0.10 pp/year; p=0.028). Azithromycin-resistant Salmonella isolates detected in 2017 equalled the cumulative total from 2011-2016. Discussion: Fluoroquinolone and tetracycline resistance rose significantly across NARMS-monitored Campylobacter species, while Salmonella showed diverging trends: declining classical multidrug resistance but rising fluoroquinolone and ceftriaxone resistance. The emerging azithromycin resistance signal threatens remaining oral treatment options. These trends support sustained investment in harmonized antimicrobial resistance surveillance and stewardship globally.

Each year, over 50 million children receive preventive malaria treatment. However, to date there has been no consensus on the most effective antimalarial drugs to use, especially given geographic differences in drug resistance. Here, we conduct a systematic review comparing the effectiveness of the most commonly used antimalarial chemopreventive regimen, sulfadoxine-pyrimethamine plus amodiaquine (SP+AQ), with other antimalarial drugs in preventing new infections. We searched MEDLINE, Embase, Global Health, PubMed and WWARN clinical trial databases until 06 December 2025 for studies satisfying the inclusion criteria. Studies were included if they were peer-reviewed, randomised-controlled studies in Africa, measuring incidence of infection or clinical episodes of Plasmodium falciparum malaria for at least 28 days post-treatment. We also compiled data on the prevalence of markers of resistance in the parasite dhfr, dhps and mdr1 genes in the study areas. We conducted meta-analyses of incidence rates, with subgroup analyses by drug resistance levels. This review is registered on PROSPERO (CRD42024577149). We identified 27 studies representing 38,252 participants in 32 sites across 13 countries. In pooled analysis, SP+AQ reduced incidence of malaria by 54.6% (95% CI: 33.8-68.8%) compared to SP alone, including significantly outperforming SP even in areas with low SP resistance. These findings suggest that countries currently using SP alone for chemoprevention should consider switching to SP+AQ. Where AQ resistance remains low, available evidence suggests SP+AQ remains efficacious for malaria chemoprevention. SP+AQ was comparable to the artemisinin-based treatment, dihydroartemisinin-piperaquine across all studies (incidence rate ratio 0.93; 95% CI 0.78-1.11). By resistance levels, SP+AQ had slightly higher efficacy in areas with low SP and AQ resistance but had comparable or slightly lower efficacy in areas with higher resistance. Using artemisinin-based treatments for chemoprevention must be balanced against the risk of worsening artemisinin resistance in Eastern and Southern Africa. This study was funded by the UK Royal Society.

Background Plasmodium vivax malaria relapses are a major source of morbidity and onward transmission of infection. The underlying mechanisms are poorly understood and current therapies sub-optimal. We examined the safety and feasibility of a controlled human malaria infection (CHMI) model for relapsing P. vivax. Methods We conducted an open-label, proof-of-concept, CHMI study of relapsing P. vivax. Healthy, malaria-naive, Duffy-positive adults aged 18-45 years with extensive CYP2D6 metaboliser phenotype and normal blood glucose-6-phosphate dehydrogenase (G6PD) levels were recruited in Oxford, UK. Mosquito-bite CHMI was performed in Nijmegen, The Netherlands, using Anopheles stephensi mosquitoes infected with PvW1, a clonal isolate of P. vivax from Thailand. All follow-up visits were conducted in Oxford, UK. Primary P. vivax infections (qPCR > 500 genome copies/mL) were treated with artemether-lumefantrine (80mg/480mg at 8, 24, 36, 48 and 60 hours). From Day 28 following CHMI, participants attended a fortnightly clinic for clinical review and qPCR blood sampling, with additional assessments performed for any reported symptoms. P. vivax relapse infections (qPCR > 500 genome copies/mL) were treated with artemether-lumefantrine as per primary infection. Definitive anti-malarial treatment with atovaquone-proguanil (1000mg/400mg once daily for three days) and primaquine (0{middle dot}5 mg/kg/day for 14 days) was administered six months following CHMI, regardless of parasitaemia or symptoms. The primary objective was to assess the safety, feasibility and frequency of relapsing P. vivax after CHMI. Remote follow-up (5 years) is ongoing. The study is registered with ISRCTN registry (ISRCTN48625883). Findings 20 participants were screened for eligibility from 21 January 2025. Five participants (median age 22 years) underwent CHMI (five infected mosquitoes per participant) on 15 April 2025. All participants developed primary P. vivax infection and experienced at least one relapse infection. Two participants experienced a second relapse. Overall incidence rate was 3{middle dot}6 relapse infections per person-year. Solicited adverse events were mild or moderate and there were no serious adverse events. Definitive anti-malarial treatment was administered to all participants. One participant experienced primaquine-induced methaemoglobinaemia, resolving with early discontinuation of treatment (total dose 5{middle dot}3 mg/kg). To date, more than six months after primaquine treatment, no further relapses have been recorded. Interpretation CHMI of relapsing P. vivax is safe and feasible, allowing exploration of the mechanisms underlying relapse infections and providing a platform for future anti-relapse efficacy studies. Funding European Union Horizon Europe programme and UK Research and Innovation (UKRI) via OptiVivax consortium; UK National Institute for Health and Care Research Biomedical Research Centre: Oxford; and UK Medical Research Council.

Background Klebsiella pneumoniae is a leading cause of sepsis in young infants. We evaluated the association of invasive K. pneumoniae disease (iKPnD) in relation to antigen-specific immunoglobulin G (IgG) and serum bactericidal activity (SBA) to four polysaccharide capsular (K) serotypes and five lipopolysaccharide (O) serotypes, as well as IgG to MrkA, in infants less than 90 days of age. Methods We conducted a retrospective case-control study in Kenyan and South African infants with blood culture-confirmed iKPnD. Serotype-specific antigen IgG concentrations of cases were compared with hospitalised controls without iKPnD. Geometric mean concentrations (GMCs) were estimated, and scaled covariate-adjusted models were used to estimate risk reduction over a grid of antibody concentrations. Results Transplacental transfer of IgG against various K. pneumoniae antigens increased with advancing gestational age. Serum IgG GMCs (expressed in RLU/mL) to disease-causing homotypic K- or O-serotypes were lower in cases compared with controls for anti-K2 (396 [95%CI: 250-628] vs 660 [95%CI: 562-776]), anti-K25 (396 [95%CI: 251-623 ] vs 1170 [95%CI: 988-1385]), anti-K149 (327 [95%CI: 204-521] vs 492 [95%CI: 435-557]); as well as anti-O1{beta},2 IgG (1282 [95%CI: 782-2101] vs 2250 [95%CI:1904-2658]). Furthermore, overall anti-MrkA IgG was lower in cases (945; 95%CI: 757-1179) compared with controls (1610; 95%CI: 1378-1880). SBA titres (expressed as IC50) did not differ between case and controls by K types, but were lower for O1{beta},2{beta} in cases (27; 95%CI: 12-63 vs. 136; 95% CI: 84-221). Conclusion Our findings provide preliminary evidence that low antibodies against three of four K-antigens, O1{beta},2{beta} and MrkA are inversely associated with iKPnD, and should be explored as potential vaccine antigens.

Background Nontuberculous mycobacteria (NTM) are an increasingly common group of pathogens that remain challenging to diagnose and treat effectively. The lack of standardization of NTM management, from identification to antibiotic resistance prediction, results in imperfect correlations between treatment and outcomes. This study characterizes the genetic heterogeneity of a previously uncharacterized NTM during a 29-month bacteremia with acquired drug resistance. Results In contrast to the initial diagnostic result identifying M. nebraskense, a rare NTM causing disease in humans, whole genome sequencing (WGS) identified Mycobacterium sp. SMC-2, a species with only one publicly available genome. High-resolution analysis of variants revealed 444 unique SNPs and 26 indels in 12 longitudinal isolates, with the highest number of low-frequency mutations between 3-5% frequency. Seven candidate drug-resistance mutations across five evolutionary trajectories showed frequency shifts that correlated with changes in minimum inhibitory concentrations to the corresponding antibiotics. These included a 23S rRNA clarithromycin-resistance SNP detected at 7% frequency when phenotypic resistance emerged, suggesting that low-frequency variants drive subpopulation evolution. Acquisition of drug resistance during therapy was associated with several low-frequency mutations in genes associated with resistance to antibiotics, including clarithromycin and quinolones, in other NTM species. Conclusion This study highlights the importance of low-frequency variants as drivers of intra-patient bacterial population diversity, allowing subpopulations to adapt to antibiotic pressure and ultimately contributing to treatment failure. Additionally, it underscores their potential implications for the development of molecular diagnostic tests for NTM resistance prediction.

Background The engagement of mucosal and systemic immunity in preventing Bordetella pertussis colonization and infection in humans, the impact of prior vaccination on host immunity and protective outcomes, and the dynamics of the host response following exposure remain poorly understood. Methods Healthy adults were challenged with increasing colony-forming units (CFUs) doses, 106-108, of B. pertussis D420 intranasally (NCT05136599). Shedding (PCR and culturing) and symptom development were monitored up to 21 days post-challenge. Serum and nasal wash IgA and IgG were measured before challenge (baseline) and up to 6 months post-challenge. Findings Antibodies increased post-challenge only in infected individuals, primarily nasal IgA. Participants who remained uninfected had higher baseline levels of filamentous hemagglutinin (FHA)- specific mucosal IgA and IgG, and higher serum IgA against fimbriae 2/3 (FIM). FHA was negatively associated with bacterial load and was a key discriminator between shedders and non-shedders, up to one week post-challenge. By day 14 post-challenge, pertussis toxin (PT) IgG and FIM IgA in both serum and mucosal samples were negatively associated with bacterial colonization. The majority (96.7%) of acellular pertussis (aP) vaccine recipients (n=23, median age 2.0 years) became infected, compared to 69.4% of those who received whole-cell pertussis vaccine (n=36; median age 32.0 years), and their antibody responses remained distinct following infection. Interpretation Nasal FHA antibodies emerged as early predictors of protection against pertussis infection, while PT IgG and FIM IgA antibodies may reflect clearance after infection. aP-primed individuals were more susceptible to infection, despite their younger age and more recent vaccination. Funding CDC Contract #75D30122C15467 and CDC IPA Agreement #24IPA2417512 Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention, US Department of Health and Human Services.

Objectives: To compare the effectiveness of povidone-iodine ear wash compared to no ear wash and oral cotrimoxazole compared to placebo given in addition to standard topical antibiotic treatment (ciprofloxacin drops) for chronic suppurative otitis media (CSOM) in Australian Aboriginal children. Methods: A randomised, parallel, 2 x 2 factorial design, assessor-blinded clinical trial in the remote Northern Territory of Australia. Aboriginal children with confirmed CSOM were eligible to be randomised into four treatment groups, allowing two primary treatment comparisons in a 2-in-1 trial approach. Participants received standard treatment (twice daily cleaning and topical ciprofloxacin drops) plus: i) either 16 weeks of pre-treatment povidone-iodine ear wash or no povidone-iodine ear wash; and ii) either 16 weeks of oral cotrimoxazole or placebo. Central randomisation with allocation concealment and triple-blinding of the oral antibiotic treatment arms was used. The relative risk (RR) and risk difference (RD) were estimated after adjustment for age, community, and the other intervention. The primary outcome was the proportion of children with any otorrhoea (clinical failure) after 16 weeks of treatment. Secondary outcomes included size of tympanic membrane (TM) perforation and amount of discharge, time to cessation of discharge, proportion of children with respiratory and other pathogens in ear discharge (at baseline and 16 weeks) and hearing levels (at 12 months). Findings: 280 children with CSOM were randomised and 270 had their primary outcome assessed. Clinical failure (presence of any ear discharge) after 16 weeks of treatment was 66/134 (49%) in the povidone-iodine group versus 69/136 (51%) in the no povidone-iodine group (RD= -1% (-12,11), p= 0.93) and 56/134 (42%) in the cotrimoxazole group versus 79/136 (58%) in the placebo group (RD=-16% (-28,-4), p=0.007). The amount of discharge, TM perforation size, the level of hearing impairment, and serious adverse events were not significantly different in both treatment comparisons. Anaerobic growth (24%), Pseudomonas aeruginosa (21%) and Haemophilus influenzae (17%) were the most common pathogens found in the ear discharge before treatment. Fungi or yeast (24%), Staphylococcus aureus (15%), and anaerobic growth (10%) were the common pathogens after 16 weeks of treatment, with no significant differences between groups. At 12 months post-randomisation, 55-60% of children had at least one discharging ear and there was no difference between treatment groups. Interpretation: Povidone-iodine ear washes did not contribute to better ear outcomes in this study. Cotrimoxazole for 16 weeks resulted in more children with clinical improvement to dry ears. Oral cotrimoxazole may play a role in reducing the burden of CSOM in populations with high rates of persistent disease.

Objective As antimicrobial resistance (AMR) continues to threaten global public health, bacteriophage therapy products (BTPs) offer a promising alternative to conventional antimicrobials. However, translation into routine clinical practice requires best practice standards for manufacturing and quality control to ensure the consistent safety, quality, and reliability of personalised BTPs produced for individual patients or small cohorts. Design A modified Delphi methodology was used to develop consensus statements, engaging experts from Australia's National Bacteriophage Therapy Regulatory Working Group across the fields of clinical microbiology, phage biology, good manufacturing practice (GMP), regulatory science, and government. The process comprised three iterative phases: (1) structured statement development, (2) an anonymous REDCap survey, and (3) a hybrid consensus meeting. The strength of evidence and recommendations was assessed using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) framework. Results Consensus was reached on 35 statements to provide best practice manufacture and quality control guidance for BTPs. These statements address requirements for phage identification and characterisation; define the point at which GMP-aligned processes commence for ubiquitous phages; outline quality control expectations for phage active pharmaceutical ingredient (pAPI) production and maintenance of BTP and host cell repositories. Additional guidance covers quality management systems, including documentation, traceability, and governance. Conclusion These consensus statements provide comprehensive best practice recommendations for the manufacture and quality control of BTPs in Australia. By promoting consistent, safe, and quality-assured approaches to personalised BTPs, they aim to facilitate clinical implementation while remaining aligned with existing international pharmacopoeial standards and regulatory frameworks.

Background: Tuberculosis (TB) treatment outcomes remain suboptimal, and standard clinical diagnostics cannot reliably identify patients at high risk of treatment failure or relapse at the time of diagnosis. While within-host Mycobacterium tuberculosis genetic diversity is hypothesized to reflect the viable bacterial burden and adaptive capacity of the infection, its clinical prognostic value remains unknown. Methods: We conducted a prospective cohort study of 364 patients with newly diagnosed, rifampicin-susceptible pulmonary TB in South Africa. Patients received standard 6-month therapy and were monitored for up to two years to ascertain composite unfavorable outcomes (treatment failure, death, or relapse). To accurately detect low-frequency (unfixed) genetic variants and eliminate reference bias artifacts, we mapped medium to high depth short-read sequences against matched, patient-specific long-read assemblies. The association between baseline pathogen genetic diversity and clinical outcomes was evaluated using multivariable Cox proportional-hazards models. Results: After bioinformatic filtering, true unfixed variants were relatively rare but significantly enriched in genes mediating pathogen adaptation and drug tolerance, including transporter proteins and two-component regulatory systems. Within-host bacterial genetic diversity (i.e., the total number of unfixed variants) ranged from 0-20, with a median of 1 per patient. In survival analysis adjusting for known clinical risk factors--including HIV status, prior TB, baseline smear positivity, and radiographic lung involvement--baseline within-host genetic diversity emerged as a strong, independent predictor of unfavorable treatment outcomes. For patients with greater than 3 unfixed variants at diagnosis, each increase of 5 unfixed variants was associated with more than double the risk of a composite unfavorable outcome (adjusted Hazard Ratio, 2.36; 95% CI, 1.27 to 4.39; p=0.007). Conclusions: Baseline within-host pathogen genetic diversity is an independent predictor of unfavorable TB treatment outcomes. As sequencing becomes increasingly integrated into routine diagnostics, quantifying unfixed variants is an accessible approach that promises to risk-stratify patients and guide the duration of individualized regimens.