Current high-throughput sequencing platforms enable routine whole-genome sequencing of bacteria via clonal amplification from primary samples, followed by DNA extraction, library preparation, and sequencing. This approach enriches bacterial DNA while removing contaminating nucleic acids, allowing efficient genomic sequencing using minimal sequencing resources per sample. However, for fastidious or obligate intracellular bacteria, such as Coxiella burnetii , cultivation is often impractical, leaving pathogen DNA heavily diluted by host and environmental nucleic acids. In such cases, sequencing requires substantially greater throughput per sample, highlighting the need for culture-independent methods to support genomic epidemiology and surveillance. Here, we report a novel two-stage DNA enrichment strategy applied to C. burnetii -contaminated sheep wool samples. Such samples feature very low pathogen biomass and a high host-DNA background. Our approach integrates (i) selective whole-genome amplification of complex DNA extracts, followed by (ii) in-solution hybridization capture using custom double-stranded DNA baits. Critically, we developed a WGA-based method to synthesize whole-genome dsDNA baits from minimal input DNA, enabling efficient bait production even in the absence of cultured isolates. Notably, we demonstrate that baits derived from C. burnetii DNA contaminated with an unrelated host (e.g., chicken) remain effective for enriching C. burnetii from samples derived from a different host species (e.g., sheep), indicating robust cross-host applicability. Using this workflow, we achieved up to 95% C. burnetii enrichment and generated six genomic assemblies suitable for downstream phylogenomic and epidemiological analyses. This cultivation-free, two-stage enrichment framework can be applied for phylogenetics and epidemiology and may significantly expand the feasibility of genomic studies for intracellular and otherwise uncultivable bacterial pathogens.
Mycetoma is a neglected tropical disease characterized by mutilating tumorous lesions in the subcutaneous tissue. The causative agents are found embedded in granules called grains. Mycetoma is either caused by bacteria (actinomycetoma) or fungi (eumycetoma). To initiate the appropriate treatment, it is important to identify the causative agent rapidly and molecular identification for eumycetoma revolutionized the time to identification. For actinomycetoma this was not possible yet. Here we developed a multiplex qPCR identification scheme for the most common causative agents of actinomycetoma in Africa. Whole genome sequencing was used to identify species-specific gene families for Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis . qPCR primers and probes were developed on these species and validated against DNA isolated from mycetoma strains and grains. Each probe was unique with no cross-reactivity with other tested species. The limit of detection ranged from 0.000013 to 0.00067 ng bacterial DNA. When the qPCRs were validated against 28 grain samples, all fungal grains remained negative and 11 out of 12 Actinomadura grains were correctly identified. This resulted in a sensitivity of 85.7% for the A. pelletieri probe and a specificity of 100%. For the A. madurae probe, a sensitivity and specificity of 100% was obtained. The actinomycetoma qPCR developed in this study can be used to identify the most common causative agents of actinomycetoma in Africa.
Aedes aegypti is the primary vector of arboviruses such as dengue, yellow fever, Zika, and chikungunya. Here, we investigate the mitochondrial DNA (mtDNA) structure and insecticide resistance of Ae. aegypti populations across Kenya. Using 175 newly sequenced Kenyan COI mtDNA fragments, along with global reference sequences, we assess maternal ancestry and phylogeographic structure. Genetic structure across Kenya was not well explained by isolation-by-distance alone, with several populations deviating from this pattern. Nairobi and Mombasa, separated by >400 km, showed greater maternal genetic similarity than expected under isolation-by-distance, a pattern that may reflect gene flow along transportation corridors. Conversely, Ukunda, around 30 km from Mombasa, was comparatively divergent, and populations in Western Kenya were genetically distinct. We found no evidence of non-African ancestry in mtDNA from Rabai or Mombasa despite its previous detection in the nuclear genomes of these populations. Instead, a subset of samples from Marigat and Ukunda showed the closest maternal affinity to West African and non-African lineages. Insecticide assays revealed high susceptibility to bendiocarb, deltamethrin, and fenitrothion across populations, but substantial resistance to permethrin, particularly at coastal sites. Genotyping of the kdr 1534C allele showed the highest resistance allele frequencies in coastal populations, broadly consistent with the phenotypic patterns. This variation in resistance was not associated with mitochondrial ancestry, suggesting that local selection pressures rather than shared maternal ancestry may be shaping resistance. Together, these findings describe the maternal genetic structure of Ae. aegypti in Kenya and show that localised resistance evolution, in the absence of organised Ae. aegypti -targeted control, underscores the need to monitor resistance mechanisms.
BACKGROUND:Melioidosis is an infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. The disease is difficult to diagnose and treat. Here, we investigated outcomes of patients with suspected or confirmed melioidosis in Laos and Thailand. METHODOLOGY:We conducted a prospective, multinational, multicenter, observational study at Mahosot Hospital (MH) in Laos, and Srinagarind Hospital (SRH) and Sunpasitthiprasong Hospital (SUH) in Thailand. We enrolled adult patients (age ≥ 18 years) with suspected or confirmed melioidosis. For suspected-melioidosis cases, patients who had septic shock, had sepsis with at least one risk factor associated with melioidosis (including diabetes mellitus, chronic kidney disease, thalassemia major, malignancy and immunosuppressive therapy) or were prescribed ceftazidime or meropenem by attending physicians were enrolled, provided they were within 24 hours of hospitalization at the study hospitals. For confirmed-melioidosis cases, patients who had a clinical specimen tested positive for B. pseudomallei by either culture or antigen-detecting immunofluorescence microscopy were enrolled. The primary endpoints were 28-day and 90-day mortality. PRINCIPAL FINDINGS:From November 2023 to October 2024, 627 patients were screened and 200 patients were enrolled (43 for suspected-melioidosis and 157 for confirmed-melioidosis). Five of 43 patients enrolled with suspected-melioidosis (12%) were subsequently diagnosed with culture-confirmed melioidosis since enrollment. 28-day and 90-day mortality were 14% (6/43) and 23% (10/43) in suspected-melioidosis patients, respectively; and 25% (39/157) and 36% (57/157) in confirmed-melioidosis patients, respectively. In multivariable Cox regression models, higher Sequential Organ Failure Assessment (SOFA) score at enrollment and intensive care unit (ICU) admission on the day of enrollment were strongly associated with mortality outcomes. Admission to SRH and longer duration of symptoms prior to enrollment were independently associated with survival outcomes. CONCLUSIONS:Future clinical trials aiming to reduce mortality in patients with suspected or confirmed melioidosis should consider stratification based on the severity of organ dysfunction at enrollment. Differences in mortality between study sites may reflect residual confounding and should be carefully considered in sample size calculations.
BACKGROUND:Classified as poverty-related diseases (PRD) leprosy and tuberculosis (TB) are long-standing mycobacterial diseases strongly linked to poverty, stigma, and social exclusion, disproportionately affecting vulnerable populations. Gender norms further shape how individuals experience illness, access to care, and coping strategies, but evidence remains fragmented and largely disease-specific. This systematic review synthesizes qualitative evidence on lived experiences of people affected by TB or leprosy worldwide and compares gender inequities across both diseases to identify shared social and structural determinants. METHODS/PRINCIPAL FINDINGS:A systematic review following PRISMA guidelines searched PubMed, Embase, Scopus, and Web of Science for peer-reviewed qualitative and mixed methods studies (1995-08/2025) reporting primary qualitative data on gendered experiences of people affected by TB or leprosy. Studies in all countries and languages were eligible. Screening and extraction were conducted using Rayyan. Inductive thematic synthesis was performed using MAXQDA. Quality was assessed using the CASP checklist. A total of 3204 records were identified, with 57 studies from 29 countries across five continents included. Across settings, women experienced greater barriers to diagnosis and care, more severe marital and familial consequences, higher stigma and social exclusion, and greater psychosocial burden than men. Men's experiences were shaped by norms of masculinity, particularly regarding economic provision and employment. Despite disease differences, psychosocial and socioeconomic impacts were similar across TB and leprosy, embedded in gendered social norms, power relations, and social determinants of health. CONCLUSIONS/SIGNIFICANCE:Gender-based inequities are pervasive across TB and leprosy globally. Comparing both diseases reveals shared social mechanisms beyond biomedical differences. Addressing them requires moving beyond biomedical models and integrating gender as a structural determinant of health. Gender-sensitive, participatory, and intersectional interventions are needed to improve early diagnosis, treatment adherence, and psychosocial and socioeconomic outcomes. REGISTRATION:Registered on Open Science Framework (OSF) on 01/09/2025.
Anthelmintic drugs are used to control soil-transmitted helminths that infect a quarter of the world’s human population. There is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. Here we describe the presence of a previously unrecognized endogenous acetylcholine (ACh) analogue, β-alanine betaine, which may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their analogues are not present in their human or animal hosts. We collected pseudocoelomic fluid from female Ascaris suum (a model for the human parasite Ascaris lumbricoides ) and subjected it to HILIC-MS and HILIC-MS/MS to reveal signals consistent with ACh, choline, and β - alanine betaine but not betaine, propionylcholine, butyrylcholine, or butyrobetaine. The presence of β-alanine betaine was confirmed by comparison with an analytical standard. Injection of betaine into female A. suum produced no effect. However, injection of β - alanine betaine produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between β - alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. RT-PCR showed that message for members of the DEG-3/DES-2 subfamily of nAChR channels, which are betaine targets, were present in the intestine and body wall of A. suum . β - alanine betaine increased intracellular Ca 2+ of the intestinal enterocytes and depolarized the membrane potential of body wall muscle cells. In N2 Caenorhabditis elegans, application of β - alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, deg-3/des-2 and lgc-41 null-mutants. These observations suggest that, in addition to ACh, β-alanine betaine - an analog of betaine produced under anaerobic conditions - may function as an endogenous ligand in anaerobic nematodes such as A. suum . An expanded repertoire of nicotinic acetylcholine receptor subfamilies in nematodes relative to mammals may reflect a corresponding need for diversification of cholinergic endogenous ligands in these organisms. This repertoire could allow their simpler neuronal system to perform more complex controls and be exploited for development of different and novel subfamily selective cholinergic anthelmintics.
Zika virus (ZIKV) is an arthropod-borne Orthoflavivirus that caused a major outbreak in Central and South America in 2015-16. During that outbreak, ZIKV infection during pregnancy was associated with high rates of adverse outcomes, including miscarriage and a spectrum of neurological birth defects collectively termed congenital Zika syndrome (CZS). Ten years later, there are still no licensed ZIKV vaccines, underscoring the need for robust experimental models to evaluate vaccine efficacy against congenital disease. Here, we evaluated a highly pathogenic nonhuman primate pregnancy model for its ability to assess vaccine-mediated protection using a Zika virus-like particle (VLP) vaccine as a test platform. First, non-pregnant rhesus macaques (RM) were vaccinated with a prime-boost method to compare control, VLP alone, and VLP plus Alhydrogel (alum) adjuvant. Vaccination plus alum elicited strong neutralizing antibody responses and reduced viral dissemination compared to controls. Next, we evaluated the vaccine's protective efficacy against adverse pregnancy complications. Two cohorts of female RM were vaccinated with ZIKV-VLP adjuvanted with alum or alum alone prior to time-mated breeding. At gestational day (GD) 30 (mid first trimester), pregnant animals were challenged with ZIKV-DAK 41524, an African-lineage strain previously shown to induce frequent first-trimester pregnancy loss. Within the vaccinated cohort, two of three animals reached the study endpoint of GD 90 with no observed adverse pregnancy outcomes. One vaccinated animal experienced early pregnancy loss despite the absence of detectable virus in fetal or placental tissues. In the sham vaccine control pregnancy cohort, severe adverse outcomes included miscarriage and hydrops fetalis with widespread viral RNA and placental pathology in two animals. These results support a high risk of early pregnancy loss following African-lineage ZIKV-DAK 41524 infection in RM. This model can be further used to understand the complexities of placental immunological features underlying miscarriage following ZIKV infection.
Japanese encephalitis (JE) virus (JEV) is a leading cause of neurological infection in the Asia-Pacific region with devastating socioeconomic consequences. We aimed to develop a target product profile (TPP) for JE diagnostics in endemic areas in the context of other flaviviruses, to better define improvements needed for patient management and public health. Thematic experts were identified from a scoping review, World Health Organization and Encephalitis International databases, as well as author networks. Initial interviews were performed with selected experts to inform a draft TPP document and start a Delphi process, aiming for consensus of ≥75% on each characteristic. A one-day meeting facilitated in-depth discussion, with breakout groups and electronic voting to develop the final TPP document. In total, 460 participants were invited. Five interviews enabled drafting a TPP. Two iterative surveys were completed by 44 respondents, including clinicians, scientists, and public health experts from 15 countries. A hybrid meeting was attended by 42 participants. Seven priority characteristics were discussed, and consensus was achieved for six; no clear consensus was reached for the preferred test sensitivity and specificity, with 73% agreement. Discussion points and expert consensus were consolidated into a roadmap for development and implementation to guide future work. A diverse group of health professionals agreed that improved diagnostics for JE are needed, and that a priority is a test for patient management that detects JE alongside other relevant flavivirus infections. There was a mandate for a rapid diagnostic test (RDT), and it was suggested that this could be implemented in a two-tiered diagnostic algorithm. The final TPP is presented in the paper; this aims to help define the updates needed to better support patient management and public health interventions, to provide a strategic reference document to drive innovation and improve clarity for JE diagnostic test developers.
BACKGROUND:Schistosomiasis and soil-transmitted helminth (STH) infections remain major neglected tropical diseases in low- and middle-income countries despite ongoing preventive chemotherapy programmes. This study assessed the prevalence, associated risk factors, and household environmental contamination related to schistosomiasis and STH infections among children in Banda District, Ghana. METHODOLOGY:A community-based cross-sectional study was conducted in four endemic communities in the Banda District, Ghana, from February to April 2025. Pre-school and school-aged children (2-15 years) were recruited using systematic sampling. Urine samples were examined with urine sedimentation for S. haematobium ova, and stool samples were analyzed using Kato-Katz for STHs and S. mansoni ova. Household soil and animal faecal samples were examined using flotation and formalin-ether concentration techniques. Risk factors were assessed through structured questionnaires. Bivariate and multivariate logistic regression analyses were performed; variables with p ≤ 0.25 were included in the multivariate model, with a p < 0.05 considered statistically significant. RESULTS:The overall prevalence of schistosomiasis was 26.3%, and the prevalence of STH infections was 28.0%. In multivariate analysis, non-school attendance (AOR = 4.59; 95% CI: 2.49-8.47; p < 0.001) and children drinking from rivers/streams (AOR = 4.58; 95% CI: 1.73 - 12.10; p = 0.009) were associated with a higher odds of schistosomiasis. For STH infections, children aged 2-9 years (AOR = 2.18; 95% CI: 1.24-3.85; p = 0.007), non-school attendance (AOR = 5.91; 95% CI: 3.41-10.24; p < 0.001) and rearing of animal within house (AOR = 4.78; 95% CI: 2.32-9.86; p < 0.001) were associated with a higher odds of infection. Both soil and animal faecal samples harbored STHs with differing species distributions between matrices (soil: 7.08% positive; animal faeces: 10.67%). CONCLUSION:This study revealed that schistosomiasis and helminth infections are prevalent among humans, animals, and the surrounding soil within shared environments. The findings support extending MDA to the community level and implement WASH, improved livestock management and health education to reduce transmission.
BACKGROUND:The emergence of Japanese encephalitis virus (JEV) in Australia raises questions about transmission potential across neighbouring Pacific Island countries and territories (PICs), where surveillance infrastructure and diagnostic capacity are limited. METHODOLOGY:We conducted a qualitative risk assessment of 22 PICs structured around three components: (1) 'release assessment', evaluating JEV introduction pathways based on geographic proximity, migratory waterbird flyway connectivity, and windborne mosquito dispersal potential; (2) 'exposure assessment', evaluating ecological transmission suitability based on vector and host competence and occurrence; and (3) 'risk characterisation', integrating release and exposure components to stratify countries into transmission risk profiles (endemic, epidemic, and low potential). Introduction potential varied across the region, with northern and western PICs having greatest connectivity to endemic regions. Ecological suitability for transmission was sufficient to support JEV transmission in 13 of the 22 PICs if introduced. Eight PICs were characterised as having endemic transmission potential (Papua New Guinea, which already has confirmed circulation, Solomon Islands, New Caledonia, Vanuatu, Fiji, Palau, Guam, and Northern Mariana Islands), based on diverse competent vector communities, high ardeid waterbird diversity, and moderate-to-high introduction potential. Five PICs (Federated States of Micronesia, Tonga, Samoa, Tuvalu, and Nauru) were characterised as having epidemic transmission potential, supported by pig densities capable of amplifying viral titres and sufficient vector diversity, but lacking ardeid populations necessary to sustain endemic cycles. The remaining nine PICs had low transmission potential due to limited vector and host communities. These risk stratifications are relative; the absolute probability of JEV establishment in any PIC remains uncertain. CONCLUSIONS:Distinct transmission profiles require differentiated public health responses, from sustained surveillance in endemic-potential settings to early detection and rapid outbreak response in epidemic-potential settings. Regional coordination through shared diagnostic networks and vaccine access pathways is critical to support preparedness before JEV becomes established across the Pacific.
BACKGROUND:Dengue remains a persistent public health challenge in eastern Guatemala, particularly in communities where irregular water supply, domestic water storage, and limited sanitation infrastructure create favorable conditions for Aedes aegypti proliferation. As concerns increase about the sustainability of chemical vector control, biological approaches such as the use of predatory copepods are receiving renewed attention. However, successful implementation depends not only on biological efficacy, but also on community awareness, acceptance, and willingness to adopt unfamiliar control methods. METHODOLOGY/PRINCIPAL FINDINGS:We conducted a cross-sectional Knowledge, Attitudes, and Practices (KAP) survey among 346 adult household representatives in eight urban and rural communities in the departments of Zacapa and Chiquimula, Guatemala. The study assessed dengue-related knowledge, household prevention practices, perceived risk, and attitudes toward potential future copepod-based biological control. Most respondents correctly identified mosquitoes as the vector of dengue, but knowledge of breeding sites and preventive measures was less comprehensive and varied by educational level. Preventive practices were inconsistent: 37% of households reported taking no action to eliminate mosquito breeding sites, and only a minority reported using multiple prevention strategies. Although prior familiarity with copepods was limited, willingness to consider their use was high when linked to clear information, technical support, and assurances regarding water safety. Concerns focused primarily on the introduction of organisms into stored water and lack of knowledge about the method, particularly in urban communities. CONCLUSIONS/SIGNIFICANCE:The findings indicate a substantial gap between basic dengue awareness and routine household prevention practices in eastern Guatemala. At the same time, the high conditional openness toward copepod-based control suggests that biological approaches may be socially acceptable if introduced through trusted local actors, transparent communication, and community engagement. These results support the further exploration of copepod-based interventions as a complementary, non-chemical vector control strategy. However, community acceptance should not be interpreted as evidence of effectiveness; rigorous field trials are needed to evaluate entomological impact, operational feasibility, safety, and sustainability under local environmental and household water-storage conditions.
BACKGROUND:Protobothrops mucrosquamatus is the primary species responsible for snakebite morbidity in Taiwan, typically resulting in severe localized tissue edema. Although freeze-dried hemorrhagic antivenom (FHAV) is the standard treatment, persistent swelling remains a frequent clinical challenge. This study aimed to identify primary edematogenic toxins and evaluate the neutralizing efficacy of FHAV and small-molecule inhibitors (SMIs) to address this therapeutic gap. METHODOLOGY/PRINCIPAL FINDINGS:Multiple linear regression of a retrospective cohort of 50 patients was used to identify predictors of edema remission. Experimentally, a murine paw edema model was established to evaluate the potency of crude venom and isolated fractions (PLA2, SVSP, and SVMP/CTL), whereas ELISA was used to determine FHAV immunoreactivity. Clinically, total FHAV dosage was the strongest independent predictor of the remission timeline (β = 0.658, T = 5.167, P < 0.001), accounting for 69.8% of the variance. Experimentally, the PLA2-enriched fraction was identified as the primary edematogenic driver, eliciting swelling at 100 μg/mL, which was statistically indistinguishable from that of crude venom, whereas the other fractions required fivefold higher concentrations to achieve similar effects. However, ELISA revealed a significant immunological mismatch: the SVMP/CTL fraction sequestered 81.49% of the FHAV, indicating that highly potent PLA2 was poorly targeted. Furthermore, SMIs (varespladib, marimastat, and batimastat) at 10 μM exhibited limited efficacy against crude venom-induced edema, suggesting that single-target interventions against the complex venom proteome are insufficient. CONCLUSIONS/SIGNIFICANCE:Persistent localized edema arises from an immunological mismatch in which FHAV recognizes primarily high-molecular-weight immunogens instead of the primary edematogenic driver, PLA2. Current clinical management necessitates increasing FHAV dosages to compensate for low specific affinity through increased total antibody volume. These findings highlight that robust prospective multicenter trials are urgently needed to transition from empirical dosing to standardized, potency-aligned FHAV protocols, potentially incorporating multimodal therapeutic cocktails to achieve comprehensive neutralization.
BACKGROUND:Using the 2025 Chikungunya fever (CHIK) outbreak in Chancheng District, Foshan City, this study applied a random forest (RF) regression model combined with SHapley Additive exPlanations (SHAP) to explore predictive associations, nonlinear relationships and potential thresholds between environmental-social factors and village/community-level cumulative incidence, to inform stratified control of mosquito-borne diseases. METHODOLOGY/PRINCIPAL FINDINGS:In this cross-sectional ecological study of 143 villages/communities, the outcome was cumulative incidence, and nine candidate covariates were assessed, including the hospitalization isolation rate, construction-site density, and population density. Multicollinearity was checked using the variance inflation factor. Model fit was evaluated by the out-of-bag (OOB) R², RMSE, and MAE, and variable importance by %IncMSE. Robustness was tested with 100 repeated runs, bootstrap thresholds from SHAP dependence plots, and a sensitivity analysis excluding the endogenous isolation rate. On the log(1 + incidence) scale, the OOB R² was 0.206, with underestimation of high-incidence areas. The hospitalization isolation rate had the highest importance (%IncMSE = 18.43) and was negatively correlated with predictions (ρ = -0.749), but this likely reflects reverse causation and is predictive only. Construction-site density was strongly positive (ρ = 0.855), with a stable threshold near 12.8 sites/km²; it remained the most robust predictor after removing the isolation rate (%IncMSE = 8.54). CONCLUSIONS/SIGNIFICANCE:Construction-site density was the most robust environmental predictor, whereas population density contributed little. These exploratory, predictive associations-not causal effects-should guide risk stratification and require prospective validation with time-matched longitudinal data.
BACKGROUND:Cervical cancer disproportionately affects people living in low- and middle-income countries (LMICs). While access to effective preventive interventions explains some of the disparity, biological causes cannot be ruled out as important contributors to the observed disparities in cervical cancer outcomes. Since chronic infection with helminths which cause immune dysregulation and suppress anti-viral responses is common in LMICs, we sought to review the available epidemiologic evidence evaluating associations between helminth infection and HPV infection and/or cervical cancer. METHODS:We searched five databases of scientific publications using search terms targeted towards journal articles published between 1990-2022 evaluating an epidemiologic association between helminth infection and HPV prevalence, persistence, or cervical cancer progression. Out of 93 reports assessed for eligibility, we identified eight relevant studies and describe them in this scoping review. RESULTS:All eight studies suggest a possible positive association between helminth infection and HPV or cervical cancer. Six studies found a positive trend in association between schistosomiasis and cervical neoplasia; two studies between hookworm infection and HPV prevalence; and 1 study between Ascaris, Trichuris, and Strongyloides and HPV prevalence. CONCLUSIONS:Published evidence suggests positive trends towards an association between helminth infection and cervical neoplasia. Given the high burden of helminth and HPV co-infection in LMICs, further evaluation of the association between helminth infection and HPV or cervical cancer is warranted.
Chikungunya virus (CHIKV) transmission in Guangdong, southern China, intensified markedly in 2025, with successive outbreak waves in Foshan and Jiangmen followed by wider dissemination across all 21 prefectures during a peak domestic travel period. By late 2025, more than 25,000 cases had been reported, suggesting a departure from the historically import-driven and geographically limited outbreaks previously observed in China. These observations raise the possibility of an early transition towards more sustained transmission, although repeated introductions followed by local amplification remain an alternative explanation. Key contributing factors include mobility-associated amplification, climate variability, flood-related expansion of mosquito breeding habitats, favourable urban ecology, and incomplete interruption of transmission during cooler months. Although winter temperatures are suboptimal for sustained transmission, vector persistence and possible vertical viral transmission may permit low-level circulation between epidemic waves. In this context, distinguishing repeated importation from local persistence becomes critical. Preventing endemic establishment will require sustained inter-epidemic surveillance, anticipatory vector control, and clearer policy pathways for deployable vaccination strategies.
BACKGROUND:Talaromycosis (TSM) is a severe opportunistic fungal infection that mostly infects patients with acquired immunodeficiency syndrome (AIDS). After antiretroviral therapy (ART), AIDS patients with TSM may develop immune reconstitution inflammatory syndrome (IRIS), leading to the paradoxical worsening of clinical symptoms. However, the immunological characteristics of TSM-related IRIS (TSM-IRIS) in AIDS patients remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS:We conducted a longitudinal cohort study of AIDS patients with TSM and analyzed the immunological features using flow cytometry, transcriptome, and multiplex cytokine assays at admission, ART initiation, and at 2, 4 weeks after ART initiation. Patients with TSM-IRIS displayed significantly higher baseline CD3 ⁺ T cells at admission and lower frequencies of PD-1 ⁺ CD4 ⁺ T cells at ART initiation than those without IRIS (non-IRIS). After ART initiation, TSM-IRIS was associated with dynamic transcriptomic changes involving inflammatory and immune-related pathway enrichment, along with sustained elevations of interleukin (IL)-17, IL-21, C-X-C motif chemokine ligand (CXCL) 5, and CXCL12. Transcriptomic analysis revealed the dynamic enrichment of pro-inflammatory pathways prior to and during the early phase of ART. CONCLUSION/SIGNIFICANCE:AIDS patients with TSM-IRIS exhibited an immune profile associated with enhanced T cell activation, characterized by accelerated immune reconstitution accompanied by heightened inflammatory activity during the early phase of ART, may potentially acting as the immune markers for prediction or diagnosis of TSM-IRIS in AIDS patients.
BACKGROUND:Cholera outbreaks remain a major public-health challenge in sub-Saharan Africa, where diagnostic capacity is limited and clinical case definitions are non-specific and re ly heavily on syndromic diagnosis. Rapid identification of Vibrio cholerae is critical, yet cholera-suspected diarrhoea can have multiple infectious causes not captured by targeted diagnostics. METHODS:We evaluated a mobile, culture-independent metagenomic sequencing workflow for on-site detection of gastrointestinal pathogens directly from faecal samples in Burundi. The offline workflow combined long-read Oxford Nanopore Technologies (ONT) sequencing with rapid, laptop-based taxonomic and antimicrobial resistance (AMR) screening and was deployed across a health centre, a district hospital, and a refugee transit camp. The frontline and real-time results were verified using both conventional culturing and in-depth bioinformatic analyses. RESULTS:V. cholerae signals were only detected in a subset of suspected cholera cases, while many samples were dominated by alternative bacterial taxa, most frequently Escherichia coli. V. cholerae abundance correlated strongly with detection of the C holera T oxin P hage CTXφ, supporting differentiation between toxigenic signal and background exposure. AMR genes were detected across samples, providing early situational insight into resistance determinants among gastrointestinal bacteria. CONCLUSIONS:Mobile, offline metagenomic sequencing enables rapid frontline characterization of gastrointestinal disease, especially cholera-suspected, in resource-limited settings and complements existing diagnostics by improving etiological resolution and outbreak response.
Dog-mediated rabies remains a preventable cause of mortality in children, particularly in low- and middle-income countries where free-roaming dogs are common. While the WHO-led "Zero by 30" strategy emphasises mass dog vaccination and access to post-exposure prophylaxis (PEP), less attention has been paid to the behavioural and communication pathways that shape children's vulnerability. This Viewpoint proposes a conceptual distinction between vulnerability to dog bites and vulnerability to rabies following exposure. Conflating these risks may contribute to diffuse messaging and missed prevention opportunities. This Viewpoint argues for a differentiated child-facing and adult-facing interventions, with schools positioned as adaptable platforms for integrating safe behaviour, disclosure, and timely care-seeking within existing systems. Aligning behavioural, educational, and biomedical components more explicitly may strengthen implementation of global rabies elimination strategies and reduce paediatric deaths.
BACKGROUND:Echinococcosis is a zoonotic disease caused by cestodes of the genus Echinococcus. Alveolar echinococcosis (AE), caused by E. multilocularis, primarily affects the liver and shows infiltrative, tumor-like growth of the metacestode stage. If untreated, AE is lethal. AE remains a neglected disease with current treatments based on albendazole or mebendazole that are parasitostatic, and not curative, underscoring the need for more effective therapies. Niclosamide is a chlorinated salicylanilide derivative with proven activities against intestinal helminths but is inactive against tissue-dwelling helminths due to poor absorption and limited bioavailability. In this study, we repurposed niclosamide ethanolamine (NEN), a formulation with improved systemic exposure, for the treatment of E. multilocularis infection in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS:We assessed the in vitro efficacy of niclosamide and NEN against E. multilocularis metacestode vesicles (IC50 < 0.2 µM) and primary parasite cells (IC50 < 0.3 µM), with active concentrations largely corresponding to NEN levels reachable in the liver. Metabolic analysis suggested that NEN acts as a mitochondrial uncoupler. Electron microscopy showed that NEN-treatments induced profound structural damage in the metacestode vesicle tissue, but mitochondrial ultrastructure was not notably affected. In mice intraperitoneally infected with E. multilocularis, NEN was orally administered during 9 weeks either alone, or in combination with albendazole. Pharmacokinetic analyses showed that NEN reached blood level concentrations above 1 µM. However, the parasite burden in NEN-treated mice was not significantly reduced under the conditions used in this study. CONCLUSIONS/SIGNIFICANCE:Although niclosamide and NEN demonstrated potent activity against E. multilocularis in vitro, this efficacy did not translate in the mouse model. The lack of in vivo activity could be attributed to several factors such as infection model, limited drug uptake by the parasite in the animal, or the rapid metabolization of the compound. Future studies should explore novel niclosamide derivatives and formulations to enhance efficacy against AE in vivo.
BACKGROUND:Dengue virus (DENV) is a mosquito transmitted arbovirus that represents a severe threat to global populations. The four serotypes of DENV often circulate endemically resulting in many discrete but overlapping transmission chains in space and time, effectively hiding the underlying patterns of transmission, and longer term consequences on local population immunity. Here we combined serotyped case data with sequence data from a single setting over 25 years to characterise the spatial dependence between cases, the number of distinct transmission chains circulating at any time and whether community transmission was followed by serotype-specific local herd immunity. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we combined geolocated case data (N = 7,487) from individuals that presented at a provincial hospital in Kamphaeng Phet Province, Thailand alongside a subset of the sequences of the viruses (N = 302) that infected them. We estimated the spatial dependence between case pairs over different temporal and spatial windows, using both serotype and sequence data, and used logistic regression to identify whether host population size or epidemic size were correlated with viral diversity. We found there was spatial dependence between cases at up to 5 km, resulting in localised serotype-specific patterns of immunity in subsequent years. Within a 78.5 km2 area there were 9.6 distinct transmission chains at any time and that each 100,000 increase in population size was associated with 9.8 additional chains. While local viral diversity was also correlated with the size of the epidemic within any month, we found diversity only had moderate predictive power to accurately recover epidemic dynamics. CONCLUSIONS/SIGNIFICANCE:These findings highlight the nuanced and spatially variable ecology of a highly endemic pathogen with substantial immunity. This study also illustrates the strengths of combining multiple data sources to shed light on the nature of DENV transmission.
