PLOS Neglected Tropical Diseases

Introduction Cutaneous leishmaniasis (CL) is a neglected tropical disease associated with reduced health-related quality of life (HRQoL) that leads to permanent scars, anatomical damage and functional impairment. We aimed to translate, culturally adapt and validate the disease specific HRQoL measure the Cutaneous Leishmaniasis Impact Questionnaire (CLIQ) into Afan Oromo. Methods The English version of the CLIQ was translated into Afan Oromo, and culturally adapted by experts with feedback from individuals affected by CL. The finalized Afan Oromo version was then administered to adults with CL. Its psychometric properties were examined using internal reliability, inter rater reliability, construct validity, and responsiveness to change. In addition, the clinical importance difference (CID) and cut-off points for the total CLIQ score were determined. Results The Afan Oromo CLIQ demonstrated acceptable content validity, with I-CVI values ranging 0.83 to1.00. One hundred and forty-four individuals with confirmed CL with a mean age of 35.5 ({+/-}16.5) years were interviewed using the Afan-Oromo version of the CLIQ. The overall median CLIQ score was 40 (IQR=24). The median score for general impacts of CL (Cluster-1), and perceptions about health services and treatment (Cluster-2) were 32 and 9 respectively. The internal consistency (Cronbach alpha= 0.87) and inter-rater reliability (ICC=0.98) were excellent. The differences in median CLIQ scores between physicians determined CL severity classifications and between small and larger lesions were significant. The Afan Oromo CLIQ was responsive to change following treatment (P = 0.037). The CID was 9 and 7 units, using distribution and anchor methods, respectively. Conclusion The Afan Oromo CLIQ is a valid and reliable disease-specific instrument to assess HRQoL of CL affected individuals.

Purpose: This study estimated out-of-pocket (OOP) expenses associated Human African Trypano-somiasis (HAT) care, in the Democratic Republic of the Congo (DRC) and explored how they influ-enced care-seeking behavior and participation in HAT control, aiming to inform effective and finan-cially accessible elimination strategies. Methods: A sequential mixed-methods study was conducted using 16 semi-structured interviews and 6 focus group discussions, followed by a structured survey of 444 recently tested participants across 6 health zones. Medical and non-medical expenditures were collected by health structure type and screening strategy (active vs. passive). Catastrophic health expenditure (CHE) was defined as OOP costs, excluding food, exceeding 10% or 25% of annual household income. Results: Payments at health facilities, transport costs and long distances delayed care-seeking, par-ticularly in passive screening (PS). Active screening (AS) was associated with minimal OOP, 93% of visits were cost-free, with a median OOP of 0.76 USD among those incurring costs. PS generated higher expenses, only 12% of PS visits were cost-free, with a median OOP of 9.08 USD among those with expenditures. Among confirmed cases, median OOP was lower through active (9.84 USD) than PS (24.23 USD). Nearly 90% of confirmed cases sold assets or borrowed money to cover expenses. CHE was uncommon under average household income(<4%), however 36% of passively detected cases exceeded the 10% threshold under minimum-wage income assumptions. Conclusion: Despite free diagnosis and treatment, accessing HAT care in rural, low-resource foci in the DRC still imposes a substantial financial burden. Reaching elimination targets and ensuring eq-uitable access will require minimizing indirect costs and logistical barriers to screening and diagno-sis. As active screening declines, routine health systems assume greater surveillance responsibilities, reducing indirect costs and logistical these barriers will be critical to sustain coverage and maintain an effective and equitable HAT elimination strategy.

BackgroundCystic echinococcosis (CE) is a neglected parasitic zoonosis that primarily affects marginalized populations in rural endemic regions. These populations have limited access to, and availability of, the appropriate infrastructure, resources, and skills required to treat this complex disease. This retrospective study reviewed and analyzed the clinical management of CE patients in Bhutan, based on hospital records from January 2020 to December 2024. MethodsHospital records of 120 patients with hepatic or pulmonary CE treated between January 2020 and December 2024 in the three hospitals caring for CE patients in Bhutan were retrospectively reviewed. Data on clinical presentation, diagnosis, treatment, and outcomes were extracted from hospital records using a standardized questionnaire. Data analysis was performed using R software (version 4.4.3). FindingsThe median age of the patients was 36 years (IQR: 21.75-53), with 60% being female. The liver was the most affected organ (70%), followed by the lungs (8%). US and CT were used in 83% for diagnostic and pre-surgical assessments. WHO-recommended CE cyst staging was performed in only 11% of cases. Pre-intervention complications were reported in 40% of patients. Treatment approaches included surgery (open or laparoscopic partial cystectomy, deroofing, and cyst drainage) combined with albendazole (ABZ) therapy (74%), PAIR in 4%, and ABZ alone in 8% of cases. Antibiotic use beyond standard perioperative prophylaxis was common (44% of cases). Post-treatment complications occurred in 23% of surgical cases, including one death; biliary leakage was the most frequent complication (55%), and more than one-third of surgical patients were discharged with drains in situ. 23% of the cohort were readmissions and 11% of the patients with hepatic CE were due to documented recurrence requiring repeat surgery. Long-term follow-up was absent, limiting the early detection and management of recurrence. Conclusion and recommendationsThe study findings show that the care for CE patients in Bhutan urgently requires implementation of US-based cyst staging and treatment allocation, the development of infrastructure and skills for the major treatment modalities recommended by WHO guidelines and long-term follow-up to improve patient outcomes, including recurrence, and to ensure quality control of CE care. Safe and proven practices, particularly in surgery, must be prioritized over diversification. Such strategies are feasible and cost-effective. Author SummaryCystic echinococcosis (CE) is a neglected parasitic disease that develops silently and can affect people for years before causing serious complications. It is common in rural, livestock-rearing regions, including Bhutan, where access and availability to appropriate care is limited. We reviewed hospital records of 120 patients treated for CE in Bhutan between 2020 and 2024 to understand current clinical practices and treatment outcomes. Most patients had large liver cysts and were diagnosed at a late stage, often only once complications had occurred. US-based cyst staging was very rarely performed, and cyst-staging was not used to inform treatment decisions. Surgery combined with anti-parasitic (albendazole) therapy was the most common treatment. Postoperative complications and disease recurrence were frequent, and most patients had no long-term follow-up to attend complications and recurrences timely. Considerable variation in surgical and medical management was observed. Developing and implementing WHO-guideline-based infrastructure, resources and training for CE patient care is urgently needed.

Abstract Introduction Onchocerciasis is targeted for elimination with community-directed treatment with ivermectin (CDTI). Alternative strategies are needed in areas where transmission persists despite long-term CDTI and/or are co-endemic with loiasis. This study assessed the efficacy of 35-day treatment with 100mg doxycycline on Wolbachia density at 6 months and microfilaridermia and palpable nodules at 30 months post-treatment. Methods A treatment follow-up study was conducted in 20 high-transmission onchocerciasis communities in a co-endemic loiasis area of South-West Cameroon. Community-based directly observed treatment with 100mg doxycycline was administered to community members aged [&ge;]9 years. Wolbachia clearance at 6-months and treatment efficacy on microfilaridermia and palpable nodules were assessed at 30-months post treatment. Factors associated with reductions in microfilaridermia post treatment, including adherence to doxycycline treatment were assessed with mixed-effects logistic regression. Results Over 92% (2835/3080) of eligible participants took 35 days of 100mg doxycycline over 5 or 6 weeks. This regimen achieved a 62.8% microfilaridermia reduction and 99% palpable nodule reduction in the 720 participants included at follow-up. Wolbachia depletion was observed in 92% of skin samples at 6 months post treatment. The most important factor associated with microfilaridermia after 30 months was having missed at least 7 doxycycline consecutive doses (OR 3.11, 95%CI: 1.17-8.26). Incomplete treatment to a lesser extent was not associated with reduced efficacy at follow-up. Conclusion This large-scale community intervention shows that a 5-week treatment with 100mg doxycycline is feasible and has high curative efficacy against adult O. volvulus as measured by the dramatic reduction in the proportion of palpable nodules at 30-months post treatment. The high efficacy shows the tremendous potential of anti-Wolbachia drugs as part of the arsenal for onchocerciasis elimination and paves the way for the next generation of anti-Wolbachia drugs with shorter treatment courses, which will facilitate the implementation of alternative strategies to accelerate onchocerciasis elimination.

Human African Trypanosomiasis (HAT) is a two-stage infection caused by Trypanosoma brucei ssp. In stage I the trypanosomes are in blood, lymph and tissue interstitial space and the infection progresses to stage II when the parasites enter the central nervous system (CNS), resulting in behavioural aberrations that proceed to coma and death. Here, we use a bioluminescent murine model of HAT to examine parasite localisation and the changes in host brain gene expression, metabolism and function, and behaviour that occur over the course of the infection. The murine HAT model reproduces the decrease in brain tryptophan seen in clinical samples, and we report for the first time an unprecedented 1.8-fold decrease in global brain glucose metabolism in stage II infection. These metabolic changes are accompanied by an 18-fold decrease in brain insulin transcripts without changes in pathways regulating the cellular responses to insulin. By contrast, genes involved in fatty acid and lipid metabolism are upregulated in the brain during stage II infection. Moreover, we show that transcriptional programmes regulating mitochondrial metabolism dynamically adapts across the time course of HAT infection, ultimately leading to a transcriptional programme that diverts host brain metabolism away from glycolysis during stage II infection. Overall, our data demonstrate a reprogramming of brain energy metabolism during stage II HAT infection that favours the utilization of fatty acids and lipids to meet the energy demands of the brain, with a reduced reliance on glucose metabolism. Despite the profound neurometabolic changes observed, host anxiety-like behaviour is unchanged and episodic learning and memory is not impaired, suggesting that brain metabolic reprogramming enables the utilisation of adipose reserves to maintain core brain functions. These finding may explain the progressive onset of neurological symptoms in HAT patients and inform the development therapeutic interventions to alleviate them. Author SummaryHuman African Trypanosomiasis is classically characterised as being a two-stage infection, stage I where the extracellular Trypanosoma brucei multiply in the blood, lymph and peripheral tissues, and stage II where parasite cross the blood-brain barrier (BBB) causing neurological symptoms and eventually death. Using a murine model of HAT we show that in stage II the key brain metabolite tryptophan is depleted and cerebral glucose utilisation is decreased, accompanied by extensive metabolic transcriptome reprogramming of the cerebral tissue during stage II infection. Despite this we see no significant change in mouse anxiety-like behaviour or learning and memory. Our data are consistent with the brain switching from glucose as the primary energy source, instead utilising the products of lipolysis to maintain essential brain functions. This new understanding of the neurometabolic changes that occur in stage II HAT may help to develop new treatments for the neurological symptoms that affect patients at this stage of the disease.

Background: Skin-related neglected tropical diseases (skin NTDs) continue to affect people living in remote communities of endemic countries, particularly in regions with limited access to dermatological care. This operational research evaluated the impact of the eSkinHealth app, a digital health tool designed to enhance case management of skin NTDs and other skin diseases in Cote d'Ivoire. The eSkinHealth app functions as a portable electronic medical record and a platform for teledermatology, connecting frontline healthcare workers to remote specialists. Methodology/Principal Findings: The study was conducted across sixteen primary health centers (PHCs) in the Sinfra and Bouafle districts, regions endemic for skin NTDs. Using a before-and-after implementation design, baseline data were collected from paper registries and compared with data captured through the app. The primary objective was to assess changes in skin disease detection and diagnosis, while also evaluating usability, acceptability, and feasibility of the tool among healthcare workers. A total of 1,766 patients were included in the analysis (mean age 22.8 years; 55% male). During the intervention period, skin NTD registrations increased significantly from 30 to 91 cases (p < 0.01). Buruli ulcer cases rose from 6 to 14 (p = 0.05), scabies from 24 to 70 (p = 0.13), and other NTDs such as leprosy, lymphatic filariasis, and yaws were newly detected and documented. In contrast, registrations of non-NTD skin diseases decreased from 662 to 472 cases (p < 0.01); however, the proportion of non-NTD cases which received diagnostic confirmation increased markedly, from 0% at baseline to 94% during the intervention period (p < 0.01). Qualitative interviews with nurses and community health workers highlighted improvements in diagnostic accuracy, patient engagement, and confidence in daily practice, while also noting persistent challenges such as stigma, transportation barriers, technical difficulties, and patient concerns about privacy. Conclusions/Significance: The integration of the eSkinHealth app into routine PHC services proved effective in enhancing diagnostic capacity for skin NTDs in resource-limited settings. However, capturing other skin diseases proved more difficult given their high prevalence. While the app demonstrated clear benefits in improving diagnostic rates and healthcare worker confidence, persistent challenges such as technical issues and patient concerns about privacy need to be addressed for future scalability. As with many digital tools, further refinement will be an ongoing process, and the lessons learnt from this study may provide valuable guidance for similar initiatives in comparable settings.

Background. Women living in leishmaniasis-endemic zones are regularly exposed to the sandfly vector in their environments. While case series and laboratory evidence consistently suggest transplacental transmission of Leishmania parasites with deleterious maternal-fetal effects, this issue has received insufficient attention, particularly in areas where the predominant Leishmania species is mainly associated with cutaneous disease (CL). Methodology/Principal Findings. We conducted an exploratory cross-sectional study in a highly endemic zone of Tabasco, Mexico, enrolling 53 women with singleton term deliveries between April 2018 and April 2020. Placental PCR was positive in 18 (34%) participants. Buccal swabs were positive in 11 (21.2%) of 52 newborns. Immunofluorescence confirmed intracellular amastigotes within macrophages near the vascular endothelium of PCR-positive placentas, with no surrounding inflammatory infiltration. Sequencing revealed homology to Leishmania mexicana or L. amazonensis. Birthweight percentile was modestly lower in the PCR-positive group (predicted mean 53.8% vs. 56.5%, p = 0.76), while small for gestational age showed a non-significant trend toward higher prevalence among PCR-positive cases (prevalence ratio = 2.06, 95% CI: 0.32-13.39, p = 0.45). Conclusions/Significance. Subclinical, dynamic transmission of Leishmania parasites typically associated with cutaneous disease was detected in this endemic zone. The presence of L. mexicana in human placentas was confirmed by immunofluorescence and sequencing, without an associated inflammatory response. These findings highlight the potential of CL-associated Leishmania species to reach the placenta and buccal mucosa of newborns, warranting further epidemiological investigation into the consequences of vertical transmission in regions with endemic CL.

Leishmaniasis, a climate-sensitive zoonotic neglected tropical disease, is transmitted by Phlebotomine sand flies and closely linked to socio-economic inequities. Understanding its spatio-temporal dynamics under environmental and social change is critical for effective control. A machine learning framework (XGBoost) was developed to map the global and European distribution of leishmaniasis, incorporating climatic indicators, land cover, elevation, and socio-economic indices (Human Development Index, AROPE). For Europe, five proven vector species (Phlebotomus perniciosus, P. ariasi, P. perfiliewi, P. neglectus, and P. tobbi) were modelled alongside cutaneous and visceral leishmaniasis. Across both analyses, land use features, particularly shrubland and forest cover, had the greatest explanatory power, reflecting their role in providing microclimates and vertebrate hosts for sand flies. Climatic factors, notably mean temperature of the coldest quarter and humidity of the warmest/driest quarters, were also influential, as these facilitate sand fly survival. Socio-economic predictors consistently improved model performance, confirming the role of poverty and inequity as determinants of disease distribution. Globally, leishmaniasis risk increased by ~17% since the 1990s, with Africa, Asia, and the Americas experiencing the greatest rise. In Europe, modest continental-scale increases (CL +1.28%; VL +2.47%) masked strong sub-national heterogeneity, including northward expansion of visceral leishmaniasis and increases in cutaneous leishmaniasis in southern and eastern regions. Sand fly projections indicated expansion of warm-adapted species (P. ariasi, P. perniciosus, P. neglectus) and contraction of species preferring cooler, more humid niches (P. perfiliewi, P. tobbi). These findings highlight climate change, land use, and inequity as interacting drivers of leishmaniasis, emphasising the need for enhanced surveillance, integrated vector management, and targeted support for vulnerable populations, including refugees and migrants.

BackgroundControl of Aedes aegypti, the primary vector of dengue and other Aedes-borne viruses, is challenged by insecticide resistance, limited efficacy of existing tools and the large and widespread epidemics. Targeted Indoor Residual Spraying (TIRS), a modification of traditional indoor residual spraying focused on Ae. aegypti resting sites, has demonstrated promising results, yet its indirect community-wide effects remain underexplored. Methodology/Principal FindingsWe conducted an entomological cluster-randomized controlled trial in Iquitos, Peru, to evaluate the direct and indirect entomological impacts of TIRS using pirimiphos-methyl. Thirty clusters were randomized to receive either TIRS (15 clusters, 898 structures) or standard Ministry of Health vector control activities (15 clusters, 1,018 structures). Aedes aegypti indoor densities were assessed in the 45 days pre-intervention and at four time points up to 255 days post-intervention using Prokopack aspiration. Generalized linear mixed models with a negative binomial link were used to estimate incidence rate ratios (IRRs) and calculate efficacy (1-IRR) for houses that received TIRS (direct effect) and untreated houses in TIRS clusters (indirect effect). Direct efficacy reached 96% at 15 days post-spraying and remained significant (40%) at 255 days post-spraying. Indirect efficacy reached 69% at 15 days and declined to 7% by 255 days post-spraying. Despite only 57% household-level TIRS coverage, both direct and indirect impacts on Ae. aegypti were significant during early post-intervention surveys, and after 8 months in TIRS clusters. Conclusions/SignificanceTIRS provided substantial and sustained reductions in indoor Ae. aegypti density, including measurable indirect effects in untreated homes within intervention clusters. These findings demonstrate the entomological value of TIRS even at moderate coverage levels and highlight its potential for both preventive and reactive vector control programs and should be considered for implementation by Ministries of Health in dengue-endemic urban settings as well as by the U.S. military when deployed to tropical or subtropical locations.

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.

The global occurrence of sexually transmitted infections (STIs) continues to rise, necessitating accurate diagnosis and treatment to curb their spread and associated complications. With the alarming increase in antimicrobial resistance (AMR) in Neisseria gonorrhoeae, effective STI management relies heavily on etiological diagnosis. The Tanzania National Standard for Medical Laboratories 2017 outlines recommended STI testing protocols based on facility levels, yet adherence to these guidelines and associated challenges remain poorly documented. This study describes the diagnostic capacity for different STIs in northern Tanzania. A cross-sectional study was conducted between May and July 2023, encompassing 14 laboratories across Moshi Municipal Council, Kilimanjaro region. The laboratories assessed were in five hospitals and nine health centres (HCs). Data regarding facility type and STI diagnostic capabilities were gathered through questionnaires administered during site visits and supplemented by observations. All five hospitals were equipped to conduct rapid diagnostic tests for HIV, syphilis, and wet preparation microscopy for Trichomonas vaginalis (TV). Only three hospitals had the capacity to perform culture and sensitivity testing using chocolate and blood agar medium, however none reported isolating Neisseria gonorrhoeae in the past year. Critical STI diagnostic tests including the Treponema pallidum particle agglutination assay (TPPA) and Treponema pallidum hemagglutination assay (TPHA) for the laboratory confirmation of syphilis, assays for Chlamydia trachomatis, Herpes Simplex virus -2, and Human papillomavirus (HPV) were absent across all five hospitals. Conversely, all health centers demonstrated proficiency in rapid treponemal tests for syphilis, together with rapid HIV test and TV testing, although one health center lacked the capacity for wet laboratory preparation for TV detection. Findings underscore a concerning lack of STI testing capacity within surveyed healthcare facilities, posing significant barriers to effective STI management and exacerbating the threat of AMR in Tanzania. In particular, the capacity for conventional microbiology culture was limited in most settings, severely compromising the ability to track and monitor AMR. Urgent investment in laboratory infrastructure and training is imperative to enhance STI diagnosis and treatment, ultimately curtailing transmission and mitigating the impact of AMR.

Shigella is a major cause of childhood diarrhea in sub-Saharan Africa. Current World Health Organization (WHO) guidelines recommend empiric antibiotics only for dysentery, yet non-dysenteric presentations account for 40-89% of Shigellainfections. We quantified the performance of existing guidelines to identify Shigella infection and evaluated enhanced syndromic criteria for improved management of Shigella cases. We leveraged data from the Enteric for Global Health (EFGH) Kenyan site, which enrolled children aged 6-35 months with medically attended diarrhea, collected rectal swabs at enrolment, and tested these for Shigella using both the culture and TaqMan-array cards (TAC). Shigella positivity was defined as either a culture-confirmed Shigella or a TAC-attributable result (CT <29.5). We compared categorical variables using the {chi}{superscript 2} test or Fishers exact as appropriate while continuous variables were compared between groups using the Wilcoxon rank-sum test. A logistic regression model was fitted to identify a parsimonious set of predictors. Out of the enrolled 1400 MAD cases, of whom 175 (12.5%) were Shigella positive, of which 134 (76.5%) being non-dysenteric. Among all enrolled children, 148 (10.6%) were dysenteric and 1,252 (89.4%) were non-dysenteric. Of the non-dysenteric cases, 57/1,252 (4.6%) and 129/1,251 (10.3%) of children were Shigella attributable by culture and TAC, respectively. Compared to Shigella negative cases, positive cases were older (Median age in months [Q1-Q3]: 20 [14-24] vs 13 [8-19], p<0.001), had higher stool frequency (5 [3-6] vs 4 [3-5], p<0.049) and were more likely to be dysenteric (42 [24%] vs 106 [8.7%], p<0.001). Contrarily, Shigella positive cases were less likely to present with vomiting (66 [37.7%] vs 587 [47.9%], p=0.014) and difficulty in breathing (0 (0%) vs 27 [2.2%], p<0.040). Dysentery alone had minimal predictive power to identify Shigella (area under the ROC curve (AUC) [95% CI]: 0.58 [0.54-0.61]), while Dysentery and Age binary (<17 months) (0.70 [0.66-0.74]), and Dysentery, Vomiting, Difficult breathing, and Age binary (0.72 [0.68-0.76]) had acceptable predictive performance. Our data shows that current guidelines miss up to 76.5% of Shigella-positive cases which are non-dysenteric. This suggests that dysentery alone has limited predictive power, but incorporating additional symptoms increases discriminatory ability by up to 14%, with age alone improving it by 12%. These findings support the need to re-evaluate the current syndromic based guidelines to better detect Shigellosis and strengthen antibiotic stewardship.

Background Rabies is a zoonotic neglected public health problem associated with animal bites, especially domestic carnivores claiming 59,000 deaths annually predominantly in developing countries of Africa and Asia. There is a high risk of exposure among rural communities endemic with animal rabies where adoption of prevention strategies is minimal. This study determined the prevalence of suspected rabies exposure, associated factors, and delayed post-exposure care-seeking among animal-bite human cases in Busia district, Uganda. Methods: This was a cross-sectional study that involved 332 consecutively sampled animal bite human cases that occurred within the period 2023 to 2024. Data for the bite cases from records were collected using a data abstraction tool. In addition, interviewer-administered semi-structured questionnaires were used to collect data on sociodemographic, animal-related and environmental characteristics. Approximate bite locations were collected using Global Positioning System (GPS) coordinates via Kobo collect. Analysis was carried out in STATA 17 using mixed effects modified Poisson regression for factors associated with suspected rabies exposure. Results: The median age of the bite cases was 18 (IQR: 9-36) with the male gender predominantly affected. The prevalence of suspected rabies exposure was 53.6% (95% Confidence interval - CI: 46.8-60.3). Factors associated were urban versus (vs) rural residence (adjusted prevalence ratio-aPR: 1.04, 95%CI: 1.00-1.08), being bitten by a stray animal (aPR: 1.28, 95% CI: 1.22-1.35) and wild animal (aPR: 1.22, 95% CI: 1.14-1.30) vs domestic animal, vaccination status of the biting animal i.e. vaccinated vs unvaccinated (aPR: 0.76, 95% CI: 0.69-0.85), provoked vs unprovoked bites (aPR: 0.82, 95% CI: 0.79-0.86), and distance to nearest river ([&ge;]5km) vs <5km (aPR: 0.93, 95% CI: 0.87-0.99). The prevalence of delayed post-exposure seeking was 23.0% (95% CI: 16.5-31.1) among the suspected rabies exposures. Conclusion: The study reveals a high prevalence of suspected rabies exposure. Factors associated are multidimensional i.e. are of human, animal and environmental origin. The one health paradigm should be emphasized during routine surveillance of rabies-related cases. The study observed that 1 in 5 bite cases delayed to seek care post bite exposure. We recommend collaborations between sectors, routine vaccination and awareness campaigns, and monitoring of wild carnivore populations and environmental dynamics in rabies-related surveillance.

Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H-ATPase (V-H+-ATPase), Ca{superscript 2}-transporting P-type ATPase (Ca2+-ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites. Author summaryProtozoan parasites such as Leishmania and Trypanosoma cause serious diseases affecting millions of people worldwide. To better understand how these parasites survive environmental changes during transmission between hosts, we studied a specialised organelle called the acidocalcisome, which stores polyphosphates and helps regulate stress responses. In this work, we used the non-pathogenic Leishmania tarentolae as a safe and cost-effective model that shares key cellular features with disease-causing species. Using a combination of CRISPR-Cas9 genome editing, proximity-based labelling (BioID), confocal microscopy, pulldown assays and AlphaFold3 structure prediction, we investigated the vacuolar transporter chaperone (VTC) complex, which synthesises and transports polyphosphate into the acidocalcisome lumen. Proximity proteomics identified several known proteins located near the VTC complex, and importantly, led us to discover three novel proteins that interact with it. These findings open new directions for exploring the organisation and regulation of the VTC complex in protozoan parasites. By revealing novel protein interactions, our study contributes to a deeper understanding of parasite biology and may help identify therapeutic targets for treating neglected tropical diseases.

Ectotherm insects growth and development are dictated by temperature and humidity. Conducive habitats and the availability of resources set ideal conditions for insect population growth. Mosquitoes require water, favorable temperature, and blood meal to survive. In this research, we picked a rapidly growing megacity, Ahmedabad, in western India, to explore and establish potential linkages between disease spread and meteorological conditions. Ahmedabad, with a population of over 8 million, is experiencing changes in rain and humidity patterns, pushing the city towards changing vector-borne disease dynamics. We examined dengue cases over ten years, 2012-22, and explored their connections with two prominent climatic variables, temperature and relative humidity. Our findings indicate that stable temperature (25-27.5 {degrees}C) and humidity (> 60%) interaction is a ruling factor in spikes in dengue cases in the city. While stable temperature ranges triggers the dengue cases, RH drives the explosive phases and sustainability of such episodes. Statistically significant increasing trends in temperatures, narrowing down of the day-night temperature ranges, and increasing night temperatures provide more stable temperature regimes in a warming world thereby likely to extend the dengue season beyond the usual monsoon season. Plain Language SummaryDengue incidences have been found to be associated with mosquito population outbreaks. Every year, thousands of lives are lost due to this deadly virus spread by mosquitoes. Particularly in the Indian subcontinent, a large proportion of these cases is associated with the monsoon season and rain patterns. In recent years, there have been abrupt spikes in dengue cases across Indian cities, particularly in western India. To understand this complex interaction of viral proliferation and local environmental conditions, the last ten years of dengue case patterns have been scanned in parallel to the climate data. Our findings suggest that stable temperature windows and humidity levels above certain thresholds trigger a rise in dengue cases. While stable temperature ranges trigger dengue cases, humidity drives such episodes explosive phases and sustainability. Our work pinpoints specific temperature-humidity combinations and suggests that local municipal corporations use them as warning indicators to initiate preventive measures.

Molecular diagnostics to detect Vibrio cholerae (Vc) may be negatively impacted by pathogen-specific lytic bacteriophage (phage) predation. To address this problem, phage detection as a proxy for pathogen detection has been proposed. However, efforts to modernize cholera diagnostics with molecular tools require addressing knowledge gaps on best practices to detect Vc and associated bacteriophages. We conducted polymerase chain reaction (PCR), quantitative PCR (qPCR), and nano-liter (nl) qPCR targeting Vc and known phages (ICP1/2/3) on stool samples collected from patients admitted at hospitals across Bangladesh. Of 4,975 patients enrolled, 2,574 diarrheal samples were collected and over 65,000 reactions were conducted, including replicates. We analyzed the results for target-specific assay alignment and then used machine learning to determine the effect of phage predation on Vc-assay alignment. Standard curve analyses were used to set qPCR-positivity thresholds at 7.3x105 CFU/mL for Vc and 1.7x103, 9.3x103, and 3.0x105 PFU/mL for ICP1, ICP2, and ICP3, respectively. Among 2,462 samples assayed by qPCR, target detection was 25.3% (623), 7.8% (193), 0.5% (13), and 5.8% (144) for Vc, ICP1, ICP2, and ICP3, respectively. There was strong alignment between assays for Vc detection ({kappa}=0.785) and moderate alignment for phage detection ({kappa}=0.609, 0.593, and 0.533 for ICP1/2/3, respectively). Phages were ranked as the first (ICP1) and third (ICP3) effectors of Vc diagnostic alignment. These findings provide insights on how to prioritize molecular methods in the cholera field as well as related less tractable diseases facing similar diagnostic challenges. IMPORTANCEThis paper presents a comprehensive comparison of molecular methods to detect Vibrio cholerae (Vc) and associated bacteriophage (phage) which can be used as a proxy for pathogen detection. This initiative is an important step towards modernizing cholera diagnostics with molecular tools. In this study, we found that quantitative polymerase chain reaction (qPCR) represents a reasonable approach to detect Vc and associated phages balancing assay performance, cost, and accessibility. A key additional finding was that phage predation was found to be a leading factor that impacts the alignment of molecular methods to detect Vc. While we recommend qPCR be added to the cholera diagnostic toolkit, the effects of phage predation need to be accounted for in the development and evaluation of cholera diagnostics. These findings have applicability to less tractable disease where diagnostics share similar vulnerabilities.

Introduction Reducing delays in leprosy case detection is essential for achieving global leprosy targets. Accurate measurement of these delays and their determinants relies largely on patient-reported data, as routine health records are often inadequate. The leprosy case detection delay (CDD) questionnaire, developed under the Post Exposure Prophylaxis for Leprosy (PEP4LEP) project, has been validated in Ethiopia, Mozambique, Tanzania, and Indonesia. However, it has not been adapted or validated for Nigeria or any major Nigerian indigenous language. This study aimed to culturally adapt and validate the CDD questionnaire for Igbo-speaking populations in Nigeria. Methodology/Principal Findings The CDD questionnaire underwent a standardized cross-cultural adaptation process. Content validity was assessed using item- and scale-level content validity indices, while construct validity was evaluated through hypothesis testing. Reproducibility was assessed using test-retest and inter-rater reliability; agreement using the Bland-Altman method and the Wilcoxon Signed-Rank test; reliability using Spearmans rank correlation coefficient and the Intraclass Correlation Coefficient (ICC); and internal consistency using Cronbachs alpha. Data were collected through face-to-face interviews with persons affected by leprosy at two time points separated by at least two weeks. Participants (n=100) had a mean age of 45.1 years (SD=18.7). Mean CDD was 77.2 months at baseline and 77.9 months at retest. The instrument demonstrated excellent content validity (I-CVI/S-CVI: 0.90-1.00), good internal consistency (Cronbachs =0.77), and excellent test-retest reliability (ICC=0.996, 95% CI: 0.994-0.997). Test and retest measurements were highly correlated ({rho}=0.985, p<0.001), with no evidence of systematic change over time (p=0.864). Seventy-two percent of participants reported identical CDD values across assessments. All items from the original English version were retained without modification. Conclusion/Significance The Igbo version of the CDD questionnaire demonstrated good validity and reliability and is suitable for assessing leprosy case detection delay among Igbo-speaking populations in Nigeria

Soil-transmitted helminth (STH) infections are a major public health burden, and there are programmes of mass drug administration that attempt to ameliorate the harm that they cause. There has been increasing use of genomics to study STH infections and other parasitic nematodes, with particular interest in whole genome sequencing (WGS). For such studies, samples are commonly stored frozen, but in settings where these infections are endemic this can be difficult, and so there would be advantages to having ambient temperature storage methods. We investigated two ambient temperature storage methods - FTA cards and DESS buffer - for infective larvae of the rat parasites Nippostrongylus brasiliensis and Strongyloides ratti, prior to DNA extraction and then WGS. Our results showed that for individual larvae stored on FTA cards or in DESS buffer, this resulted in a lower proportion of sequence reads that mapped to the reference genomes, compared to the frozen control samples. Generally, for individual larvae, DESS-storage resulted in better sequencing results than FTA-storage. However, for pools of 10 or 50 larvae, then these ambient temperature storage methods generally resulted in comparable sequence read mapping to the frozen control samples.

Fasciolosis caused by Fasciola hepatica is an economically important disease in sheep and cattle. Knowledge of the population genetic structure of F. hepatica is important for understanding gene flow and informing disease control. In the present study, we designed, developed, and validated a multilocus sequence typing (MLST) scheme based on six markers. These markers were selected by aligning newly sequenced whole-genome sequence (WGS) data with available reference genomes and selecting variable regions with five or more single-nucleotide polymorphisms SNPs from different scaffolds of the F. hepatica reference genome Fasciola 10x pilon (GCA_900302435.1). Twenty markers were initially identified, of which 12 were multiplexed for deep amplicon sequencing after validation on worm and faecal eggs DNA; six markers were ultimately retained for downstream population genetics analysis. These markers were used to investigate population genetic structure in 15 cattle- and 27 sheep-derived F. hepatica populations in UK. A total of 53 unique alleles from six MLST markers were identified from 30 faecal (cattle = 13, sheep = 17) and 12 adult worm (cattle = 2, sheep = 10) populations. Shared alleles were observed in sheep- and cattle-derived populations. The highest allelic variation was observed in the Scottish Borders, Southern Scotland, and South-West England, and the lowest in North-West England. Minimal genetic differentiation was observed between cattle- and sheep-derived populations, with most genetic structuring within rather than between populations. Five markers showed high allelic polymorphism, whereas one marker showed low levels of allelic polymorphism, highlighting the importance of multilocus approaches. Overall, this six MLST-marker panel provides a tool for population genetic studies, revealing high gene flow and clonal expansion of F. hepatica across hosts and regions in the UK.

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.