Mary Elizabeth Miranda

Viral genomic data can resolve rabies transmission and guide outbreak response, but surveillance is often limited less by sequencing capacity than by getting suitable specimens from remote collection sites to a laboratory, a process that can take months. Here, we show that lateral flow devices (LFDs) used for rabies diagnosis can also serve as genomic specimens, integrating diagnosis and genomic sampling into a single workflow without additional specimen collection. We evaluated LFDs and FTA cards for rabies virus genome recovery under laboratory and field conditions. During surveillance across the island provinces of MIMAROPA, Philippines, the Bionote LFD had 99.1% sensitivity and 100% specificity against the gold-standard direct fluorescent antibody test (n = 319), with results available a median of 1 day after sampling versus 5 days for DFAT confirmation. Under laboratory conditions simulating field storage (ambient temperature or 37°C for up to 21 days), both residual LFD material and FTA cards supported near-complete genome sequencing, although genome recovery declined with storage duration, particularly at elevated temperatures. Consistent with this, field-collected LFDs yielded near-complete genomes up to 23 days of ambient storage but only partial genomes after 29-77 days, indicating a practical window of around three weeks for use. Repurposing LFDs already embedded in diagnostic workflows offers a practical route to decentralised genomic surveillance without additional sample collection or a cold chain. FTA cards provide a complementary preservation option, while further optimisation of specimen preparation, extraction and storage could improve genome recovery and extend the sequencing window.

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Southeast Asia and is thought to circulate between Culex mosquitoes, amplifying pig hosts, and ardeid birds, with humans acting as incidental dead-end hosts. However, multiple mosquito species may contribute to transmission, making community composition, abundance, and feeding behaviour important for understanding human exposure risk. This study investigated mosquito diversity, abundance, and blood feeding patterns across 24 sites in three Philippine provinces with a history of JEV cases. Mosquitoes were collected using CO₂-baited BG-Pro traps during five surveys between August 2024 and February 2026, and associations with environmental and host-density variables were assessed using generalised linear mixed-effects models. A total of 8,595 mosquitoes were collected, including 5,378 females representing eight genera and 13 species. Culex dominated collections (86.5%), with Culex quinquefasciatus the most abundant species. Mosquito abundance, diversity, and blood feeding activity were higher during the rainy season, while abundance increased with temperature and chicken density and diversity was greatest in high NDVI and low human dense environments. Genus-specific analyses showed overall abundance patterns were largely driven by Culex, whereas Aedes and Armigeres exhibited distinct environmental responses. Of 274 blood meals, 88.3% originated from chickens, primarily from Cx quinquefasciatus, highlighting the critical need to study the potential role of chickens in JEV ecology.

Background: Understanding mosquito blood-feeding preferences can help infer vector-borne pathogen transmission dynamics and improve intervention strategies for important human and animal pathogens. The gold-standard tests for identifying mosquito blood meals are expensive, time-consuming methods and decrease in accuracy as blood meals are digested. Mid-infrared spectroscopy with machine-learning (MIRS-ML) can offer a rapid, cost-effective alternative approach to identifying host blood meals but has never been used to analyse blood meals from Culex spp., an increasingly important genus of vectors of several high-priority multi-host pathogens such as orthoflaviviruses and filariasis. Methods: In this study, laboratory-reared Culex quinquefasciatus were fed blood from six hosts (human, pig, chicken, cattle, dog and horse). Dried bloodspots were created 6, 12, 24 and 30 h after digestion in the mosquito by rolling abdominal contents onto filter paper and then scanned using a Bruker ALPHA Fourier-transform infrared spectrometer. The spectra generated were used to train a multilayer perceptron algorithm to predict blood meals from these six host species and then tested on field-collected mosquito blood meals. Results: For laboratory-reared mosquitoes, the model was 93.6% accurate in predicting host species in comparison to 16.7% random classification, with a small decline in accuracy after 30 h of digestion (6%). While the accuracy for field-collected bloodspots was lower (67.9%), this likely reflects differences in blood source from wild mosquitoes, such as no anticoagulants and direct feeding from hosts rather than artificial feeders. Model performance was further enhanced by applying a probability threshold, which increased accuracy to 78% in field samples while removing 21% samples. Conclusions: This study demonstrates the potential application of MIRS-ML with dried bloodspots as a practical, scalable tool for blood meal analysis with scope for improving accuracy through further model development. This method may be of particular use for large-scale surveillance programmes in low-resource settings due to low running and labour costs. Further development of this method using samples from field-collected mosquitoes could enhance classification accuracy and expand the range of identifiable host species.

Background: Zoonotic disease outbreaks are a growing global health concern, highlighting the need for tools to strengthen outbreak responses. Process mapping is a systems thinking approach that allows the complex multilevel, multisectoral network of actors and processes involved in an outbreak response to be clearly visualised and analysed using a One Health lens. We applied process mapping to a rabies outbreak in the previously rabies-free Philippine province of Romblon to demonstrate its utility. Methods: Key stakeholders were interviewed to inform the development of a provisional map of the actors, processes and challenges involved in the outbreak response. During a facilitated workshop, stakeholders worked in multisectoral, multilevel groups to refine the map and co-develop solutions to the challenges identified. Results: Process mapping revealed multiple challenges within rabies surveillance and outbreak response, particularly relating to case testing, recording and reporting, as well as intersectoral communication, community sensitisation and dog vaccination. Many of these issues were exacerbated by structural factors such as the Philippines’ archipelagic geography and decentralised governance of animal and human healthcare delivery. Broader issues linked with rabies’ endemic and neglected status were also identified: insufficiently One Health-orientated national guidance on outbreak declaration and response; resource constraints exacerbated by political devolution; and persistent weaknesses in intersectoral coordination and data sharing, compounded by gaps in international guidance on rabies and One Health practice. Suggested solutions that were possible to implement quickly with minimal resources, such as staff training, were undertaken. Recommendations requiring structural change, like enhancing reporting systems, were communicated to the provincial, regional and national government as policy briefs. Conclusion: Our research demonstrates the potential value of process mapping for enhancing One Health disease surveillance and outbreak response, strengthening health systems, and improving preparedness. To address the more systemic challenges revealed, outbreak response guidance must be improved, for rabies specifically and within One Health frameworks more generally, and the implications of political devolution on the control of neglected tropical diseases should be explored. These steps will be essential to achieving the global target of zero human deaths from dog-mediated rabies by 2030.