Shirlee Wohl

Much remains unknown about cholera transmission dynamics in Africa. Applying both traditional phylogeographic techniques and innovative methods for combining epidemiologic and genomic data, we identified three to four distinct “cholera transmission units” within which cross-border cholera transmission has been more frequent. These units can be grouped into two major transmission clusters, one in Western Africa and bordering countries and one spanning the Central, Eastern, and Southern regions. Simulation studies show that these clusters can be used to assess the risk of international spread of introduced cholera strains and identify heterogeneities in the speed of international spread based on outbreak location. Our methods further allow us to infer cholera incidence attributable to specific lineages in the absence of sequencing data. This work has clear implications for cholera control.

Background: During the 2014 Ebola outbreak in Sierra Leone, widespread infections among healthcare workers at Kenema Government Hospital severely disrupted clinical operations. Whether these infections primarily arose from repeated patient exposures or from transmission among colleagues remained unclear, particularly as patient admissions exceeded the hospital’s capacity for safe isolation and care. Methods: We revisited the outbreak using expanded viral surveillance and classical genomic analysis, Bayesian transmission inference with juniper , and firsthand epidemiologic accounts from hospital staff. We analyzed 279 Ebola virus genomes from Kenema District, including 35 from infected healthcare workers, and 514 genomes in the broader transmission reconstruction, explicitly accounting for unsampled cases. Results: The 35 healthcare worker genomes comprised 28 haplotypes, including five shared by multiple cases. In the reconstructed transmission network, 22 candidate healthcare worker-to-healthcare worker links exceeded a posterior probability threshold of 5%, representing 6.1-fold enrichment over expectation. Healthcare workers accounted for 33.2% of expected onward transmissions at their peak in the two-week window beginning June 23, 2014. Firsthand anecdotal accounts situated inferred relationships within specific wards, clinical procedures, and working relationships of KGH, including care of infected colleagues and infections among laboratory, pharmacy, and other personnel beyond routine bedside care. Conclusions: Ebola transmission involved interconnected groups of healthcare workers across multiple hospital departments during a period of severe resource constraints and expanding patient demand. These findings underscore the importance of protecting and surveilling healthcare workers as an interconnected workforce during emerging outbreaks.

Cholera is caused by toxigenic Vibrio cholerae serogroups O1 or O139 and is transmitted through contaminated water or food. Conventional detection methods for V. cholerae in environmental water samples, including membrane filtration and culture, typically require about three days, specialized equipment, and technical expertise. A simple and rapid method for detecting the V. cholerae O1 in water could support outbreak prevention, environmental surveillance, and studies of persistence and transmission. Here, we describe a low-cost, streamlined method for detecting toxigenic V. cholerae O1 in environmental water which can detect V. cholerae O1 within 24 hours. We filter one liter of the water through gauze, incubate the gauze in alkaline peptone water (APW) overnight at 37 C, and test the APW sample with a serogroup O1-specific rapid diagnostic test (RDT). To preserve nucleic acid material from APW broth, we place two drops on each circle of a filter paper (Whatman 903), dry it, and later analyze the DNA using PCR and whole genome sequencing. We evaluated this method using 95 environmental samples collected from 17 sites in Lagos, Nigeria. 54 samples were RDT positive and each of these samples were confirmed by culture and by PCR targeting ompW and ctxA as toxigenic V. cholerae O1. Whole-genome sequencing identified these isolates as belonging to the AFR12 lineage. Among the RDT-negative samples, PCR detected 24 samples positive for ompW but negative for ctxA , consistent with non-toxigenic non-O1 V. cholerae , and 11 of these were further identified as non- toxigenic serogroup O139 by qPCR using a commercial assay (Bioperfectus). The cost for the RDT test was <$2.50 per water sample. This low-cost APW enrichment and RDT-based approach enables identification of environmental water contaminated with toxigenic V. cholerae O1 within 24 hours and enables the collection of samples useful for further bacterial molecular characterization. Author Summary: Standard methods to identify water samples contaminated with V. cholerae serotype O1 are commonly used, but their use for public health practice has been limited due cost, time, and resource requirements. This study describes a convenient, efficient and inexpensive method to detect V. cholerae O1 from environmental water samples and evaluates this method using water samples collected from 17 sites in Lagos, Nigeria during 2025. The method involves filtering one liter of water through a gauze pad, followed by overnight incubation of the gauze in alkaline peptone water (APW) enrichment broth. The APW is then tested for cholera using a rapid diagnostic test (RDT) specific for V. cholerae O1. Among 95 environmental water samples, 54 were positive for V. cholerae O1 using the RDTs. This method provides results within one day at a cost of <$2.50 USD per sample. If there is need for additional studies, RDT positive samples can be sent for culture, and nucleic acid from the APW can be saved by spotting the APW on filter paper. The filter paper can then be stored at room temperature indefinitely. All of the RDT positive samples were confirmed by culture and PCR Among the PCR negative samples, 24 samples were PCR positive for non-O1 V. cholerae, including 11 that were positive for non-toxigenic V. cholerae O139 by qPCR. Whole genome sequencing of the samples found the RDT positive samples to be of the AFR12 lineage, and the DNA contained many genetic markers of antibiotic resistance. Identifying cholera contaminated water quickly provides information to health authorities which can facilitate interventions to reduce transmission and can also provide samples for further epidemiologic and molecular studies.

Despite the existence of an effective vaccine, the United States continues to experience outbreaks of hepatitis A, including in Massachusetts (MA) and New Hampshire (NH) in 2018 and again in MA in 2023. To clarify the relationship between these outbreaks and better understand their drivers, we generated hepatitis A virus whole genome sequences from reported cases and analyzed them using open-source genotyping tools developed and released as part of this study. We found that the 2018 and 2023 outbreaks were caused by distinct viral strains, despite affecting individuals with similar demographic characteristics and reported risk factors. Detailed analysis of genomic and epidemiologic data further resolved transmission patterns within and across outbreaks, showing that experiencing homelessness and prior use of drugs were associated with increased transmission while also revealing transmission between individuals with and without these risk factors, as well as spread across state borders. Together, these findings demonstrate the value of broadly accessible genomic tools for understanding hepatitis A outbreaks and illustrate how whole genome analysis can complement epidemiological investigation by resolving transmission patterns and outbreak drivers that can inform public health interventions.

Cholera remains a significant public health burden in many countries in sub-Saharan Africa, though the exact mechanisms of bacterial emergence and spread remain largely undefined. We generated genomic data from 763 Vibrio cholerae O1 isolates predominantly collected between 2019-2024 to create the largest dataset of V. cholerae genomes sequenced locally in Africa. This dataset enabled us to interrogate recent patterns of spread, including the rapid circulation of the AFR15 lineage associated with unusually large outbreaks in Southern Africa. We provide evidence for the movement of the AFR15 lineage into new African Union Member States and confirm previously observed differences in V. cholerae transmission dynamics in West versus East Africa, though cross-border transmission is prevalent on both sides of the continent. Despite observed differences, evolutionary processes are similar across lineages and we find no evidence for significant changes in antimicrobial resistance genotypes. Overall, our findings emphasize the importance of regionally coordinated cross-border surveillance and interventions, while also demonstrating the critical role of locally generated genomic data in understanding the spread of cholera in Africa.

Much remains unknown about cholera transmission dynamics in Africa. Applying both traditional phylogeographic techniques and novel methods for combining epidemiologic and genomic data, we identified 3-4 distinct ‘cholera transmission units’ within which cross-border cholera transmission has been more frequent. These units can be grouped into two major transmission clusters, one in Western Africa and bordering countries, and one spanning the Central, Eastern, and Southern regions. Simulation studies show that these clusters can be used to assess the risk of international spread of introduced cholera strains and identify heterogeneities in the speed of international spread based on outbreak location. Our methods further allow us to infer cholera incidence attributable to specific lineages in the absence of sequencing data. This work has clear implications for cholera control.

Understanding respiratory virus transmission requires integrating pathogen genomic data with detailed epidemiological context. We analyzed >85,000 SARS-CoV-2 genomes linked to epidemiological data collected in Massachusetts from November 2021 to January 2023. Collected in 666 settings, including schools, colleges, and skilled nursing facilities, this dataset enabled direct comparison of within-setting transmission to community-wide risk. Elevated transmission was concentrated in specific age-defined subpopulations, older adolescents in schools, undergraduates in colleges, and residents in nursing facilities, with little additional risk among other groups, notably including staff. Young adults, particularly in colleges, played a disproportionate role in spread, and new viral lineages consistently expanded first within this group. Viral spread followed reproducible urban-to-rural gradients, and vaccination was associated with reduced onward transmission. We further estimated sequencing thresholds required for timely detection of emerging variants. Together, these findings reveal consistent demographic and spatial structure in SARS-CoV-2 transmission that can inform future viral respiratory pathogen genomic surveillance.

Seventh pandemic Vibrio cholerae was first identified in Cameroon in 1971, causing several sporadic disease clusters with few cases. More recent years have seen larger cholera outbreaks, but the mechanism behind these periodic outbreaks is poorly understood, and it is unclear the degree to which antibiotic resistant strains contribute to disease burden and spread. We used whole genome sequencing to characterize 13 V. cholerae isolates from the 2018–2019, 2020, and 2021–2023 cholera epidemics in Cameroon. All these isolates belonged to the T12 lineage, and most showed the same antimicrobial resistance (AMR) pattern regardless of year. This suggests that cholera outbreaks in Cameroon are, at least in part, a continuation of the outbreaks previously reported in 2018 and as far back as 2012. This finding has important implications for cholera management since it suggests the ongoing presence of pathogenic cholera even in years with few reported cases. Similarly, the AMR results suggest the need for new treatment approaches, as resistance to many common antibiotics was found even within our limited sample set. As such, whole genome sequencing should be implemented in low-income countries such as Cameroon to improve disease surveillance and to detect and predict pathogen antibiotic resistance profiles.

Highly Pathogenic Avian Influenza strain H5N1 has caused a multi-state outbreak among US dairy cattle, spreading across 15 states and infecting hundreds of herds since its onset. We rapidly developed and optimized PCR-based detection assays and sequencing protocols to support H5N1 molecular surveillance. Using 214 retail milk from 20 states for methods development, we found that H5N1 concentrations by digital PCR strongly correlated with qPCR cycle threshold (Ct) values, with dPCR exhibiting greater sensitivity. We also found that metagenomic sequencing after hybrid selection was best for higher concentration samples while amplicon sequencing performs best for lower concentrations. By establishing these methods, we were able to support the creation of a statewide surveillance program to test bulk milk samples monthly from all cattle dairy farms within Massachusetts, which remain negative to date. The methods, workflow, and recommendations described here provide a framework for others aiming to conduct H5N1 surveillance efforts.

Cholera remains a significant public health burden in many countries in sub-Saharan Africa, though the exact mechanisms of bacterial emergence and spread remain largely undefined. We generated genomic data from 763 Vibrio cholerae O1 isolates predominantly collected between 2019-2024 to create the largest dataset of V. cholerae genomes sequenced locally in Africa. This dataset enabled us to interrogate recent patterns of spread, including the rapid circulation of the AFR15 lineage associated with unusually large outbreaks in Southern Africa. We provide evidence for the movement of the AFR15 lineage into new African Member States and confirm previously observed differences in V. cholerae transmission dynamics in West versus East Africa, though cross-border transmission is prevalent on both sides of the continent. Despite observed differences, evolutionary processes are similar across lineages and we find no evidence for significant changes in antimicrobial resistance genotypes. Overall, our findings emphasize the importance of regionally coordinated cross-border surveillance and interventions, while also demonstrating the critical role of locally generated genomic data in understanding the spread of cholera in Africa.