Background/Objectives: Cholera remains a significant global health challenge. Shanchol (ShantaBiotech, India), one of the WHO prequalified Oral Cholera Vaccines (OCVs) available until recently, has been used to immunize people as a two-dose regimen (14 days apart, on day 0 and 14). However, growing evidence suggests that a single-dose strategy may mediate short-term protection, especially in those over 5 years of age. Hence, it is crucial to design a suitable and effective administration scheme for Shanchol, particularly in cholera-endemic regions. Methods: In this study, adult volunteers were vaccinated with either a single dose, a two-dose regimen with a 14-day interval, or a two-dose regiment with a 30-day interval. We studied the antigen-specific helper memory (CD4+CD45RO+) and cytotoxic memory (CD8+CD45RO+) T cells responses of vaccinees along with the specific mucosal immune responses to gut-homing ß7 lipopolysaccharides (LPSs). Results: By day 7 post-vaccination, Shanchol induced robust helper and cytotoxic memory T cell responses to V. cholerae membrane protein (AKI-MP) following a single dose of vaccination. In the two-dose groups, we observed a significant elevation of AKI-MP-specific responses after the 2nd dose. We found that circulatory gut homing (β7+) LPS-specific IgA responses of antibody-secreting cells (ASCs) peaked at D7 among all vaccine groups. Moreover, we observed that β7+ LPS-specific ASCs at D7 significantly correlated with the LPS-specific antibody titer in plasma. Conclusions: These findings suggest that a single dose of OCV in adults induces immune responses comparable to a two-dose regimen, suggesting a single-dose vaccination may be adequate to mediate protection against cholera in cholera endemic zones—especially in reactive campaigns.

Abstract Background Between 200–300 severe malaria cases are diagnosed annually in the U.S. Intravenous artesunate (IVAS), the FDA-approved 1st-line therapy for severe malaria since 2020, is commercially available but at high cost. Uncertainty regarding where people with severe malaria will seek medical care poses supply chain challenges. We identified hospitals where people with severe malaria were treated and mapped proximity to Level 1 trauma centers as a proxy for hospitals that provide complex clinical care, which could assist with planning for IVAS distribution. Location identification and manual adjudication process for hospitals that hospitalized people with severe malaria as reported to CDC from 2012–2018. Of the original 1,912 cases of severe malaria reported to the CDC from 2012-2018, we excluded 379 cases that were not admitted or were admitted but missing a hospital name. Of the remaining 1,533 severe malaria cases that were eligible for adjudication, 1,237 were an exact match with an existing hospital location. Of the 296 cases that were not an exact match, we manually adjudicated 229 cases by clarifying a hospital name that was abbreviated, incomplete, or had a change in name; only 49 cases could not be identified given multiple possible sites with the same name, and 18 cases with no name that matched a hospital site. Six additional cases were excluded because of occurring at a site with no Level 1 trauma center within driving distance (i.e., Alaska and US Virgin Islands). Methods Using 2012–2018 data reported to the Centers for Disease Control and Prevention (CDC), we analyzed free text of U.S. hospital names where people were treated as inpatients for severe malaria. We identified these hospitals using Google Maps Geocoding Application Programming Interface (API) followed by manual adjudication. We excluded cases when data were incomplete or ambiguous or if a Level 1 trauma center was inaccessible (e.g., Alaska or U.S. Virgin Islands). We then mapped hospitals to U.S. counties and estimated travel times between hospitals and the nearest Level 1 trauma center as per 2014–2016, using Google Maps Distance Matrix API. Average numbers of severe malaria cases at identified hospitals per US county annually as reported to the CDC during 2012–2018. Panel A shows the geographic distribution and average number of severe malaria cases at identified hospitals per year by county reported to CDC during 2012–2018; darker color shows counties with more severe malaria cases reported. Panel B shows that the distribution is highly skewed to 2 counties that reported more than 10 inpatient cases annually; 2,824 counties (pale peach) reported no cases of severe malaria admitted to an identified hospital during 2012–2018. Results After excluding 379 cases with no recorded hospital name from 1,912 severe malaria cases, we successfully adjudicated hospitals for 1,460/1,533 cases (Fig 1). Two counties reported >10 inpatient cases per year (Bronx County, NY and New York County, NY), and 2,824 counties never reported a hospitalized severe malaria case (Fig 2). Almost 35% of inpatient cases occurred at Level 1 trauma centers (508/1,460 [34.8%]) (Fig 3). Of the 952/1,460 (65.2%) inpatient cases not at Level 1 trauma centers, 891/952 (93.5%) were at hospitals located < 2h estimated driving time of a Level 1 trauma center. Only 61/1,460 (4.2%) cases were at locations >2h from a Level 1 trauma center. Limitations include that the analysis did not consider the original site of clinical presentation with malaria or locations of people with severe malaria not hospitalized. Histogram of modeled travel times between each severe malaria case at an identified hospital and the nearest Level 1 trauma center. Of severe malaria cases reported to CDC in 2012–2018 and hospitalized at an identified hospital, 34.8% were treated at Level 1 trauma centers (red bar); for severe malaria cases that were not at Level 1 trauma centers, we found that modeled transit times between treatment site and Level 1 trauma center were less than 2 hours except in 4.2% of severe malaria cases at an identified hospital (right of black dashed vertical line). Conclusion More than 95% of people admitted for severe malaria at identified hospitals reported to the CDC in 2012–2018 were at or within 2h estimated driving time of a Level 1 trauma center, which may suggest priorities for implementing IVAS distribution. Disclosures Alison Ridpath, MD, MPH, Abbvie Inc.: Stocks/Bonds (Public Company)|Amarin Corperation PLC: Stocks/Bonds (Public Company)|Amgen Inc: Stocks/Bonds (Public Company)|BioNTech: Stocks/Bonds (Public Company)|Immunic Inc: Stocks/Bonds (Public Company)|infinity Pharmaceutical Companies: Stocks/Bonds (Public Company)|IQVIA Holdings Inc: Stocks/Bonds (Public Company)|Johnson and Johnson: Stocks/Bonds (Public Company)|Merck and Co inc: Stocks/Bonds (Public Company)|Pfizer Inc.: Stocks/Bonds (Public Company)|Protagonist Therapeutics: Stocks/Bonds (Public Company)

BACKGROUND:Global travel plays a role in the spread of infectious diseases. Existing travel surveillance programs collect data before and after trips, resulting in data incompleteness and recall bias. We developed the Travel Healthy mobile app to address these gaps, by enabling U.S. travelers to report daily symptom surveys including GPS location. The app offers traveler tools, including outbreak notices, a travel wallet, and a malaria medication reminder. METHODS:We developed Travel Healthy following a user-centric approach. We recruited study participants through an online platform and at the Travelers' Advice and Immunization Center at Massachusetts General Hospital, between July 2023 and August 2024. We analyzed demographic, GPS, and self-reported symptom data from the first 50 participants. Data were collected starting one day before the trip and ending three days after. A post-travel feedback survey was performed. RESULTS:Participants visited 204 locations in Asia, Africa, the Americas, and Europe. Mean age was 33 years and 66 % were female. The most common purposes of travel were leisure and/or business, with 46 (92 %) of participants listing these as traveling reasons. A total of 755 daily symptom surveys were entered, with 105 reporting symptoms, corresponding to 29 of the 50 (58 %) participants. Among all symptoms with GPS data, 58 % were upper respiratory symptoms, 25 % were gastrointestinal (clustered in South Asia), and 17 % were other. Post-travel questionnaires showed that participants found the application easy to use. CONCLUSION:This pilot study underscores the potential of participatory surveillance tools to complement traditional public health surveillance methods for travel-related illness.

Mass oral cholera vaccination campaigns targeted at subnational areas with high incidence are central to global cholera elimination efforts. Serological surveillance offers a complementary approach to address gaps in clinical surveillance in these regions. However, similar immune responses from vaccination and infection can lead to overestimates of the incidence of infection. To address this, we analyzed antibody dynamics in infected and vaccinated individuals to refine seroincidence estimation strategies for partially vaccinated populations. We tested 757 longitudinal serum samples from confirmed Vibrio cholerae O1 cases and uninfected contacts in Bangladesh as well as vaccinees from Bangladesh and Haiti, using a multiplex bead assay to measure IgG, IgM, and IgA binding to five cholera-specific antigens. Infection elicited stronger and broader antibody responses than vaccination, with rises in cholera toxin B-subunit (CTB) and toxin-coregulated pilus A (TcpA) antibodies uniquely associated with infection. Previously proposed random forest models frequently misclassified vaccinated individuals as recently infected (over 20% at some time points) during the first 4 months post-vaccination. To address this, we developed new random forest models incorporating vaccinee data, which kept false-positive rates among vaccinated (1%) and unvaccinated (6%) individuals low without a significant loss in sensitivity. Simulated serosurveys demonstrated that unbiased seroincidence estimates could be achieved within 21 days of vaccination campaigns by ascertaining the vaccination status of participants or applying updated models. These approaches to overcome biases in serological surveillance enable reliable seroincidence estimation even in areas with recent vaccination campaigns enhancing the utility of serological surveillance as an epidemiologic tool in moderate-to-high cholera incidence settings.IMPORTANCESerological surveillance can improve how we monitor cholera in high-burden areas where clinical surveillance is limited. However, vaccination can produce immune responses similar to infection, leading to overestimates in seroincidence. This study extends seroincidence estimation techniques using machine learning models to partially vaccinated populations. We analyzed antibody dynamics from vaccinated and infected individuals to develop methods that reduce the misclassification of vaccinated individuals as recently infected. These methods enable reliable seroincidence estimates in areas with recent vaccination campaigns, providing a step toward better epidemiologic monitoring in the context of global cholera control initiatives. Studies in other populations are needed to further validate our results and understand their generalizability.

BackgroundBoth SARS-CoV-2 infection and COVID-19 vaccines elicit immunological responses. However, it is difficult to distinguish responses generated after vaccination versus natural infection.MethodsWe investigated SARS-CoV-2 spike receptor-binding domain (RBD) and nucleocapsid-specific IgG and RBD specific IgG subclasses (IgG1, IgG2, IgG3 and IgG4) responses using ELISA in four different groups; (1) COVID-19 patients (n=39) with varying disease severity and (2) COVID-19 vaccinated individuals (n=24, both adenovirus/mRNA based) (3) vaccinated after infection (n=39) and (4) patients experienced breakthrough infection (n=14), in Bangladesh.ResultsBoth COVID-19 patients and vaccinees developed robust RBD-specific IgG responses. In contrast, nucleocapsid specific IgG responses were found in patients but not in vaccine recipients. A distinct IgG subclass antibody response was observed in COVID-19 patients compared to COVID-19-vaccinated individuals. Specifically, COVID-19 patients exhibited elevated levels of both IgG1 and IgG3, with IgG3 dominating in the early phase of infection (days 1-7) followed by a subsequent increase in IgG1. Conversely, COVID-19 vaccination predominantly induced IgG1 responses without a concurrent rise in IgG3. This effect was more evident when a significant rise of IgG1 but not IgG3 was observed in patients who received COVID-19 vaccines after 90 days of infection. However, following breakthrough infection, we observed an increase in both IgG1 and IgG3. All of these findings collectively indicate that COVID-19 vaccination predominantly induces IgG1, whereas natural infection can elicit responses in both IgG1 and IgG3 subclasses.ConclusionThe findings highlight RBD-specific IgG3 as well as nucleocapsid IgG as crucial markers for differentiating between vaccination and natural infection and suggest these assays have utility for longitudinal monitoring of vaccinations and for establishing SARS-CoV-2 correlates of protection.

Background:Cholera is a public health threat in resource-limited settings and is responsible for causing over 3 million cases globally. Mucosal immune responses play an important role in protecting against Vibrio cholerae infection, a non-invasive mucosal pathogen, yet traditional plasma-based assays are invasive and logistically challenging, particularly during outbreaks in low- and middle-income countries (LMICs). Saliva offers a unique window into mucosal immunity and may serve as a non-invasive alternative for seroprevalence and vaccine immunogenicity studies. Methods:We conducted a cross-sectional antibody profiling study to analyse cholera-specific antibodies in saliva and plasma samples from 74 participants upon presenting to the cholera treatment centres. These were collected from four treatment centres in Lusaka during Zambia's most severe cholera outbreak in 2024 caused by Vibrio cholerae O1 Ogawa. Levels of total IgG, IgG1-3, IgM, secretory IgA, and IgA1-2 isotypes were used to compare the biomarker profile between the two sample types. Results:Saliva and plasma antibody profiles were comparable, with elevated IgA1 and IgA2 responses to cholera toxin-B (CtxB), sialidase, HlyA, and TcpA in saliva. Broader systemic responses were seen in plasma, including high CtxB-specific IgM, IgA1, and total IgG levels. Notably, biomarkers such as HlyA, Ogawa O-specific polysaccharide (OSP), and sialidase exhibited significant positive correlations between plasma and saliva. Elevated biomarker levels of HlyA, Ogawa O-specific polysaccharide (OSP), and sialidase in people living with HIV/AIDS (PLWHA) suggested immunological differences that warrant further exploration. Conclusion:We demonstrate that saliva is a viable, non-invasive alternative for cholera antibody-based profiling, offering practical advantages in resource-constrained settings. Given its strong correlation with systemic antibody profiles, saliva may be a practical sample for sero-surveillance in resource-limited settings. Future studies should investigate the duration of these salivary responses to further substantiate their use in estimating disease burden and immunity.

Bacteriophages (phages) likely play a critical role in modulating transmission dynamics of diarrheal pathogens. This study investigated the role of phages in modulating the prevalence and seasonal patterns of major diarrheal pathogens, Vibrio cholerae O1 (VCO1), enterotoxigenic Escherichia coli (ETEC), Shigella spp., and Salmonella spp. in diarrheal patients and environmental wastewater specimens collected from six different sites in Dhaka, Bangladesh, in 2024. VCO1, ETEC, Shigella, and Salmonella were detected in 10.1%, 7.8%, 1.7%, and 2.4% of diarrheal specimens, respectively. In contrast, phages targeting these pathogens were more frequently isolated, with detection rates of 20% for VCO1, 30% for ETEC, 57% for Shigella, and 9.2% for Salmonella-specific phages. Adults showed a significantly higher burden of VCO1 and corresponding phages compared with children <5 years (P < 0.001). Seasonal analysis revealed significant correlations between VCO1 (37.3%) and corresponding phages (57.6%) peaking in late September in both clinical (r = 0.53, P < 0.0001) and environmental wastewater specimens (r = 0.65, P < 0.001). The highest correlation (r = 0.68) was found between the increased rate of wastewater phages in the preceding week and a rise in cholera cases in the following week. ETEC and ETEC phages isolated from wastewater also showed strong correlations (r = 0.65, P < 0.001). Cross-specificity analysis demonstrated that VCO1 phages were highly specific to their targets, whereas ETEC and Shigella phages exhibited broader host ranges, with some Shigella phages capable of infecting ETEC and Salmonella spp. Overall, these findings support the hypothesis that Vibrio phages could serve as an alternative or complementary tool for cholera surveillance.IMPORTANCEUnderstanding the dynamics between phages and their bacterial hosts is critical for elucidating disease burden; however, their potential for surveillance remains underexplored. To our knowledge, this is the first study that longitudinally investigated major diarrheal pathogens and their phages in both clinical and environmental sources to assess the potential of bacteriophages as a tool to improve diarrheal surveillance. The high frequency of phages compared to the host bacterial counterparts suggests a valuable, yet underutilized, role for phages in surveillance systems. Strong seasonal alignment between V. cholerae O1 and its phages, both peaking in late September, suggests that phage dynamics may reflect pathogen transmission. These preliminary observations raise the possibility that wastewater-derived Vibrio phages could function as early indicators of cholera burden. Future research should aim to explore the complex and poorly understood interactions between phages and their bacterial hosts, particularly how these dynamics shape pathogen populations in endemic settings.

Enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea, is defined by heat-stable (ST) and/or heat-labile (LT) toxins and associated colonization factors (CFs). However, there is still a knowledge gap in understanding ETEC's evolution, particularly in endemic regions like Bangladesh. This study investigates the genomic attributes contributing to the rise of ETEC-associated diarrhea in Bangladesh during 2022-2023. Whole genome sequencing of 325 ETEC isolates (2022-2023), compared with historical strains (1980-2021), revealed significant evolutionary changes. Our findings showed a significant shift in ETEC toxin from LT to ST over the period 2013-2023. The most frequent virulence profile during this period was CFA/I + CS21 compared with previous years (1980-2021). The emergence of CS23-positive ETEC was reported for the first time in Bangladesh, which was considered a less common CF in previous studies. Notably, we report the four novel lineages "L26-L29" in this study through phylogenetic analysis, partly encompassing emergent CS23-positive ETEC strains. Additionally, the high prevalence of multi-drug-resistant ETEC strains and the presence of ESBL-CTX-M-resistant gene during 2022-2023 are a matter of great concern, underscoring the need for preventive measures. The switch of distinct toxin and CF combinations, the rapid emergence of CS23, ESBL-CTX-M resistance, and novel lineages may be the reason behind the increased number of ETEC diarrheal cases between 2022 and 2023. These findings highlight the rapid ETEC evolution that underscores the necessity of continued genomic surveillance to track ongoing changes.IMPORTANCEThis study expands on previous evidence, demonstrating a remarkable genomic diversity in ETEC strains from 2022 to 2023, particularly in virulence factors and AMR genes. The combined findings from these studies will be important for mitigating future diarrheal outbreaks by informing preventive measures, including future vaccine targets, and implementing antibiotic stewardship programs against ETEC infection. Importantly, this research underscores the necessity of continued genomic surveillance to track ongoing changes in ETEC. Such monitoring is essential for understanding the pathogen's evolving population structure, transmission dynamics, and resistance mechanisms.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that the V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from ten cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than co-infection with divergent V. cholerae O1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in the V. cholerae genome decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract.

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCVSf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

ABSTRACT Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

Abstract Background Extended spectrum beta-lactamase producing Enterobacterales (ESBL-PE) present a risk to public health by limiting the efficacy of multiple classes of beta-lactam antibiotics against infection. International travellers may acquire these organisms and identifying individuals at high risk of acquisition could help inform clinical treatment or prevention strategies. Methods We used data collected from a cohort of 528 international travellers enrolled in a multicentre US-based study to derive a clinical prediction rule (CPR) to identify travellers who developed ESBL-PE colonization, defined as those with new ESBL positivity in stool upon return to the United States. To select candidate features, we used data collected from pre-travel and post-travel questionnaires, alongside destination-specific data from external sources. We utilized LASSO regression for feature selection, followed by random forest or logistic regression modelling, to derive a CPR for ESBL acquisition. Results A CPR using machine learning and logistic regression on 10 features has an internally cross-validated area under the receiver operating characteristic curve (cvAUC) of 0.70 (95% confidence interval 0.69–0.71). We also demonstrate that a four-feature model performs similarly to the 10-feature model, with a cvAUC of 0.68 (95% confidence interval 0.67–0.69). This model uses traveller’s diarrhoea, and antibiotics as treatment, destination country waste management rankings and destination regional probabilities as predictors. Conclusions We demonstrate that by integrating traveller characteristics with destination-specific data, we could derive a CPR to identify those at highest risk of acquiring ESBL-PE during international travel.

International travel can cause new illness or exacerbate existing conditions. Because primary care providers are frequent sources of health advice to travelers, they should be familiar with destination-specific disease risks, be knowledgeable about travel and routine vaccines, be prepared to prescribe chemoprophylaxis and self-treatment regimens, and be aware of travel medicine resources. Primary care providers should recognize travelers who would benefit from referral to a specialized travel clinic for evaluation. Those requiring yellow fever vaccination, immunocompromised hosts, pregnant persons, persons with multiple comorbid conditions, or travelers with complex itineraries may warrant specialty referral.

Background Culture-based studies have shown that acquisition of extended-spectrum beta-lactamase-producing Enterobacterales is common during international travel; however, little is known about the role of the gut microbiome before and during travel, nor about acquisition of other antimicrobial-resistant organisms. We aimed to identify (1) whether the gut microbiome provided colonisation resistance against antimicrobial-resistant organism acquisition, (2) the effect of travel and travel behaviours on the gut microbiome, and (3) the scale and global heterogeneity of antimicrobial-resistant organism acquisition. Methods In this metagenomic analysis, participants were recruited at three US travel clinics (Boston, MA; New York, NY; and Salt Lake City, UT) before international travel. Participants had to travel internationally between Dec 8, 2017, and April 30, 2019, and have DNA extractions for stool samples both before and after travel for inclusion. Participants were excluded if they had at least one low coverage sample (<1 million read pairs). Stool samples were collected at home before and after travel, sent to a clinical microbiology laboratory to be screened for three target antimicrobial-resistant organisms (extended-spectrum beta-lactamase-producing Enterobacterales, carbapenem-resistant Enterobacterales, and mcr-mediated colistin-resistant Enterobacterales), and underwent DNA extraction and shotgun metagenomic sequencing. We profiled metagenomes for taxonomic composition, antibiotic-resistant gene content, and characterised the Escherichia coli population at the strain level. We analysed pre-travel samples to identify the gut microbiome risk factors associated with acquisition of the three targeted antimicrobial resistant organisms. Pre-travel and post-travel samples were compared to identify microbiome and resistome perturbation and E coli strain acquisition associated with travel. Findings A total of 368 individuals travelled between the required dates, and 296 had DNA extractions available for both before and after travel. 29 travellers were excluded as they had at least one low coverage sample, leaving a final group of 267 participants. We observed a perturbation of the gut microbiota, characterised by a significant depletion of microbial diversity and enrichment of the Enterobacteriaceae family. Metagenomic strain tracking confirmed that 67% of travellers acquired new strains of E coli during travel that were phylogenetically distinct from their pre-travel strains. We observed widespread enrichment of antibiotic-resistant genes in the gut, with a median 15% (95% CI 10-20, p<1 x 10(-10)) increase in burden (reads per kilobase per million reads). This increase included antibiotic-resistant genes previously classified as threats to public health, which were 56% (95% CI 36-91, p=2 x 10(-11)) higher in abundance after travel than before. Fluoroquinolone antibiotic-resistant genes were aquired by 97 (54%) of 181 travellers with no detected pre-travel carriage. Although we found that visiting friends or relatives, travel to south Asia, and eating uncooked vegetables were risk factors for acquisition of the three targeted antimicrobial resistant organisms, we did not observe an association between the pre-travel microbiome structure and travel-related antimicrobial-resistant organism acquisition. Interpretation This work highlights a scale of E coli and antimicrobial-resistant organism acquisition by US travellers not apparent from previous culture-based studies, and suggests that strategies to control antimicrobial-resistant organisms addressing international traveller behaviour, rather than modulating the gut microbiome, could be worthwhile.

International travel can cause new illness or exacerbate existing conditions. Because primary care providers are frequent sources of health advice to travelers, they should be familiar with destination-specific disease risks, be knowledgeable about travel and routine vaccines, be prepared to prescribe chemoprophylaxis and self-treatment regimens, and be aware of travel medicine resources. Primary care providers should recognize travelers who would benefit from referral to a specialized travel clinic for evaluation. Those requiring yellow fever vaccination, immunocompromised hosts, pregnant persons, persons with multiple comorbid conditions, or travelers with complex itineraries may warrant specialty referral.

We announce the coding-complete genomes of four different strains of SARS-CoV-2 Omicron lineages, XBB.1.16, XBB.2.3, FL.4 (alias of XBB.1.9.1.4), and XBB.3. These strains were obtained between October 2022 and May 2023 from nasopharyngeal swabs of four Bangladeshi individuals, while one of them had a travel history. Genomic data were produced by implementing ARTIC Network-based amplicon sequencing using the Oxford Nanopore Technology.

Glycoconjugate vaccines are important additions to the existing means for prevention of diseases caused by bacterial and viral pathogens. Conjugating carbohydrates to proteins is a crucial step in the development of these vaccines. Traditional mass spectrometry techniques, such as MALDI-TOF and SELDI-TOF, have difficulties in detecting glycoconjugates with high molecular masses. Mass photometry (MP) is a single-molecule technique that has been recently developed, which allows mass measurements of individual molecules and generates mass distributions based on hundreds to thousands of these measurements. In this study, we evaluated the performance of MP in monitoring carbohydrate-protein conjugation reactions and characterization of conjugates. Three different glycoconjugates were prepared from carrier protein BSA, and one from a large protein complex, a virus capsid with 3.74 MDa molecular mass. The masses measured by MP were consistent with those obtained by SELDI-TOF-MS and SEC-MALS. The conjugation of BSA dimer to carbohydrate antigen was also successfully characterized. This study shows that the MP technique is a promising alternative to methods developed earlier for monitoring glycoconjugation reactions and characterization of glycoconjugates. It measures intact molecules in solution and it is highly accurate over a wide mass range. MP requires only a very small amount of sample and has no specific buffer constraints. Other MP advantages include minimal cost of consumables and rapid data collection and analysis. Its advantages over other methods make it a valuable tool for researchers in the glycoconjugation field.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

BACKGROUND:Vibriocidal antibodies are currently the best characterised correlate of protection against cholera and are used to gauge immunogenicity in vaccine trials. Although other circulating antibody responses have been associated with a decreased risk of infection, the correlates of protection against cholera have not been comprehensively compared. We aimed to analyse antibody-mediated correlates of protection from both V cholerae infection and cholera-related diarrhoea. METHODS:We conducted a systems serology study that analysed 58 serum antibody biomarkers as correlates of protection against V cholerae O1 infection or diarrhoea. We used serum samples from two cohorts: household contacts of people with confirmed cholera in Dhaka, Bangladesh, and cholera-naive volunteers who were recruited at three centres in the USA, vaccinated with a single dose of CVD 103-HgR live oral cholera vaccine, and then challenged with V cholerae O1 El Tor Inaba strain N16961. We measured antigen-specific immunoglobulin responses against antigens using a customised Luminex assay and used conditional random forest models to examine which baseline biomarkers were most important for classifying individuals who went on to develop infection versus those who remained uninfected or asymptomatic. V cholerae infection was defined as having a positive stool culture result on days 2-7 or day 30 after enrolment of the household's index cholera case and, in the vaccine challenge cohort, was the development of symptomatic diarrhoea (defined as two or more loose stools of ≥200 mL each, or a single loose stool of ≥300 mL over a 48-h period). FINDINGS:In the household contact cohort (261 participants from 180 households), 20 (34%) of the 58 studied biomarkers were associated with protection against V cholerae infection. We identified serum antibody-dependent complement deposition targeting the O1 antigen as the most predictive correlate of protection from infection in the household contacts, whereas vibriocidal antibody titres ranked lower. A five-biomarker model predicted protection from V cholerae infection with a cross-validated area under the curve (cvAUC) of 79% (95% CI 73-85). This model also predicted protection against diarrhoea in unvaccinated volunteers challenged with V cholerae O1 after vaccination (n=67; area under the curve [AUC] 77%, 95% CI 64-90). Although a different five-biomarker model best predicted protection from the development of cholera diarrhoea in the challenged vaccinees (cvAUC 78%, 95% CI 66-91), this model did poorly at predicting protection against infection in the household contacts (AUC 60%, 52-67). INTERPRETATION:Several biomarkers predict protection better than vibriocidal titres. A model based on protection against infection among household contacts was predictive of protection against both infection and diarrhoeal illness in challenged vaccinees, suggesting that models based on observed conditions in a cholera-endemic population might be more likely to identify broadly applicable correlates of protection than models trained on single experimental settings. FUNDING:National Institute of Allergy and Infectious Diseases and National Institute of Child Health and Human Development, National Institutes of Health.

ABSTRACT. There is a need for next-generation cholera vaccines that provide high-level and durable protection in young children in cholera-endemic areas. A cholera conjugate vaccine (CCV) is in development to address this need. This vaccine contains the O-specific polysaccharide (OSP) of Vibrio cholerae O1 conjugated via squaric acid chemistry to a recombinant fragment of the tetanus toxin heavy chain (OSP:rTTHc). This vaccine has been shown previously to be immunogenic and protective in mice and found to be safe in a recent preclinical toxicological analysis in rabbits. We took advantage of excess serum samples collected as part of the toxicological study and assessed the immunogenicity of CCV OSP:rTTHc in rabbits. We found that vaccination with CCV induced OSP-, lipopolysaccharide (LPS)-, and rTTHc-specific immune responses in rabbits, that immune responses were functional as assessed by vibriocidal activity, and that immune responses were protective against death in an established virulent challenge assay. CCV OSP:rTTHc immunogenicity in two animal model systems (mice and rabbits) is encouraging and supports further development of this vaccine for evaluation in humans.