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Kenya has experienced cholera outbreaks since 1971, with the most recent wave beginning in late 2014. Between 2015-2020, 32 of 47 counties reported 30,431 suspected cholera cases. The Global Task Force for Cholera Control (GTFCC) developed a Global Roadmap for Ending Cholera by 2030, which emphasizes the need to target multi-sectoral interventions in priority cholera burden hotspots. This study utilizes the GTFCC's hotspot method to identify hotspots in Kenya at the county and sub-county administrative levels from 2015 through 2020. 32 of 47 (68.1%) counties reported cholera cases during this time while only 149 of 301 (49.5%) sub-counties reported cholera cases. The analysis identifies hotspots based on the mean annual incidence (MAI) over the past five-year period and cholera's persistence in the area. Applying a MAI threshold of 90th percentile and the median persistence at both the county and sub-county levels, we identified 13 high risk sub-counties from 8 counties, including the 3 high risk counties of Garissa, Tana River and Wajir. This demonstrates that several sub-counties are high level hotspots while their counties are not. In addition, when cases reported by county versus sub-county hotspot risk are compared, 1.4 million people overlapped in the areas identified as both high-risk county and high-risk sub-county. However, assuming that finer scale data is more accurate, 1.6 million high risk sub-county people would have been misclassified as medium risk with a county-level analysis. Furthermore, an additional 1.6 million people would have been classified as living in high-risk in a county-level analysis when at the sub-county level, they were medium, low or no-risk sub-counties. This results in 3.2 million people being misclassified when county level analysis is utilized rather than a more-focused sub-county level analysis. This analysis highlights the need for more localized risk analyses to target cholera intervention and prevention efforts towards the populations most vulnerable.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's and travelers' diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.

Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections.

BackgroundAfter a multi-country Asian outbreak of cholera due to Vibrio cholerae serogroup O139 which started in 1992, it is rarely detected from any country in Asia and has not been detected from patients in Africa.Methodology/principal findingsWe extracted surveillance data from the Dhaka and Matlab Hospitals of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to review trends in isolation of Vibrio cholerae O139 in Bangladesh. Data from the Dhaka Hospital is a 2% sample of > 100,000 diarrhoeal patients treated annually. Data from the Matlab Hospital includes all diarrhoeal patients who hail from the villages included in the Matlab Health and Demographic Surveillance System. Vibrio cholerae O139 was first isolated in Dhaka in 1993 and had been isolated every year since then except for a gap between 2005 and 2008. An average of thirteen isolates was detected annually from the Dhaka Hospital during the last ten years, yielding an estimated 650 cases annually at this hospital. During the last ten years, cases due to serogroup O139 represented 0.47% of all cholera cases; the others being due to serogroup O1. No cases with serogroup O139 were identified at Matlab since 2006. Clinical signs and symptoms of cholera due to serogroup O139 were similar to cases due to serogroup O1 though more of the O139 cases were not dehydrated. Most isolates of O139 remained sensitive to tetracycline, ciprofloxacin, and azithromycin, but they became resistant to erythromycin starting in 2009.Conclusions/significanceCholera due to Vibrio cholerae serogroup O139 continues to cause typical cholera in Dhaka, Bangladesh.

Nigeria is endemic for cholera since 1970, and Kano State report outbreaks annually with high case fatality ratios ranging from 4.98%/2010 to 5.10%/2018 over the last decade. However, interventions focused on cholera prevention and control have been hampered by a lack of understanding of hotspot Local Government Areas (LGAs) that trigger and sustain yearly outbreaks. The goal of this study was to identify and categorize cholera hotspots in Kano State to inform a national plan for disease control and elimination in the State. We obtained LGA level confirmed and suspected cholera data from 2010 to 2019 from the Nigeria Centre for Disease Control (NCDC) and Kano State Ministry of Health. Data on inland waterbodies and population numbers were obtained from online sources and NCDC, respectively. Clusters (hotspots) were identified using SaTScan through a retrospective analysis of the data for the ten-year period using a Poisson discrete space-time scan statistic. We also used a method newly proposed by the Global Task Force on Cholera Control (GTFCC) to identify and rank hotspots based on two epidemiological indicators including mean annual incidence per 100 000 population of reported cases and the persistence of cholera for the study period. In the ten-year period, 16,461 cholera cases were reported with a case fatality ratio of 3.32% and a mean annual incidence rate of 13.4 cases per 100 000 population. Between 2010 and 2019, the most severe cholera exacerbations occurred in 2014 and 2018 with annual incidence rates of 58.01 and 21.52 cases per 100 000 inhabitants, respectively. Compared to 2017, reported cases and deaths increased by 214.56% and 406.67% in 2018. The geographic distribution of outbreaks revealed considerable spatial heterogeneity with the widest in 2014. Space-time clustering analysis identified 18 out of 44 LGAs as high risk for cholera (hotspots) involving both urban and rural LGAs. Cholera clustered around water bodies, and the relative risk of having cholera inside the hotspot LGA were 1.02 to 3.30 times higher than elsewhere in the State. A total of 4,894,144 inhabitants were in these hotspots LGAs. Of these, six LGAs with a total population of 1.665 million had a relative risk greater than 2 compared to the state as a whole. The SaTScan (statistical) and GTFCC methods were in agreement in hotspots identification. This study identified cholera hotspots LGAs in Kano State from 2010-2019. Hotspots appeared in both urban and rural settings. Focusing control strategies on these hotspots will facilitate control and eliminate cholera from the State.

Recent studies have gained a better appreciation of the potential impacts of enteric infections beyond symptomatic diarrhea. It is recognized that infections by several enteropathogens could be associated with growth deficits in children and intestinal and systemic inflammation may play an important underlying role. With enterotoxigenic E. coli (ETEC) being one of the leading causes of diarrhea among children in the developing world and important contributor to stunting, a better understanding of the impact of ETEC infection beyond diarrhea is timely and greatly needed. To address this, we evaluated if ETEC infection induces intestinal and systemic inflammation and its impact on colonization and immune responses to ETEC vaccine-specific antigens in a dose descending experimental human challenge model using ETEC strain H10407. This study demonstrates that the concentrations of myeloperoxidase (MPO) in stool and intestinal fatty acid-binding protein (an indicator of compromised intestinal epithelial integrity) in serum, significantly increased following ETEC infection in both diarrhea and asymptomatic cases and the magnitudes and kinetics of MPO are dose and clinical outcome dependent. Cytokines IL-17A and IFN-γ were significantly increased in serum post-ETEC challenge. In addition, higher pre-challenge concentrations of cytokines IL-10 and GM-CSF were associated with protection from ETEC diarrhea. Interestingly, higher MPO concentrations were associated with higher intestinal colonization of ETEC and lower seroconversions of colonization factor I antigen, but the reverse was noted for seroconversions to heat-labile toxin B-subunit. Together this study has important implications for understanding the acute and long-term negative health outcomes associated with ETEC infection.

Objectives To evaluate the quality and coverage of the campaign to distribute oral cholera vaccine (OCV) during a cholera outbreak in Hoima, Uganda to guide future campaigns of cholera vaccine.Design Survey of communities targeted for vaccination to determine vaccine coverage rates and perceptions of the vaccination campaign, and a separate survey of vaccine staff who carried out the campaign.Setting Hoima district, Uganda.Participants Representative clusters of households residing in the communities targeted for vaccination and staff members who conducted the vaccine campaign.Results Among 209 households (1274 individuals) included in the coverage survey, 1193 (94%; 95% CI 92% to 95%) reported receiving at least one OCV dose and 998 (78%; 95% CI 76% to 81%) reported receiving two doses. Among vaccinated individuals, minor complaints were reported by 71 persons (5.6%). Individuals with ‘some’ education (primary school or above) were more knowledgeable regarding the required OCV doses compared with non-educated (p=0.03). Factors negatively associated with campaign implementation included community sensitisation time, staff payment and problems with field transport. Although the campaign was carried out quickly, the outbreak was over before the campaign started. Most staff involved in the campaign (93%) were knowledgeable about cholera control; however, 29% did not clearly understand how to detect and manage adverse events following immunisation.Conclusion The campaign achieved high OCV coverage, but the surveys provided insights for improvement. To achieve high vaccine coverage, more effort is needed for community sensitisation, and additional resources for staff transportation and timely payment for campaign staff is required. Pretest and post-test assessment of staff training can identify and address knowledge and skill gaps.

Introduction In 2017, amidst insecurity and displacements posed by Boko Haram armed insurgency, cholera outbreak started in the Muna Garage camp for Internally Displaced Persons (IDPs) in Borno State, Nigeria. In response, the Borno Ministry of Health and partners determined to provide oral cholera vaccine (OCV) to about 1 million people in IDP camps and surrounding communities in six Local Government Areas (LGAs) including Maiduguri, Jere, Konduga, Mafa, Dikwa, and Monguno. As part of Monitoring and Evaluation, we described the coverage achieved, adverse events following immunisation (AEFI), non-vaccination reasons, vaccination decisions as well as campaign information sources.Methods We conducted two-stage probability cluster surveys with clusters selected without replacement according to probability-proportionate-to-population-size in the six LGAs targeted by the campaign. Individuals aged ≥1 years were the eligible study population. Data sources were household interviews with vaccine card verification and memory recall, if no card, as well as multiple choice questions with an open-ended option.Results Overall, 12 931 respondents participated in the survey. Overall, 90% (95% CI: 88 to 92) of the target population received at least one dose of OCV, range 87% (95% CI: 75 to 94) in Maiduguri to 94% (95% CI: 88 to 97) in Monguno. The weighted two-dose coverage was 73% (95% CI: 68 to 77) with a low of 68% (95% CI: 46 to 86) in Maiduguri to a high of 87% (95% CI: 74 to 95) in Dikwa. The coverage was lower during first round (76%, 95% CI: 71 to 80) than second round (87%, 95% CI: 84 to 89) and ranged from 72% (95% CI: 42 to 89) and 82% (95% CI: 82 to 91) in Maiduguri to 87% (95% CI: 75 to 95) and 94% (95% CI: 88 to 97) in Dikwa for the respective first and second rounds. Also, coverage was higher among females of age 5 to 14 and ≥15 years than males of same age groups. There were mild AEFI with the most common symptoms being fever, headache and diarrhoea occurring up to 48 hours after ingesting the vaccine. The most common actions taken after AEFI symptoms included ‘did nothing’ and ‘self-medicated at home’. The top reason for taking vaccine was to protect from cholera while top reason for non-vaccination was travel/work. The main source of campaign information was a neighbour. An overwhelming majority (96%, 95% CI: 95% to 98%) felt the campaign team treated them with respect. While 43% (95% CI: 36% to 50%) asked no questions, 37% (95% CI: 31% to 44%) felt the team addressed all their concerns.Conclusion The campaign achieved high coverage using door-to-door and fixed sites strategies amidst insecurity posed by Boko Haram. Additional studies are needed to improve how to reduce non-vaccination, especially for the first round. While OCV provides protection for a few years, additional actions will be needed to make investments in water, sanitation and hygiene infrastructure.

Introduction In August 2017, a cholera outbreak started in Muna Garage Internally Displaced Persons camp, Borno state, Nigeria and >5000 cases occurred in six local government areas. This qualitative study evaluated perspectives about the emergency response to this outbreak.Methods We conducted 39 key informant interviews and focus group discussions, and reviewed 21 documents with participants involved with surveillance, water, sanitation, hygiene, case management, oral cholera vaccine (OCV), communications, logistics and coordination. Qualitative data analysis used thematic techniques comprising key words in context, word repetition and key sector terms.Results Authorities were alerted quickly, but outbreak declaration took 12 days due to a 10-day delay waiting for culture confirmation. Outbreak investigation revealed several potential transmission channels, but a leaking latrine around the index cases’ house was not repaired for more than 7 days. Chlorine was initially not accepted by the community due to rumours that it would sterilise women. Key messages were in Hausa, although Kanuri was the primary local language; later this was corrected. Planning would have benefited using exercise drills to identify weaknesses, and inventory sharing to avoid stock outs. The response by the Rural Water Supply and Sanitation Agency was perceived to be slow and an increased risk from a religious festival was not recognised. Case management was provided at treatment centres, but some partners were concerned that their work was not recognised asking, ‘Who gets the glory and the data?’ Nearly one million people received OCV and its distribution benefited from a robust infrastructure for polio vaccination. There was initial anxiety, rumour and reluctance about OCV, attributed by many to lack of formative research prior to vaccine implementation. Coordination was slow initially, but improved with activation of an emergency operations centre (EOC) that enabled implementation of incident management system to coordinate multisectoral activities and meetings held at 16:00 hours daily. The synergy between partners and government improved when each recognised the government’s leadership role.Conclusion Despite a timely alert of the outbreak, delayed laboratory confirmation slowed initial response. Initial responses to the outbreak were not well coordinated but improved with the EOC. Understanding behaviours and community norms through rapid formative research should improve the effectiveness of the emergency response to a cholera outbreak. OCV distribution was efficient and benefited from the polio vaccine infrastructure.

BackgroundGlobal estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination.Methodology/principal findingsWhole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor.ConclusionsWGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas.

BACKGROUND:Household contacts of cholera patients are at a 100 times higher risk of developing cholera than the general population. The objective of this study was to examine the incidence of V. cholerae infections among household contacts of cholera patients in a rural setting in Bangladesh, to identify risk factors for V. cholerae infections among this population, and to investigate transmission pathways of V. cholerae using multilocus variable-number tandem-repeat analysis (MLVA). METHODOLOGY/PRINCIPAL FINDINGS:Stool from household contacts, source water and stored water samples were collected from cholera patient households on Day 1, 3, 5, and 7 after the presentation of the index patient at a health facility. Two hundred thirty clinical and water V. cholerae isolates were analyzed by MLVA. Thirty seven percent of households had at least one household contact with a V. cholerae infection. Thirteen percent of households had V. cholerae in their water source, and 27% had V. cholerae in stored household drinking water. Household contacts with V. cholerae in their water source had a significantly higher odds of symptomatic cholera (Odds Ratio (OR): 5.49, 95% Confidence Interval (CI): 1.07, 28.08). Contacts consuming street vended food had a significantly higher odds of a V. cholerae infection (OR: 9.45, 95% CI: 2.14, 41.72). Older age was significantly associated with a lower odds of a V. cholerae infection (OR: 0.96, 95% CI: 0.93, 0.99). Households with both water and clinical V. cholerae-positive samples all had isolates that were closely related by MLVA. CONCLUSIONS/SIGNIFICANCE:These findings emphasize the need for interventions targeting water treatment and food hygiene to reduce V. cholerae infections.

BACKGROUND:For almost 50 years sub-Saharan Africa, including Uganda, has experienced several outbreaks due to Vibrio cholerae. Our aim was to determine the genetic relatedness and spread of strains responsible for cholera outbreaks in Uganda. METHODOLOGY/PRINCIPAL FINDINGS:Sixty-three V. cholerae isolates collected from outbreaks in Uganda between 2014 and 2016 were tested using multiplex polymerase chain reaction (PCR), multi-locus variable number of tandem repeat analysis (MLVA) and whole genome sequencing (WGS). Three closely related MLVA clonal complexes (CC) were identified: CC1, 32% (20/63); CC2, 40% (25/63) and CC3, 28% (18/63). Each CC contained isolates from a different WGS clade. These clades were contained in the third wave of the 7th cholera pandemic strain, two clades were contained in the transmission event (T)10 lineage and other in T13. Analysing the dates and genetic relatedness revealed that V. cholerae genetic lineages spread between districts within Uganda and across national borders. CONCLUSION:The V. cholerae strains showed local and regional transmission within Uganda and the East African region. To prevent, control and eliminate cholera, these countries should implement strong cross-border collaboration and regional coordination of preventive activities.

A reliable and effective human challenge model is needed to help down-select the most promising ETEC vaccines currently under development. Such a model would need to reliably induce diarrhea in a high proportion of volunteers using the lowest possible inoculum to maximize safety and sensitivity. Previously we validated a challenge model that utilized a dose of 2x107 CFU of ETEC strain H10407 (LT+, ST+, CFA/I+ and O78+) to induce attack rates for moderate to severe diarrhea (MSD) of ~60-70%. Here we detail efforts to further refine the model in an attempt to determine if a lower challenge dose of H10407 can be used. Thirty subjects were randomized 1:1 to receive an oral administration of H10407 at doses of 106 or 105 CFU in bicarbonate buffer. After challenge, subjects were monitored for signs and symptoms of enteric illness and stool samples were collected to detect shedding of the challenge strain. Systemic and mucosal immune responses were measured using serum, antibody in lymphocyte supernatant and fecal samples. The attack rate was 13.3% (2/15) and 26.7% (4/15) for MSD in the 105 and 106 groups, respectively. Four MSD cases met criteria for early antibiotic treatment. All subjects but one shed the challenge strain in fecal samples. The frequency and magnitude of anti-LT toxin, CFA/I and LPS O78 immune responses were antigen, dose, severity of diarrhea and shedding levels dependent. Notably, although of lower magnitude, there were considerable immune responses in the subjects with no diarrhea. This may indicate that immune responses to asymptomatic infections of ETEC in children in the endemic countries may contribute to protection. Based on this and our prior studies, we conclude that a dose of 2x107 H10407 remains the lowest practical dose for use in future volunteer studies evaluating candidate vaccines and other preventive or therapeutic ETEC interventions.ClinicalTrials.gov NCT00844493.

To examine rates of Shigella infections in household contacts of pediatric shigellosis patients, we followed contacts and controls prospectively for 1 week after the index patient obtained care. Household contacts of patients were 44 times more likely to develop a Shigella infection than were control contacts (odds ratio 44.7, 95% CI 5.5–361.6); 29 (94%) household contacts of shigellosis patients were infected with the same species and serotype as the index patient’s. Pulsed-field gel electrophoresis showed that 14 (88%) of 16 with infected contacts had strains that were indistinguishable from or closely related to the index patient’s strain. Latrine area fly counts were higher in patient households compared with control households, and 2 patient household water samples were positive for Shigella. We show high susceptibility of household contacts of shigellosis patients to Shigella infections and found environmental risk factors to be targeted in future interventions.

BACKGROUND:Vibrio cholerae causes over 2 million cases of cholera and 90,000 deaths each year. Serosurveillance can be a useful tool for estimating the intensity of cholera transmission and prioritizing populations for cholera control interventions. Current methods involving venous blood draws and downstream specimen storage and transport methods pose logistical challenges in most settings where cholera strikes. To overcome these challenges, we developed methods for determining cholera-specific immune responses from dried blood spots (DBS). METHODOLOGY/PRINCIPAL FINDINGS:As conventional vibriocidal assay methods were unsuitable for DBS eluates from filter paper, we adopted a drop-plate culture method. We show that DBS collected from volunteers in South Sudan, and stored for prolonged periods in field conditions, retained functional vibriocidal antibodies, the titers of which correlated with paired serum titers determined by conventional spectrophotometric methods (r = 0.94, p = 0.00012). We also showed that eluates from DBS Serum Separator cards could be used with conventional spectrophotometric vibriocidal methods, and that they correlated with paired serum at a wide range of titers (r = 0.96, p

Despite advance in science and technology for prevention, detection and treatment of cholera, this infectious disease remains a major public health problem in many countries in sub-Saharan Africa, Uganda inclusive. The aim of this study was to identify cholera hotspots in Uganda to guide the development of a roadmap for prevention, control and elimination of cholera in the country.We obtained district level confirmed cholera outbreak data from 2011 to 2016 from the Ministry of Health, Uganda. Population and rainfall data were obtained from the Uganda Bureau of Statistics, and water, sanitation and hygiene data from the Ministry of Water and Environment. A spatial scan test was performed to identify the significantly high risk clusters. Cholera hotspots were defined as districts whose center fell within a significantly high risk cluster or where a significantly high risk cluster was completely superimposed onto a district. A zero-inflated negative binomial regression model was employed to identify the district level risk factors for cholera. In total 11,030 cases of cholera were reported during the 6-year period. 37(33%) of 112 districts reported cholera outbreaks in one of the six years, and 20 (18%) districts experienced cholera at least twice in those years. We identified 22 districts as high risk for cholera, of which 13 were near a border of Democratic Republic of Congo (DRC), while 9 districts were near a border of Kenya. The relative risk of having cholera inside the high-risk districts (hotspots) were 2 to 22 times higher than elsewhere in the country. In total, 7 million people were within cholera hotspots. The negative binomial component of the ZINB model shows people living near a lake or the Nile river were at increased risk for cholera (incidence rate ratio, IRR = 0.98, 95% CI: 0.97 to 0.99, p < .01); people living near the border of DRC/Kenya or higher incidence rate in the neighboring districts were increased risk for cholera in a district (IRR = 0.99, 95% CI: 0.98 to 1.00, p = .02 and IRR = 1.02, 95% CI: 1.01 to 1.03, p < .01, respectively). The zero inflated component of the ZINB model yielded shorter distance to Kenya or DRC border, higher incidence rate in the neighboring districts, and higher annual rainfall in the district were associated with the risk of having cholera in the district.The study identified cholera hotspots during the period 2011-2016. The people located near the international borders, internationally shared lakes and river Nile were at higher risk for cholera outbreaks than elsewhere in the country. Targeting cholera interventions to these locations could prevent and ultimately eliminate cholera in Uganda.

BACKGROUND:Rotavirus vaccines are now globally recommended by the World Health Organization (WHO), but in early 2009 WHO's Strategic Advisory Group of Experts on Immunization reviewed available data and concluded that there was no evidence for the efficacy or effectiveness of a two-dose schedule of the human rotavirus vaccine (HRV; Rotarix) given early at 6 and 10 wk of age. Additionally, the effectiveness of programmatic rotavirus vaccination, including possible indirect effects, has not been assessed in low-resource populations in Asia. METHODS AND FINDINGS:In Bangladesh, we cluster-randomized (1:1) 142 villages of the Matlab Health and Demographic Surveillance System to include two doses of HRV with the standard infant vaccines at 6 and 10 wk of age or to provide standard infant vaccines without HRV. The study was initiated November 1, 2008, and surveillance was conducted concurrently at Matlab Diarrhoea Hospital and two community treatment centers to identify children less than 2 y of age presenting with acute rotavirus diarrhea (ARD) through March 31, 2011. Laboratory confirmation was made by enzyme immunoassay detection of rotavirus antigen in stool specimens. Overall effectiveness of the HRV vaccination program (primary objective) was measured by comparing the incidence rate of ARD among all children age-eligible for vaccination in villages where HRV was introduced to that among such children in villages where HRV was not introduced. Total effectiveness among vaccinees and indirect effectiveness were also evaluated. In all, 6,527 infants were age-eligible for vaccination in 71 HRV villages, and 5,791 in 71 non-HRV villages. In HRV villages, 4,808 (73.7%) infants received at least one dose of HRV. The incidence rate of ARD was 4.10 cases per 100 person-years in non-HRV villages compared to 2.8 per 100 person-years in HRV villages, indicating an overall effectiveness of 29.0% (95% CI, 11.3% to 43.1%). The total effectiveness of HRV against ARD among vaccinees was 41.4% (95% CI, 23.2% to 55.2%). The point estimate for total effectiveness was higher against ARD during the first year of life than during the second (45.2% versus 28.9%), but estimates for the second year of life lacked precision and did not reach statistical significance. Indirect effects were not detected. To check for bias in presentation to treatment facilities, we evaluated the effectiveness of HRV against acute diarrhea associated with enterotoxigenic Escherichia coli; it was 4.0% (95% CI, -46.5% to 37.1%), indicating that bias likely was not introduced. Thirteen serious adverse events were identified among recipients of HRV, but none were considered related to receipt of study vaccine. The main limitation of this study is that it was an open-label study with an observed-only control group (no placebo). CONCLUSIONS:The two-dose HRV rotavirus vaccination program significantly reduced medically attended ARD in this low-resource population in Asia. Protection among vaccinees was similar to that in other low-resource settings. In low-resource populations with high rotavirus incidence, large-scale vaccination across a wide population may be required to obtain the full benefit of rotavirus vaccination, including indirect effects. TRIAL REGISTRATION:ClinicalTrials.gov NCT00737503.

Abstract Objective To describe and analyse the characteristics of oral cholera vaccination campaigns; including location, target population, logistics, vaccine coverage and delivery costs. Methods We searched PubMed, the World Health Organization (WHO) website and the Cochrane database with no date or language restrictions. We contacted public health personnel, experts in the field and in ministries of health and did targeted web searches. Findings A total of 33 documents were included in the analysis. One country, Viet Nam, incorporates oral cholera vaccination into its public health programme and has administered approximately 10.9 million vaccine doses between 1997 and 2012. In addition, over 3 million doses of the two WHO pre-qualified oral cholera vaccines have been administered in more than 16 campaigns around the world between 1997 and 2014. These campaigns have either been pre-emptive or reactive and have taken place under diverse conditions, such as in refugee camps or natural disasters. Estimated two-dose coverage ranged from 46 to 88% of the target population. Approximate delivery cost per fully immunized person ranged from 0.11–3.99 United States dollars. Conclusion Experience with oral cholera vaccination campaigns continues to increase. Public health officials may draw on this experience and conduct oral cholera vaccination campaigns more frequently.

Even though cholera has existed for centuries and many parts of the country have sporadic, endemic and epidemic cholera, it is still an under-recognized health problem in India. A Cholera Expert Group in the country was established to gather evidence and to prepare a road map for control of cholera in India. This paper identifies cholera burden hotspots and factors associated with an increased risk of the disease.We acquired district level data on cholera case reports of 2010-2015 from the Integrated Disease Surveillance Program. Socioeconomic characteristics and coverage of water and sanitation was obtained from the 2011 census. Spatial analysis was performed to identify cholera hotspots, and a zero-inflated Poisson regression was employed to identify the factors associated with cholera and predicted case count in the district. 27,615 cholera cases were reported during the 6-year period. Twenty-four of 36 states of India reported cholera during these years, and 13 states were classified as endemic. Of 641 districts, 78 districts in 15 states were identified as "hotspots" based on the reported cases. On the other hand, 111 districts in nine states were identified as "hotspots" from model-based predicted number of cases. The risk for cholera in a district was negatively associated with the coverage of literate persons, households using treated water source and owning mobile telephone, and positively associated with the coverage of poor sanitation and drainage conditions and urbanization level in the district.The study reaffirms that cholera continues to occur throughout a large part of India and identifies the burden hotspots and risk factors. Policymakers may use the findings of the article to develop a roadmap for prevention and control of cholera in India.

Vaccinating a buffer of individuals around a case (ring vaccination) has the potential to target those who are at highest risk of infection, reducing the number of doses needed to control a disease. We explored the potential vaccine effectiveness (VE) of oral cholera vaccines (OCVs) for such a strategy.This analysis uses existing data from a cluster-randomized clinical trial in which OCV or placebo was given to 71,900 participants in Kolkata, India, from 27 July to 10 September 2006. Cholera surveillance was then conducted on 144,106 individuals living in the study area, including trial participants, for 5 y following vaccination. First, we explored the risk of cholera among contacts of cholera patients, and, second, we measured VE among individuals living within 25 m of cholera cases between 8 and 28 d after onset of the index case. For the first analysis, individuals living around each index case identified during the 5-y period were assembled using a ring to define cohorts of individuals exposed to cholera index cases. An index control without cholera was randomly selected for each index case from the same population, matched by age group, and individuals living around each index control were assembled using a ring to define cohorts not exposed to cholera cases. Cholera attack rates among the exposed and non-exposed cohorts were compared using different distances from the index case/control to define the rings and different time frames to define the period at risk. For the VE analysis, the exposed cohorts were further stratified according to the level of vaccine coverage into high and low coverage strata. Overall VE was assessed by comparing the attack rates between high and low vaccine coverage strata irrespective of individuals' vaccination status, and indirect VE was assessed by comparing the attack rates among unvaccinated members between high and low vaccine coverage strata. Cholera risk among the cohort exposed to cholera cases was 5-11 times higher than that among the cohort not exposed to cholera cases. The risk gradually diminished with an increase in distance and time. The overall and indirect VE measured between 8 and 28 d after exposure to a cholera index case during the first 2 y was 91% (95% CI 62%-98%) and 93% (95% CI 44%-99%), respectively. VE persisted for 5 y after vaccination and was similar whether the index case was a young child (

BACKGROUND:Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC) and to develop a disease score. METHODS:Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials. RESULTS:Data on 264 subjects receiving seven ETEC strains at doses from 1x105 to 1x1010 cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65); however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects. CONCLUSION:Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.

Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4-6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5-95.3) sensitivity and 100% (95% CI: 94.4-100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2-93.6) for culture performed on site and 72.2% (95% CI: 54.8-85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7-100) and 100% (95% CI: 94.5-100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited.

BACKGROUND:Vibrio cholerae is endemic in South Asia and Africa where outbreaks of cholera occur widely and are particularly associated with poverty and poor sanitation. Knowledge of the genetic diversity of toxigenic V. cholerae isolates, particularly in Africa, remains scarce. The constraints in improving this understanding is not only the lack of regular cholera disease surveillance, but also the lack of laboratory capabilities in endemic countries to preserve, store and ship isolates in a timely manner. We evaluated the use of simplified sample preservation methods for molecular characterization using multi-locus variable-number tandem-repeat analysis (MLVA) for differentiation of Vibrio cholerae genotypes. METHODS AND FINDINGS:Forty-seven V. cholerae isolates and 18 enriched clinical specimens (e.g. stool specimens after enrichment in broth) from cholera outbreaks in Cameroon were preserved on Whatman filter paper for DNA extraction. The samples were collected from two geographically distinct outbreaks in the Far North of Cameroon (FNC) in June 2014 and October 2014. In addition, a convenience sample of 14 isolates from the Philippines and 8 from Mozambique were analyzed. All 87 DNAs were successfully analyzed including 16 paired samples, one a cultured isolate and the other the enriched specimen from which the isolate was collected. Genotypic results were identical between 15 enriched specimens and their culture isolates and the other pair differed at single locus. Two closely related, but distinct clonal complexes were identified among the Cameroonian specimens from 2014. CONCLUSIONS:Collecting V. cholerae using simplified laboratory methods in remote and low-resource settings allows for subsequent advanced molecular characterization of V. cholerae O1. These simplified DNA preservation methods identify V. cholerae and make possible timely information regarding the genetic diversity of V. cholerae; our results set the stage for continued molecular epidemiological research to better understand the transmission and dissemination of V. cholerae in Africa and elsewhere worldwide.

BackgroundIn 2013, a stockpile of oral cholera vaccine (OCV) was created for use in outbreak response, but vaccine availability remains severely limited. Innovative strategies are needed to maximize the health impact and minimize the logistical barriers to using available vaccine. Here we ask under what conditions the use of one dose rather than the internationally licensed two-dose protocol may do both.Methods and findingsUsing mathematical models we determined the minimum relative single-dose efficacy (MRSE) at which single-dose reactive campaigns are expected to be as or more effective than two-dose campaigns with the same amount of vaccine. Average one- and two-dose OCV effectiveness was estimated from published literature and compared to the MRSE. Results were applied to recent outbreaks in Haiti, Zimbabwe, and Guinea using stochastic simulations to illustrate the potential impact of one- and two-dose campaigns. At the start of an epidemic, a single dose must be 35%-56% as efficacious as two doses to avert the same number of cases with a fixed amount of vaccine (i.e., MRSE between 35% and 56%). This threshold decreases as vaccination is delayed. Short-term OCV effectiveness is estimated to be 77% (95% CI 57%-88%) for two doses and 44% (95% CI -27% to 76%) for one dose. This results in a one-dose relative efficacy estimate of 57% (interquartile range 13%-88%), which is above conservative MRSE estimates. Using our best estimates of one- and two-dose efficacy, we projected that a single-dose reactive campaign could have prevented 70,584 (95% prediction interval [PI] 55,943-86,205) cases in Zimbabwe, 78,317 (95% PI 57,435-100,150) in Port-au-Prince, Haiti, and 2,826 (95% PI 2,490-3,170) cases in Conakry, Guinea: 1.1 to 1.2 times as many as a two-dose campaign. While extensive sensitivity analyses were performed, our projections of cases averted in past epidemics are based on severely limited single-dose efficacy data and may not fully capture uncertainty due to imperfect surveillance data and uncertainty about the transmission dynamics of cholera in each setting.ConclusionsReactive vaccination campaigns using a single dose of OCV may avert more cases and deaths than a standard two-dose campaign when vaccine supplies are limited, while at the same time reducing logistical complexity. These findings should motivate consideration of the trade-offs between one- and two-dose campaigns in resource-constrained settings, though further field efficacy data are needed and should be a priority in any one-dose campaign.

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.

BACKGROUND:The global burden of cholera is largely unknown because the majority of cases are not reported. The low reporting can be attributed to limited capacity of epidemiological surveillance and laboratories, as well as social, political, and economic disincentives for reporting. We previously estimated 2.8 million cases and 91,000 deaths annually due to cholera in 51 endemic countries. A major limitation in our previous estimate was that the endemic and non-endemic countries were defined based on the countries' reported cholera cases. We overcame the limitation with the use of a spatial modelling technique in defining endemic countries, and accordingly updated the estimates of the global burden of cholera. METHODS/PRINCIPAL FINDINGS:Countries were classified as cholera endemic, cholera non-endemic, or cholera-free based on whether a spatial regression model predicted an incidence rate over a certain threshold in at least three of five years (2008-2012). The at-risk populations were calculated for each country based on the percent of the country without sustainable access to improved sanitation facilities. Incidence rates from population-based published studies were used to calculate the estimated annual number of cases in endemic countries. The number of annual cholera deaths was calculated using inverse variance-weighted average case-fatality rate (CFRs) from literature-based CFR estimates. We found that approximately 1.3 billion people are at risk for cholera in endemic countries. An estimated 2.86 million cholera cases (uncertainty range: 1.3m-4.0m) occur annually in endemic countries. Among these cases, there are an estimated 95,000 deaths (uncertainty range: 21,000-143,000). CONCLUSION/SIGNIFICANCE:The global burden of cholera remains high. Sub-Saharan Africa accounts for the majority of this burden. Our findings can inform programmatic decision-making for cholera control.

BACKGROUND: Pregnancy is a known risk factor for malaria which is associated with increased maternal and infant mortality and morbidity in areas of moderate-high malaria transmission intensity where Plasmodium falciparum predominates. The nature and impact of malaria, however, is not well understood in pregnant women residing in areas of low, unstable malaria transmission where P. falciparum and P. vivax co-exist. METHODS: A large longitudinal active surveillance study of malaria was conducted in the Chittagong Hill Districts of Bangladesh. Over 32 months in 2010-2013, the period prevalence of asymptomatic P. falciparum infections was assessed by rapid diagnostic test and blood smear and compared among men, non-pregnant women and pregnant women. A subset of samples was tested for infection by PCR. Hemoglobin was assessed. Independent risk factors for malaria infection were determined using a multivariate logistic regression model. RESULTS: Total of 34 asymptomatic P. falciparum infections were detected by RDT/smear from 3,110 tests. The period prevalence of asymptomatic P. falciparum infection in pregnant women was 2.3%, compared to 0.5% in non-pregnant women and 0.9% in men. All RDT/smear positive samples that were tested by PCR were PCR-positive, and PCR detected additional 35 infections that were RDT/smear negative. In a multivariate logistic regression analysis, pregnant women had 5.4-fold higher odds of infection as compared to non-pregnant women. Malaria-positive pregnant women, though asymptomatic, had statistically lower hemoglobin than those without malaria or pregnancy. Asymptomatic malaria was found to be evenly distributed across space and time, in contrast to symptomatic infections which tend to cluster. CONCLUSION: Pregnancy is a risk factor for asymptomatic P. falciparum infection in the Chittagong Hill Districts of Bangladesh, and pregnancy and malaria interact to heighten the effect of each on hemoglobin. The even distribution of asymptomatic malaria, without temporal and spatial clustering, may have critical implications for malaria elimination strategies.

BackgroundMalaria is endemic in 13 of 64 districts of Bangladesh, representing a population at risk of about 27 million people. The highest rates of malaria in Bangladesh occur in the Chittagong Hill Districts, and Plasmodium falciparum (predominately chloroquine resistant) is the most prevalent species.MethodsThe objective of this research was to describe the epidemiology of symptomatic P. falciparum malaria in an area of Bangladesh following the introduction of a national malaria control program. We carried out surveillance for symptomatic malaria due to P. falciparum in two demographically defined unions of the Chittagong Hill Districts in Bangladesh, bordering western Myanmar, between October 2009 and May 2012. The association between sociodemographics and temporal and climate factors with symptomatic P. falciparum infection over two years of surveillance data was assessed. Risk factors for infection were determined using a multivariate regression model.Results472 cases of symptomatic P. falciparum malaria cases were identified among 23,372 residents during the study period. Greater than 85% of cases occurred during the rainy season from May to October, and cases were highly clustered geographically within these two unions with more than 80% of infections occurring in areas that contain approximately one-third of the total population. Risk factors statistically associated with infection in a multivariate logistic regression model were living in the areas of high incidence, young age, and having an occupation including jhum cultivation and/or daily labor. Use of long lasting insecticide-treated bed nets was high (89.3%), but its use was not associated with decreased incidence of infection.ConclusionHere we show that P. falciparum malaria continues to be hypoendemic in the Chittagong Hill Districts of Bangladesh, is highly seasonal, and is much more common in certain geographically limited hot spots and among certain occupations.