Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI.

Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm-far exceeding the recommended limit of 0.003 ppm-were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl2. 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.

Analyses of drinking water quality during a protracted cholera epidemic in Malawi - a cross-sectional study of key physicochemical and microbiological parameters.

Anecdotal evidence and available literature indicated that contaminated water played a major role in spreading the prolonged cholera epidemic in Malawi from 2022 to 2023. This study assessed drinking water quality in 17 cholera-affected Malawi districts from February to April 2023. Six hundred and thirty-three records were analysed. The median counts/100 ml for thermotolerant coliform was 98 (interquartile range (IQR): 4-100) and that for Escherichia coli was 0 (IQR: 0-9). The drinking water in all (except one) districts was contaminated by thermotolerant coliform, while six districts had their drinking water sources contaminated by E. coli. The percentage of contaminated drinking water sources was significantly higher in shallow unprotected wells (80.0% for E. coli and 95.0% for thermotolerant coliform) and in households (55.8% for E. coli and 86.0% for thermotolerant coliform). Logistic regression showed that household water has three times more risk of being contaminated by E. coli and two and a half times more risk of being contaminated by thermotolerant coliform compared to other water sources. This study demonstrated widespread contamination of drinking water sources during a cholera epidemic in Malawi, which may be the plausible reason for the protracted nature of the epidemic.

Spatiotemporal dynamics of cholera epidemics in Ethiopia: 2015-2021.

Since the onset of the seventh cholera pandemic, Ethiopia has been affected by recurrent epidemics. However, the epidemiology of cholera in this country remains poorly understood. This study aimed to describe cholera outbreak characteristics in Ethiopia from 2015 to 2021. During this period, Ethiopia experienced four epidemic waves. The first wave involved nationwide outbreaks during the second half of 2016 followed by outbreaks predominantly affecting Somali Region in 2017. The second wave primarily affected Tigray and Afar Regions. During the third wave, multiple smaller-scale outbreaks occurred during 2019. The fourth wave was limited to Bale Zone (Oromia Region) in 2021. Overall, a north to south shift was observed over the course of the study period. Major cholera transmission factors included limited access to safe water and sanitation facilities. Severe weather events (drought and flooding) appear to aggravate cholera diffusion. Cholera transmission between Ethiopia and nearby countries (Kenya and Somalia), likely plays a major role in regional cholera dynamics. Overall, this study provides the first understanding of recent spatiotemporal cholera dynamics in Ethiopia to inform cholera control and elimination strategies.

Comparison of analysis methods to classify cholera hotspots in Ethiopia from 2015 to 2021.

Cholera continues to represent a major public health concern in Ethiopia. The country has developed a Multi-sectoral National Cholera Elimination Plan in 2022, which targets prevention and control interventions in cholera hotspots. Multiple methods to classify cholera hotspots have been used in several countries. Since 2014, a classification method developed by United Nations Children's Fund has been applied to guide water, sanitation and hygiene interventions throughout Sub-Saharan Africa based on three outbreak parameters: frequency, duration and standardized attack rate. In 2019, the Global Task Force on Cholera Control (GTFCC) proposed a method based on two parameters: average annual cholera incidence and persistence. In 2023, an updated GTFCC method for multisectoral interventions considers three epidemiological indicators (cumulative incidence, cumulative mortality and persistence,) and a cholera-case confirmation indicator. The current study aimed to classify cholera hotspots in Ethiopia at the woreda level (equivalent to district level) applying the three methods and comparing the results to optimize the hotspot targeting strategy. From 2015 to 2021, cholera hotspots were located along major routes between Addis Ababa and woredas adjacent to the Kenya and Somalia borders, throughout Tigray Region, around Lake Tana, and in Afar Region. The multi-method comparison enables decision makers to prioritize interventions according to a sub-classification of the highest-priority areas.

Early use of oral cholera vaccines as a prime control measure during outbreaks: Necessary but not sufficient.

INTRODUCTION: Despite being a preventable and treatable disease, cholera remains a public health problem in Sudan. The objective of the outbreak investigation was to identify associated risk factors that would help institute appropriate control measures. MATERIAL AND METHODS: A case control study design was chosen to identify the risk factors for cholera in Gadarif State. RESULTS: Multi-variate analysis of identified two risk factors and three preventive factors for cholera in Gadarif City. RISK FACTORS: Buying foods or drinks from street vendors (OR = 71.36), 95 % CI: 16.58-307.14), living in an urban setting (Gadarif City) (OR = 5.38), 95 % CI: 2.10-13.81); and the preventive factors were: Washing hands with water after defecation but without soap (OR = 0.16), 95 % CI: 0.04-0.63) or with soap (OR = 0.01), 95 % CI: 0.00-0.03), washing hands before eating (OR = 0.15), 95 % CI: 0.05-0.51) and taking Oral Cholera Vaccine (OCV) (OR = 0.19, 95 % CI: 0.08-0.44). The effectiveness of OCV (VE) was (Unadjusted VE: 80 %, 95 % CI: 69 %-87 %) or (Adjusted VE = 81.0 %, 95 % CI: 56.0 %-92.0 %). DISCUSSION: Cholera outbreaks, especially in the setting of a complex humanitarian crises, can spread rapidly, resulting in many deaths, and quickly become a public health crisis. Implementation of a community-wide vaccination campaign using OCV as early as possible during the outbreak while implementing other control measures to target hotspots and at-risk populations would expedite halting outbreaks of cholera and save lives.

In Commemoration of Dr. Mostafa Pourtaghva Shahrestani, a Pioneer in Infectious Disease Research.

It is important to honor the contributions of scientific leaders who have dedicated their lives to advancing knowledge and serving their country. One way is to document their experiences and personalities in a documentary format, which can serve as a historical record and an inspiration for future generations. Dr. Mostafa Pourtaghva Shahrestani, a renowned physician and specialist in infectious diseases and tropical medicine, has made significant contributions to public health in Iran. He has played a crucial role in controlling infectious diseases such as smallpox, tuberculosis, rabies, plague, and cholera. Throughout his career, he has held various executive positions, including the head of Pasteur Hospital and the director of the Pasteur Institute of Iran. Dr. Pourtaghva's life is a testament to his unwavering dedication to public health services, as evidenced by his continuous effort, love, and interest in honest work. His inspiring story can serve as a model for those who seek to follow in his footsteps.

Safety and immunogenicity of the Euvichol-S oral cholera vaccine for prevention of Vibrio cholerae O1 infection in Nepal: an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial.

BACKGROUND: In October, 2017, WHO launched a strategy to eliminate cholera by 2030. A primary challenge in meeting this goal is the limited global supply capacity of oral cholera vaccine and the worsening of cholera outbreaks since 2021. To help address the current shortage of oral cholera vaccine, a WHO prequalified oral cholera vaccine, Euvichol-Plus was reformulated by reducing the number of components and inactivation methods. We aimed to evaluate the immunogenicity and safety of Euvichol-S (EuBiologics, Seoul, South Korea) compared with an active control vaccine, Shanchol (Sanofi Healthcare India, Telangana, India) in participants of various ages in Nepal. METHODS: We did an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial at four hospitals in Nepal. Eligible participants were healthy individuals aged 1-40 years without a history of cholera vaccination. Individuals with a history of hypersensitivity reactions to other preventive vaccines, severe chronic disease, previous cholera vaccination, receipt of blood or blood-derived products in the past 3 months or other vaccine within 4 weeks before enrolment, and pregnant or lactating women were excluded. Participants were randomly assigned (1:1:1:1) by block randomisation (block sizes of two, four, six, or eight) to one of four groups (groups A-D); groups C and D were stratified by age (1-5, 6-17, and 18-40 years). Participants in groups A-C were assigned to receive two 1·5 mL doses of Euvichol-S (three different lots) and participants in group D were assigned to receive the active control vaccine, Shanchol. All participants and site staff (with the exception of those who prepared and administered the study vaccines) were masked to group assignment. The primary immunogenicity endpoint was non-inferiority of immunogenicity of Euvichol-S (group C) versus Shanchol (group D) at 2 weeks after the second vaccine dose, measured by the seroconversion rate, defined as the proportion of participants who had achieved seroconversion (defined as ≥four-fold increase in V cholerae O1 Inaba and Ogawa titres compared with baseline). The primary immunogenicity endpoint was assessed in the per-protocol analysis set, which included all participants who received all their planned vaccine administrations, had no important protocol deviations, and who provided blood samples for all immunogenicity assessments. The primary safety endpoint was the number of solicited adverse events, unsolicited adverse events, and serious adverse events after each vaccine dose in all ages and each age stratum, assessed in all participants who received at least one dose of the Euvichol-S or Shanchol. Non-inferiority of Euvichol-S compared with Shanchol was shown if the lower limit of the 95% CI for the difference between the seroconversion rates in Euvichol-S group C versus Shanchol group D was above the predefined non-inferiority margin of -10%. The trial was registered at ClinicalTrials.gov, NCT04760236. FINDINGS: Between Oct 6, 2021, and Jan 19, 2022, 2529 healthy participants (1261 [49·9%] males; 1268 [50·1%] females), were randomly assigned to group A (n=330; Euvichol-S lot number ES-2002), group B (n=331; Euvichol-S ES-2003), group C (n=934; Euvichol-S ES-2004]), or group D (n=934; Shanchol). Non-inferiority of Euvichol-S versus Shanchol in seroconversion rate for both serotypes at 2 weeks after the second dose was confirmed in all ages (difference in seroconversion rate for V cholerae O1 Inaba -0·00 [95% CI -1·86 to 1·86]; for V cholerae O1 Ogawa -1·62 [-4·80 to 1·56]). Treatment-emergent adverse events were reported in 244 (9·7%) of 2529 participants in the safety analysis set, with a total of 403 events; 247 events were reported among 151 (9·5%) of 1595 Euvichol-S recipients and 156 events among 93 (10·0%) of 934 Shanchol recipients. Pyrexia was the most common adverse event in both groups (57 events among 56 [3·5%] of 1595 Euvichol-S recipients and 37 events among 35 [3·7%] of 934 Shanchol recipients). No serious adverse events were deemed to be vaccine-related. INTERPRETATION: A two-dose regimen of Euvichol-S vaccine was non-inferior to the active control vaccine, Shanchol, in terms of seroconversion rates 2 weeks after the second dose. The simplified formulation and production requirements of the Euvichol-S vaccine have the potential to increase the supply of oral cholera vaccine and reduce the gap between the current oral cholera vaccine supply and demand. FUNDING: The Bill & Melinda Gates Foundation. TRANSLATION: For the Nepali translation of the abstract see Supplementary Materials section.

Genomic characteristics of clinical non-toxigenic Vibrio cholerae isolates in Switzerland: a cross-sectional study.

STUDY AIMS: Although non-toxigenic Vibrio cholerae lack the ctxAB genes encoding cholera toxin, they can cause diarrhoeal disease and outbreaks in humans. In Switzerland, V. cholerae is a notifiable pathogen and all clinical isolates are analysed at the National Reference Laboratory for Enteropathogenic Bacteria and Listeria. Up to 20 infections are reported annually. In this study, we investigated the population structure and genetic characteristics of non-toxigenic V. cholerae isolates collected over five years. METHODS:  V. cholerae isolates were serotyped and non-toxigenic isolates identified using a ctxA-specific PCR. Following Illumina whole-genome sequencing, genome assemblies were screened for virulence and antibiotic resistance genes. Phylogenetic analyses were performed in the context of 965 publicly available V. cholerae genomes. RESULTS: Out of 33 V. cholerae infections reported between January 2017 and January 2022 in Switzerland, 31 were caused by ctxA-negative isolates. These non-toxigenic isolates originated from gastrointestinal (n = 29) or extraintestinal (n = 2) sites. They were phylogenetically diverse and belonged to 29 distinct sequence types. Two isolates were allocated to the lineage L3b, a ctxAB-negative but tcpA-positive clade previously associated with regional outbreaks. The remaining 29 isolates were placed in lineage L4, which is associated with environmental strains. Genes or mutations associated with reduced susceptibility to the first-line antibiotics fluoroquinolones and tetracyclines were identified in 11 and 3 isolates, respectively. One isolate was predicted to be multidrug resistant. CONCLUSIONS:  V. cholerae infections in Switzerland are rare and predominantly caused by lowly virulent ctxAB-negative and tcpA-negative strains. As V. cholerae is not endemic in Switzerland, cases are assumed to be acquired predominantly during travel. This assumption was supported by the phylogenetic diversity of the analysed isolates.

Analysis of environmental factors influencing endemic cholera risks in sub-Saharan Africa.

The recurring cholera outbreaks in sub-Saharan Africa are of growing concern, especially considering the potential acceleration in the global trend of larger and more lethal cholera outbreaks due to the impacts of climate change. However, there is a scarcity of evidence-based research addressing the environmental and infrastructure factors that sustain cholera recurrence in Africa. This study adopts a statistical approach to investigate over two decades of endemic cholera outbreaks and their relationship with five environmental factors: water provision, sanitation provision, raising temperatures, increased rainfall and GDP. The analysis covers thirteen of the forty-two countries in the mainland sub-Saharan region, collectively representing one-third of the region's territory and half of its population. This breadth enables the findings to be generalised at a regional level. Results from all analyses consistently associate water provision with cholera reduction. The stratified model links increased water provision with a reduction in cholera risk that ranged from 4.2 % to 84.1 % among eight countries (out of 13 countries) as well as a reduction of such risk that ranged from 9.8 % to 68.9 % when there is increased sanitation provision, which was observed in nine countries (out of 13). These results indicate that the population's limited access to water and sanitation, as well as the rise in temperatures, are critical infrastructure and environmental factors contributing to endemic cholera and the heightened risk of outbreaks across the sub-Saharan region. Therefore, these are key areas for targeted interventions and cross-border collaboration to enhance resilience to outbreaks and lead to the end of endemic cholera in the region. However, it is important to interpret the results of this study with caution; hence, further investigation is recommended to conduct a more detailed analysis of the impact of infrastructure and environmental factors on reducing cholera risk.

Spatiotemporal dynamics of cholera hotspots in the Democratic Republic of the Congo from 1973 to 2022.

BACKGROUND: Since the early 1970s, cholera outbreaks have been a major public health burden in the Democratic Republic of Congo (DRC). Cholera cases have been reported in a quasi-continuous manner in certain lakeside areas in the Great Lakes Region. As these cholera-endemic health zones constitute a starting point for outbreaks and diffusion towards other at-risk areas, they play a major role in cholera dynamics in the country. Monitoring the spatiotemporal dynamics of cholera hotspots and adjusting interventions accordingly thus reduces the disease burden in an efficient and cost-effective manner. METHODS: A literature review was conducted to describe the spatiotemporal dynamics of cholera in the DRC at the province level from 1973 to 1999. We then identified and classified cholera hotspots at the provincial and health zone levels from 2003 to 2022 and described the spatiotemporal evolution of hotspots. We also applied and compared three different classification methods to ensure that cholera hotspots are identified and classified according to the DRC context. RESULTS: According to all three methods, high-priority hotspots were concentrated in the eastern Great Lakes Region. Overall, hotspots largely remained unchanged over the course of the study period, although slight improvements were observed in some eastern hotspots, while other non-endemic areas in the west experienced an increase in cholera outbreaks. The Global Task Force on Cholera Control (GTFCC) and the Department of Ecology and Infectious Disease Control (DEIDC) methods largely yielded similar results for the high-risk hotspots. However, the medium-priority hotspots identified by the GTFCC method were further sub-classified by the DEIDC method, thereby providing a more detailed ranking for priority targeting. CONCLUSIONS: Overall, the findings of this comprehensive study shed light on the dynamics of cholera hotspots in the DRC from 1973 to 2022. These results may serve as an evidence-based foundation for public health officials and policymakers to improve the implementation of the Multisectoral Cholera Elimination Plan, guiding targeted interventions and resource allocation to mitigate the impact of cholera in vulnerable communities.

Congruity of genomic and epidemiological data in modelling of local cholera outbreaks.

Cholera continues to be a global health threat. Understanding how cholera spreads between locations is fundamental to the rational, evidence-based design of intervention and control efforts. Traditionally, cholera transmission models have used cholera case-count data. More recently, whole-genome sequence data have qualitatively described cholera transmission. Integrating these data streams may provide much more accurate models of cholera spread; however, no systematic analyses have been performed so far to compare traditional case-count models to the phylodynamic models from genomic data for cholera transmission. Here, we use high-fidelity case-count and whole-genome sequencing data from the 1991 to 1998 cholera epidemic in Argentina to directly compare the epidemiological model parameters estimated from these two data sources. We find that phylodynamic methods applied to cholera genomics data provide comparable estimates that are in line with established methods. Our methodology represents a critical step in building a framework for integrating case-count and genomic data sources for cholera epidemiology and other bacterial pathogens.

Sporadic regional re-emergent cholera: a 19th century problem in the 21st century.

Cholera, caused by Vibrio cholerae, is a severe diarrheal disease that necessitates prompt diagnosis and effective treatment. This review comprehensively examines various diagnostic methods, from traditional microscopy and culture to advanced nucleic acid testing like polymerase spiral reaction and rapid diagnostic tests, highlighting their advantages and limitations. Additionally, we explore evolving treatment strategies, with a focus on the challenges posed by antibiotic resistance due to the activation of the SOS response pathway in V. cholerae. We discuss promising alternative treatments, including low-pressure plasma sterilization, bacteriophages, and selenium nanoparticles. The paper emphasizes the importance of multidisciplinary approaches combining novel diagnostics and treatments in managing and preventing cholera, a persistent global health challenge. The current re-emergent 7th pandemic of cholera commenced in 1961 and shows no signs of abeyance. This is probably due to the changing genetic profile of V. cholerae concerning bacterial pathogenic toxins. Given this factor, we argue that the disease is effectively re-emergent, particularly in Eastern Mediterranean countries such as Lebanon, Syria, etc. This review considers the history of the current pandemic, the genetics of the causal agent, and current treatment regimes. In conclusion, cholera remains a significant global health challenge that requires prompt diagnosis and effective treatment. Understanding the history, genetics, and current treatments is crucial in effectively addressing this persistent and re-emergent disease.