BACKGROUND: In Addis Ababa, Ethiopia, open ditches along innner roads in residential areas serve to convey domestic wastewater and rainwater away from residences. Contamination of drinking water by wastewater through faulty distribution lines could expose households to waterborne illnesses. This prompted the study to assess the microbiological safety of wastewater and drinking water in Addis Ababa, identify the pathogens therein, and determine their antibiotic resistance patterns.
UNASSIGNED: O1, mainly Hikojima serotype, was isolated from 23 wastewater and 16 drinking water samples. Similarly, 19 wastewater and 10 drinking water samples yielded Escherichia coli O157:H7. V. cholerae O1 were 100% resistant to the penicillins (Amoxacillin and Ampicillin), and 51-82% were resistant to the cephalosporins. About 44% of the V. cholerae O1 isolates in this study were Extended Spectrum Beta-Lactamase (ESBL) producers. Moreover, 26% were resistant to Meropenem. Peperacillin/Tazobactam was the only effective β-lactam antibiotic against V. cholerae O1. V. cholerae O1 isolates showed 37 different patterns of multiple resistance ranging from a minimum of three to a maximum of ten antimicrobials. Of the E. coli O157:H7 isolates, 71% were ESBL producers. About 96% were resistant to Ampicillin. Amikacin and Gentamicin were very effective against E. coli O157:H7 isolates. The isolates from wastewater and drinking water showed multiple antibiotic resistance against three to eight antibiotic drugs.
CONCLUSIONS: Open ditches for wastewater conveyance along innner roads in residence areas and underground faulty municipal water distribution lines could be possible sources for V. cholerae O1 and E. coli O157:H7 infections to surrounding households and for dissemination of multiple drug resistance in humans and, potentially, the environment.

OBJECTIVE: Globally, there are estimated to be 2.9 million cholera cases annually. Early detection of cholera outbreaks is crucial for resource allocation for case management and for targeted interventions to be delivered to stop the spread of cholera. In resource limited settings such as Eastern Democratic Republic of the Congo (DRC), there is often limited laboratory capacity for analysing stool samples for cholera by bacterial culture. Therefore, rapid diagnostic tests (RDTs) for cholera present a promising tool to rapidly test stool samples in a health facility setting for cholera. Our objective is to evaluate the Crystal VC O1 RDT for cholera detection compared with bacterial culture and polymerase chain reaction (PCR) for Vibrio cholerae.
METHODS: From March 2020 to December 2022, stool samples were collected from 644 diarrhoea patients admitted to 94 health facilities in Bukavu in Eastern DRC. Patient stool samples were analysed by Crystal VC O1 RDT for cholera and by bacterial culture and PCR for V. cholerae O1.
RESULTS: Twenty six percent of diarrhoea patients (166/644) had stool samples positive for cholera by RDT, and 24% (152/644) had stool samples positive for V. cholerae O1 by bacterial culture or PCR. The overall specificity and sensitivity of the Crystal VC O1 RDT by direct testing was 94% (95% confidence interval [CI]: 92%-96%) and 90% (95% CI, 84%-94%), respectively, when compared with either a positive result by bacterial culture or PCR.
CONCLUSIONS: Our findings suggest that the Crystal VC O1 RDT presents a promising tool for cholera surveillance in this cholera endemic setting in sub-Saharan Africa.

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.
RESULTS: 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.
CONCLUSIONS: 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.
BACKGROUND: The Bill & Melinda Gates Foundation.
UNASSIGNED: For the Nepali translation of the abstract see Supplementary Materials section.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Seventh pandemic Vibrio choleare O1 El Tor strain is responsible for the on-going pandemic outbreak of cholera globally. This strain evolved from non-pathogenic V. cholerae by acquiring seventh pandemic gene (VC 2346), pandemic Islands (VSP1 and VSP2), pathogenicity islands (VP1 and VP2) and CTX prophage region. The cholera toxin production is mainly attributed to the presence of ctx gene in these strains. However, several variants of this strain emerged as hybrid strains or atypical strains. The present study aimed to assess the aquatic environment of Cochin, India, over a period of 5 years for the emergence of multidrug resistant V. cholerae and its similarity with seventh pandemic strain. The continuous surveillance and monitoring resulted in the isolation of ctx negative, O1 positive V. cholerae isolate (VC6) from coastal water, Cochin, Kerala. The isolate possessed the biotype specific O1 El Tor tcpA gene and lacked other biotype specific ctx, zot, ace and rst genes. Whole genome analysis revealed the isolate belongs to pandemic sequence type (ST) 69 with the possession of pandemic VC2346 gene, pathogenic island VPI1, VPI2, and pandemic island VSP1 and VSP2. The isolate possessed several insertion sequences and the SXT/R391 family related Integrative Conjugative Elements (ICEs). In addition to this, the isolate genome carried virulence genes such as VgrG, mshA, ompT, toxR, ompU, rtxA, als, VasX, makA, and hlyA and antimicrobial resistance genes such as gyrA, dfrA1, strB, parE, sul2, parC, strA, VC1786ICE9-floR, and catB9. Moreover, the phylogenetic analysis suggests that the isolate genome is more closely related to seventh pandemic V. cholerae O1 N16961 strain. This study reports the first incidence of environmental ctx negative seventh pandemic V. choleare O1 El Tor isolate, globally and its presence in the aquatic system likely to induce toxicity in terms of public health point of view. The presence of this isolate in the aquatic environment warns the strict implementation of the epidemiological surveillance on the occurrence of emerging strains and the execution of flagship program for the judicious use of antibiotics in the aquatic ecosystem.

Vibrio cholerae can form biofilms in the aquatic environment and in the human intestine, facilitating the release of hyper-infectious aggregates. Due to the increasing antibiotic resistance, alternatives need to be found. One of these alternatives is antimicrobial peptides, including polymyxin B (PmB). In this study, we first investigated the resistance of V. cholerae O1 El Tor strain A1552 to various antimicrobials under aerobic and anaerobic conditions. An increased resistance to PmB is observed in anaerobiosis, with a 3-fold increase in the dose required for 50 % growth inhibition. We then studied the impact of the PmB on the formation and the degradation of V. cholerae biofilms to PmB. Our results show that PmB affects more efficiently biofilm formation under anaerobic conditions. On the other hand, preformed biofilms are susceptible to degradation by PmB at concentrations close to the minimal inhibitory concentration. At higher concentrations, we observe an opacification of the biofilm structures within 20 min post-treatment, suggesting a densification of the structure. This densification does not seem to result from the overexpression of matrix genes but rather from DNA release through massive cell lysis, likely forming a protective shield that limits the penetration of the PmB into the biofilm.

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that spreads locally and globally in epidemics and pandemics. Although it was discovered that fish harbor V. cholerae strains in their intestines, most investigations revealed non-toxic V. cholerae serogroups in fish. Due to the rarity of toxigenic V. cholerae serogroups, it is difficult to cultivate these strains from environmental samples. Hence, here we aimed to uncover evidence of the occurrence of toxigenic V. cholerae in the intestines and spleens of various fish species. By using molecular detection tools, we show that V. cholerae O1 and strains positive for the cholera toxin inhabit both healthy and diseased fish intestines and spleens, suggesting that fish may serve as intermediate vectors of toxigenic V. cholerae. No significant differences were found between the abundance of toxigenic V. cholerae (either O1 or cholera toxin positive strains) in the healthy and the diseased fish intestines or spleens. In conclusion, a variety of fish species may serve as potential vectors and reservoirs of toxigenic V. cholerae as they form a link between the other reservoirs of V. cholerae (chironomids, copepods, and waterbirds). Similarly, they may aid in the spread of this bacterium between water bodies.

UNASSIGNED: Cholera is an acute intestinal infection caused by Vibrio cholera (V. cholera). The development of antibodies against specific V. cholerae may have a therapeutic effect. In the present research, we investigated the protective effect of egg yolk Immunoglobulin (IgY), which was produced by immunizing hens with formaldehyde-killed V. cholerae O1 and subsequently the isolated IgY was orally administrated to the V. cholerae O1 infected mice for evaluation of its immunizing capability.
UNASSIGNED: In the current study, hens were immunized three times with formaldehyde-killed V. cholerae O1 (1.5×107 CFU/ml) and an equal volume of adjuvant. The IgY was isolated from egg yolk by polyethylene glycol method. The validity and activity of isolated IgY were confirmed with SDS-PAGE and ELISA methods, respectively. Subsequently IgY was orally administered to suckling mice following challenge with V. cholerae O1. ELISA results showed high antibody titer in the serum and egg yolk. Also, SDS-PAGE analysis showed successful purification of IgY and anti-V. cholerae IgY prevented the death of mice infected with V. cholerae O1. The anti-V. cholera IgY was administered at 2, 4, 6 hr intervals after 3 hr of inoculation of mice with V. cholerae O1.
UNASSIGNED: Results showed that the rate of surviving mice (2 mg/ml of IgY) were 60% after 4 hr and 40% after 6 hr and the rate of surviving mice (5 mg/ml of IgY) was 70% after 4 hr and 60% after 6 hr.
UNASSIGNED: The findings suggested the egg yolk-driven IgY as a natural antibacterial protein, could be effective in the prevention and treatment of cholera disease.

BACKGROUND: Cholera is a potentially lethal diarrheal disease produced by Vibrio cholerae serotypes O1 El Tor and O139. Known since antiquity, the condition causes epidemics in many areas, particularly in Asia, Africa, and South America. Left untreated, the mortality may reach 50%. The crucial therapeutic intervention is intravenous or oral rehydration and correction of acidosis, dyselectrolytemia, and renal impairment. Antibiotic use represents the main pharmacological intervention.
OBJECTIVE: What are the milestones of the antibiotics use recommended by experts for the pharmacological management of cholera in the past century?
METHODS: To determine the changes in the experts\' approach to the management of cholera and particularly the use of antibiotics as presented in a widely used textbook in the United States.
METHODS: The chapters describing the management of cholera in the 26 editions of Cecil Textbook of Medicine published from 1927 through 2020.
RESULTS: Sulfonamides were recommended in 1947, followed by the introduction of tetracyclines, chloramphenicol, and furazolidone in 1955. The options were restricted in 2000 to doxycycline. In the past decade, patients infected with strains known to have a degree a resistance to tetracyclines were treated with azithromycin or ciprofloxacin. Antibiotic use decreases the volume of stool and the duration of diarrhea but has not been considered lifesaving. Drugs with antimotility, antiemetic, or antisecretory properties are not useful.
CONCLUSIONS: The utility of antibiotic use in cholera has been endorsed by experts, but only as an adjunct to rapid and complete fluid and electrolyte replacement.

OBJECTIVE: It is well recognized that only Vibrio cholerae O1 causes cholera pandemics. However, not all O1 strains cause pandemic-level disease. In this study, we analyzed non-pandemic O1 V. cholerae isolates from the 1960s to the 1990s from China and found that they fell into three lineages, one of which shared the most recent common ancestor with pandemic O1 strains. Each of these non-pandemic O1 lineages has unique properties that contribute to their capacity to cause cholera. The findings of this study enhanced our understanding of the emergence and evolution of both pandemic and non-pandemic O1 V. cholerae.