ABSTRACT
OBJECTIVES: As of December, 1st, 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, resulted in more than 1 472 917 deaths worldwide and death toll is still increasing exponentially. Many COVID-19 infected people are asymptomatic or experience moderate symptoms and recover without medical intervention. However, older people and those with comorbid hypertension, diabetes, obesity, or heart disease are at higher risk of mortality. Because current therapeutic options for COVID-19 patients are limited specifically for this elderly population at risk, Biophytis is developing BIO101 (20-hydroxyecdysone, a Mas receptor activator) as a new treatment option for managing patients with SARS-CoV-2 infection at the severe stage. The angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2. Interaction between ACE2 and SARS-CoV2 spike protein seems to alter the function of ACE2, a key player in the renin-angiotensin system (RAS). The clinical picture of COVID-19 includes acute respiratory distress syndrome (ARDS), cardiomyopathy, multiorgan dysfunction and shock, all of which might result from an imbalance of the RAS. We propose that RAS balance could be restored in COVID-19 patients through MasR activation downstream of ACE2 activity, with 20-hydroxyecdysone (BIO101) a non-peptidic Mas receptor (MasR) activator. Indeed, MasR activation by 20-hydroxyecdysone harbours anti-inflammatory, anti-thrombotic, and anti-fibrotic properties. BIO101, a 97% pharmaceutical grade 20-hydroxyecdysone could then offer a new therapeutic option by improving the respiratory function and ultimately promoting survival in COVID-19 patients that develop severe forms of this devastating disease. Therefore, the objective of this COVA study is to evaluate the safety and efficacy of BIO101, whose active principle is 20-hydroxyecdysone, in COVID-19 patients with severe pneumonia. TRIAL DESIGN: Randomized, double-blind, placebo-controlled, multi-centre, group sequential and adaptive which will be conducted in 2 parts. Part 1: Ascertain the safety and tolerability of BIO101 and obtain preliminary indication of the activity of BIO101, in preventing respiratory deterioration in the target population Part 2: Re-assessment of the sample size needed for the confirmatory part 2 and confirmation of the effect of BIO101 observed in part 1 in the target population. The study is designed as group sequential to allow an efficient run-through, from obtaining an early indication of activity to a final confirmation. And adaptive - to allow accumulation of early data and adapt sample size in part 2 in order to inform the final design of the confirmatory part of the trial. PARTICIPANTS: Inclusion criteria 1. Age: 45 and above 2. A confirmed diagnosis of COVID-19 infection, within the last 14 days, prior to randomization, as determined by PCR or other approved commercial or public health assay, in a specimen as specified by the test used. 3. Hospitalized, in observation or planned to be hospitalized due to COVID-19 infection symptoms with anticipated hospitalization duration ≥3 days 4. With evidence of pneumonia based on all of the following: a. Clinical findings on a physical examination b. Respiratory symptoms developed within the past 7 days 5. With evidence of respiratory decompensation that started not more than 4 days before start of study medication and present at screening, meeting one of the following criteria, as assessed by healthcare staff: a. Tachypnea: ≥25 breaths per minute b. Arterial oxygen saturation ≤92% c. A special note should be made if there is suspicion of COVID-19-related myocarditis or pericarditis, as the presence of these is a stratification criterion 6. Without a significant deterioration in liver function tests: a. ALT and AST ≤ 5x upper limit of normal (ULN) b. Gamma-glutamyl transferase (GGT) ≤ 5x ULN c. Total bilirubin ≤ 5×ULN 7. Willing to participate and able to sign an informed consent form (ICF). Or, when relevant, a legally authorized representative (LAR) might sign the ICF on behalf of the study participant 8. Female participants should be: at least 5 years post-menopausal (i.e., persistent amenorrhea 5 years in the absence of an alternative medical cause) or surgically sterile; OR a. Have a negative urine pregnancy test at screening b. Be willing to use a contraceptive method as outlined in inclusion criterion 9 from screening to 30 days after last dose. 9. Male participants who are sexually active with a female partner must agree to the use of an effective method of birth control throughout the study and until 3 months after the last administration of the investigational product. (Note: medically acceptable methods of contraception that may be used by the participant and/or partner include combined oral contraceptive, contraceptive vaginal ring, contraceptive injection, intrauterine device, etonogestrel implant, each supplemented with a condom, as well as sterilization and vasectomy). 10. Female participants who are lactating must agree not to breastfeed during the study and up to 14 days after the intervention. 11. Male participants must agree not to donate sperm for the purpose of reproduction throughout the study and until 3 months after the last administration of the investigational product. 12. For France only: Being affiliated with a European Social Security. Exclusion criteria 1. Not needing or not willing to remain in a healthcare facility during the study 2. Moribund condition (death likely in days) or not expected to survive for >7 days - due to other and non-COVID-19 related conditions 3. Participant on invasive mechanical ventilation via an endotracheal tube, or extracorporeal membrane oxygenation (ECMO), or high-flow Oxygen (delivery of oxygen at a flow of ≥16 L/min.). 4. Participant is not able to take medications by mouth (as capsules or as a powder, mixed in water). 5. Disallowed concomitant medication: Consumption of any herbal products containing 20-hydroxyecdysone and derived from Leuzea carthamoides; Cyanotis vaga or Cyanotis arachnoidea is not allowed (e.g. performance enhancing agents). 6. Any known hypersensitivity to any of the ingredients, or excipients of the study medication, BIO101. 7. Renal disease requiring dialysis, or known renal insufficiency (eGFR≤30 mL/min/1.73 m2, based on Cockcroft & Gault formula). 8. In France only: a. Non-affiliation to compulsory French social security scheme (beneficiary or right-holder). b. Being under tutelage or legal guardianship. Participants will be recruited from approximately 30 clinical centres in Belgium, France, the UK, USA and Brazil. Maximum patients' participation in the study will last 28 days. Follow-up of participants discharged from hospital will be performed through post-intervention phone calls at 14 (± 2) and 60 (± 4) days. INTERVENTION AND COMPARATOR: Two treatment arms will be tested in this study: interventional arm 350 mg b.i.d. of BIO101 (AP 20-hydroxyecdysone) and placebo comparator arm 350 mg b.i.d of placebo. Administration of daily dose is the same throughout the whole treatment period. Participants will receive the study medication while hospitalized for up to 28 days or until a clinical endpoint is reached (i.e., 'negative' or 'positive' event). Participants who are officially discharged from hospital care will no longer receive study medication. MAIN OUTCOMES: Primary study endpoint: The proportion of participants with 'negative' events up to 28 days. 'Negative' events are defined as respiratory deterioration and all-cause mortality. For the purpose of this study, respiratory deterioration will be defined as any of the following: Requiring mechanical ventilation (including cases that will not be intubated due to resource restrictions and triage). Requiring extracorporeal membrane oxygenation (ECMO). Requiring high-flow oxygen defined as delivery of oxygen at a flow of ≥16 L/min. Only if the primary endpoint is significant at the primary final analysis the following Key secondary endpoints will be tested in that order: Proportion of participants with events of respiratory failure at Day 28 Proportion of participants with 'positive' events at Day 28. Proportion of participants with events of all-cause mortality at Day 28 A 'positive' event is defined as the official discharge from hospital care by the department due to improvement in participant condition. Secondary and exploratory endpoints: In addition, a variety of functional measures and biomarkers (including the SpO2 / FiO2 ratio, viral load and markers related to inflammation, muscles, tissue and the RAS / MAS pathways) will also be collected. RANDOMIZATION: Randomization is performed using an IBM clinical development IWRS system during the baseline visit. Block-permuted randomization will be used to assign eligible participants in a 1:1 ratio. In part 1, randomization will be stratified by RAS pathway modulator use (yes/no) and co-morbidities (none vs. 1 and above). In Part 2, randomization will be stratified by centre, gender, RAS pathway modulator use (yes/no), co-morbidities (none vs. 1 and above), receiving Continuous Positive Airway Pressure/Bi-level Positive Airway Pressure (CPAP/BiPAP) at study entry (Yes/No) and suspicion of COVID-19 related myocarditis or pericarditis (present or not). BLINDING (MASKING): Participants, caregivers, and the study team assessing the outcomes are blinded to group assignment. All therapeutic units (TU), BIO101 b.i.d. or placebo b.i.d., cannot be distinguished in compliance with the double-blind process. An independent data-monitoring committee (DMC) will conduct 2 interim analyses. A first one based on the data from part 1 and a second from the data from parts 1 and 2. The first will inform about BIO101 safety, to allow the start of recruitment into part 2 followed by an analysis of the efficacy data, to obtain an indication of activity. The second interim analysis will inform about the sample size that will be required for part 2, in order to achieve adequate statistical power. Numbers to be randomised (sample size) Number of participants randomized: up to 465, in total Part 1: 50 (to obtain the proof of concept in COVID-19 patients). Part 2: 310, potentially increased by 50% (up to 465, based on interim analysis 2) (to confirm the effects of BIO101 observed in part 1). TRIAL STATUS: The current protocol Version is V 10.0, dated on 24.09.2020. The recruitment that started on September 1st 2020 is ongoing and is anticipated to finish for the whole study by March2021. TRIAL REGISTRATION: The trial was registered before trial start in trial registries: EudraCT , No. 2020-001498-63, registered May 18, 2020; and Clinicaltrials.gov, identifier NCT04472728 , registered July 15, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.
Subject(s)
COVID-19 Drug Treatment , Ecdysterone/therapeutic use , Respiratory Insufficiency/drug therapy , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/physiopathology , Disease Progression , Double-Blind Method , Extracorporeal Membrane Oxygenation/statistics & numerical data , Hospitalization , Humans , Hypoxia/physiopathology , Middle Aged , Mortality , Oxygen Inhalation Therapy/statistics & numerical data , Proto-Oncogene Mas , Proto-Oncogene Proteins/metabolism , Randomized Controlled Trials as Topic , Receptors, Coronavirus/metabolism , Receptors, G-Protein-Coupled/metabolism , Renin-Angiotensin System , Respiration, Artificial/statistics & numerical data , Respiratory Insufficiency/physiopathology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Tachypnea/physiopathology , Treatment OutcomeABSTRACT
Developing countries continue to contribute significantly to the global burden of childhood mortality due to infectious diseases. Infections leading to diseases like diarrhoea, pneumonia and meningitis account for millions of deaths annually. Most of these diseases are preventable by vaccination and therefore global vaccination rates have risen substantially with clear benefits. But paradoxically, the vaccines have demonstrated lower immunogenicity in developing countries as compared to their industrialised counterparts. Malnutrition in resource poor settings along with repeated polymicrobial infections at early age are some of the reasons for the differences in vaccine efficacy in different settings. Recent studies indicate that the gastrointestinal microbiota possibly influences maturation of immune system as well as vaccine efficacy. In this review we discuss evidences from in vitro, animal and human studies showing that probiotics can positively modulate gut microbiota composition and exert immunomodulatory effects on the host. We also discuss how they should be evaluated for their ability to improve vaccine performance especially in low resource settings.
Subject(s)
Developing Countries , Gastrointestinal Microbiome/immunology , Probiotics , Vaccines/immunology , Animals , Child , Humans , Immunogenicity, Vaccine , Immunomodulation , Probiotics/administration & dosage , Probiotics/pharmacology , Vaccination , Vaccines/administration & dosageABSTRACT
The 2 biotypes of Vibrio cholerae O1 serogroup strains-classical and El Tor-use glucose in distinct ways. Classical biotype strains perform organic acid-producing fermentation and eventually lose viability due to the self-induced creation of an acidic environment, whereas El Tor biotype strains use an alternative neutral fermentation pathway, which confers them with survival advantages. However, we report that the neutral fermentation pathway has only been recruited in prototype Wave 1 El Tor biotype strains, which have not been isolated since the mid-1990s. Current Wave 2 and Wave 3 atypical El Tor strains contain a single-base deletion in a gene that directs bacteria toward neutral fermentation, resulting in the loss of neutral fermentation and an appearance that is similar to classical biotype strains. Moreover, when sufficient glucose was supplied, Wave 1 El Tor strains maintained their use of acid-producing fermentation, in parallel with neutral fermentation, and thus lost viability in the late stationary phase. The global replacement of Wave 1 El Tor strains by Wave 2 and 3 atypical El Tor strains implies that the acidic fermentation pathway may not be disadvantageous to V. cholerae. The characteristics that we have reported might improve oral rehydration in the treatment of cholera.
Subject(s)
Glucose/metabolism , Vibrio cholerae O1/metabolism , Batch Cell Culture Techniques , Glucose/pharmacology , Hydrogen-Ion Concentration , Microbial Viability/drug effects , Serogroup , Vibrio cholerae O1/drug effects , Vibrio cholerae O1/genetics , Vibrio cholerae O1/growth & developmentABSTRACT
A total of 45 strains of Vibrio cholerae O1 isolated from 10 different places in India where they were associated with cases of cholera between the years 2007 and 2008 were examined by molecular methods. With the help of phenotypic and genotypic tests the strains were confirmed to be O1 El Tor biotype strains with classical ctxB gene. Polymerase chain reaction (PCR) analysis by double - mismatch amplification mutation assay PCR showed 16 of these strains carried the ctxB-7 allele reported in Haitian strains. Sequencing of the ctxB gene in all the 45 strains revealed that in 16 strains the histidine at the 20th amino acid position had been replaced by asparagine and this single nucleotide polymorphism did not affect cholera toxin production as revealed by beads enzyme-linked immunosorbent assay. This study shows that the new ctxB gene sequence was circulating in different places in India. Seven representatives of these 45 strains analysed by pulsed - field gel electrophoresis showed four distinct Not I digested profiles showing that multiple clones were causing cholera in 2007 and 2008.
Subject(s)
Cholera Toxin/genetics , Vibrio cholerae O1/classification , Vibrio cholerae O1/genetics , Bacterial Typing Techniques , Electrophoresis, Gel, Pulsed-Field , Enzyme-Linked Immunosorbent Assay , Genotype , Haiti , India , Sequence Analysis, DNAABSTRACT
Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.
Subject(s)
Cholera Toxin/metabolism , Cholera/epidemiology , Cholera/microbiology , Vibrio cholerae O1/isolation & purification , Blotting, Western , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Feces/microbiology , Genotype , Humans , Molecular Sequence Data , Molecular Typing , Sequence Analysis, DNA , Tanzania/epidemiology , Vibrio cholerae O1/pathogenicityABSTRACT
Culture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as 'no known aetiology'. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower C(t) values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated.
Subject(s)
Bacteriological Techniques/methods , Coinfection/epidemiology , Cross Infection/epidemiology , Diarrhea/epidemiology , Real-Time Polymerase Chain Reaction/methods , Bacterial Load , Campylobacter/classification , Campylobacter/isolation & purification , Coinfection/diagnosis , Coinfection/microbiology , Cross Infection/diagnosis , Cross Infection/microbiology , Diarrhea/diagnosis , Diarrhea/microbiology , Escherichia coli/classification , Escherichia coli/isolation & purification , Feces/microbiology , Humans , India/epidemiology , Prevalence , Shigella/classification , Shigella/isolation & purification , Vibrio/classification , Vibrio/isolation & purificationABSTRACT
Rotavirus is a common viral cause of severe diarrhoea. For the underlying cause of rotavirus seasonality, the meteorological factor has been suspected, whereas quantitative correlation between seasonality and meteorological factor has not been fully investigated. In this study, we investigated the correlation of temporal patterns of the isolation rate of rotavirus with meteorological condition (temperature, relative humidity, rainfall) in Kolkata, India. We used time-series analysis combined with spectral analysis and least squares method. A 1-year cycle explained underlying variations of rotavirus and meteorological data. The 1-year cycle for rotavirus data was correlated with an opposite phase to that for meteorological data. Relatively high temperature could be associated with a low value of isolation rate of rotavirus in the monsoon season. Quantifying a correlation of rotavirus infections with meteorological conditions might prove useful in predicting rotavirus epidemics and health services could plan accordingly.
Subject(s)
Diarrhea/epidemiology , Rotavirus Infections/epidemiology , Rotavirus Infections/virology , Diarrhea/virology , Hot Temperature , Humans , Humidity , Incidence , India/epidemiology , Prevalence , Rain , Rotavirus/physiology , Seasons , TemperatureABSTRACT
Helicobacter pylori is one of the most diverse bacterial species that chronically infects more than 70% of Indian population. Interestingly, data showing microdiversity of the H. pylori strains within a particular gastric niche remained scarce. To understand the extent of genetic diversity among H. pylori strains within a given host, 30 patients with gastro-duodenal problems were subjected to endoscopy and from each patient 10 single colonies were isolated. Characterization of each of these 10 single colonies by DNA fingerprinting as well as genotyping of several important genetic markers viz. cagA, vacA, iceA, vapD, cag PAI empty site, IS605, RFLP and two other genetic segments within cag PAI revealed that all of the 30 patients were infected with more than one strain and sometimes strains with 5 to 6 types of genetic variants. Analyses of certain genetic loci showed the microdiversity among the colonies from single patient, which may be due to the recombination events during long-term carriage of the pathogen. These results suggest that most of the patients have acquired H. pylori due to repeated exposure to this pathogen with different genetic make-up, which may increase the possibility of super infections. Genetic exchanges between these unrelated H. pylori strains may support certain H. pylori variant to grow better in a given host than the parental strain and thereby increasing the possibility for the severity of the infection.
Subject(s)
Helicobacter Infections/epidemiology , Helicobacter Infections/microbiology , Helicobacter pylori/metabolism , Antigens, Bacterial , Biodiversity , DNA Primers/genetics , Duodenum/microbiology , Endoscopy/methods , Genetic Markers , Genetic Variation , Genotype , Humans , Polymerase Chain Reaction/methods , Polymorphism, Genetic , Recombination, Genetic , Sequence Analysis, DNA , Stomach/microbiology , VirulenceABSTRACT
Microbially-unsafe water is still a major concern in most developing countries. Although many water-purification methods exist, these are expensive and beyond the reach of many people, especially in rural areas. Ayurveda recommends the use of copper for storing drinking-water. Therefore, the objective of this study was to evaluate the effect of copper pot on microbially-contaminated drinking-water. The antibacterial effect of copper pot against important diarrhoeagenic bacteria, including Vibrio cholerae O1, Shigella flexneri 2a, enterotoxigenic Escherichia coli, enteropathogenic E. coli, Salmonella enterica Typhi, and Salmonella Paratyphi is reported. When drinking-water (pH 7.83 +/- 0.4; source: ground) was contaminated with 500 CFU/mL of the above bacteria and stored in copper pots for 16 hours at room temperature, no bacteria could be recovered on the culture medium. Recovery failed even after resuscitation in enrichment broth, followed by plating on selective media, indicating loss of culturability. This is the first report on the effect of copper on S. flexneri 2a, enteropathogenic E. coli, and Salmonella Paratyphi. After 16 hours, there was a slight increase in the pH of water from 7.83 to 7.93 in the copper pots while the other physicochemical parameters remained unchanged. Copper content (177 +/- 16 ppb) in water stored in copper pots was well within the permissible limits of the World Health Organization. Copper holds promise as a point-of-use solution for microbial purification of drinking-water, especially in developing countries.
Subject(s)
Copper/pharmacology , Diarrhea/prevention & control , Drinking Water/microbiology , Enterobacteriaceae/growth & development , Water Microbiology , Water Purification/methods , Developing Countries , Diarrhea/microbiology , Drinking Water/analysis , Drinking Water/chemistry , Enterobacteriaceae/drug effects , HumansABSTRACT
New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.
Subject(s)
Cholera/microbiology , DNA, Bacterial/genetics , Molecular Typing , Vibrio cholerae O1/classification , Vibrio cholerae O1/genetics , Cholera Toxin/genetics , Cluster Analysis , Electrophoresis, Gel, Pulsed-Field , Evolution, Molecular , Genomic Islands , Genotype , Humans , Multigene Family , Open Reading Frames , Vibrio cholerae O1/isolation & purification , ZambiaABSTRACT
OBJECTIVES: In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. METHODS: The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. RESULTS: Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. CONCLUSIONS: This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages.
Subject(s)
Bacteriophage N4/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Genome, Viral , Vibrio cholerae O1/virology , Base Composition , Cluster Analysis , Molecular Sequence Data , Nucleic Acid Hybridization , Open Reading Frames , Phylogeny , Sequence Analysis, DNAABSTRACT
BACKGROUND & OBJECTIVES: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. METHODS: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. RESULTS: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6')-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), bla TEM-1 (35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. INTERPRETATION & CONCLUSIONS: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria.
Subject(s)
DNA Gyrase/genetics , Diarrhea/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Enterotoxigenic Escherichia coli/genetics , Escherichia coli Infections/microbiology , Genes, MDR/genetics , Integrons/genetics , Anti-Bacterial Agents/pharmacology , DNA Gyrase/drug effects , DNA Topoisomerase IV/drug effects , DNA Topoisomerase IV/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Diarrhea/drug therapy , Diarrhea/epidemiology , Disease Outbreaks , Drug Resistance, Multiple, Bacterial/drug effects , Enterotoxigenic Escherichia coli/isolation & purification , Enterotoxigenic Escherichia coli/pathogenicity , Escherichia coli/classification , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli Infections/drug therapy , Escherichia coli Infections/epidemiology , Escherichia coli Infections/genetics , Fluoroquinolones/pharmacology , Humans , India/epidemiology , Microbial Sensitivity Tests , Mutation/drug effects , Mutation/genetics , Quinolones/pharmacologyABSTRACT
BACKGROUND & OBJECTIVES: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. METHODS: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. RESULTS: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. INTERPRETATION & CONCLUSIONS: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).
Subject(s)
Chimera/genetics , Cholera/epidemiology , Cholera/microbiology , Vibrio cholerae O1/classification , Vibrio cholerae O1/isolation & purification , Atypical Bacterial Forms/genetics , Bacterial Typing Techniques/methods , Cholera/genetics , Cholera Toxin/genetics , DNA, Bacterial/genetics , Genetic Variation , Genotype , Humans , Molecular Epidemiology/methods , Phenotype , Polymorphism, Restriction Fragment Length/genetics , Thailand/epidemiology , Vibrio cholerae O1/geneticsABSTRACT
In Bangladesh, one of the main causes of waterborne diseases is related to the use of contaminated surface water. This pilot study was conducted to determine the acceptability and effectiveness of a recently developed surface water purifying mixture to prevent diarrhoeal diseases in a rural community in Bangladesh. The mixture, using a combination of alum potash, bleaching powder and lime, is added to 15 l of surface water and mixed; the water becomes suitable for drinking after 30 min. A total of 420 households from 15 villages were provided with the mixture and were taught how to use it. Episodes of diarrhoeal disease from study families were determined from hospital records of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) in Matlab and were compared with diarrhoea episodes among 1613 control families who were not provided with the mixture. A total of 83 diarrhoeal patients were treated at Matlab Hospital from 1613 control families, but only one patient was treated for diarrhoea from among the intervention families. Among the intervention families, 73 families decided to shift from using tube well water to surface water using the mixture. The mixture could be used as a cheaper, easier and simpler point-of-use water treatment strategy in Bangladesh.
Subject(s)
Diarrhea/prevention & control , Water Purification/standards , Adolescent , Adult , Alum Compounds/chemistry , Bangladesh/epidemiology , Calcium Compounds/chemistry , Child , Child, Preschool , Chlorine Compounds , Diarrhea/epidemiology , Diarrhea/microbiology , Female , Health Knowledge, Attitudes, Practice , Humans , Infant , Male , Oxides/chemistry , Pilot Projects , Rural Health , Water Purification/methods , Water Supply/standards , Young AdultABSTRACT
Acute diarrhoea remains a major public health challenge in developing countries. We examined the role of a probiotic in the prevention of acute diarrhoea to discover if there was an effect directed towards a specific aetiology. A double-blind, randomized, controlled field trial involving 3758 children aged 1-5 years was conducted in an urban slum community in Kolkata, India. Participants were given either a probiotic drink containing Lactobacillus casei strain Shirota or a nutrient drink daily for 12 weeks. They were followed up for another 12 weeks. The primary outcome of this study was the occurrence of first episodes of diarrhoea. We assessed this during 12 weeks of intake of study agent and also for 12 weeks of follow-up. There were 608 subjects with diarrhoea in the probiotic group and 674 subjects in the nutrient group during the study period of 24 weeks. The level of protective efficacy for the probiotic was 14% (95% confidence interval 4-23, P<0·01 in adjusted model). The reduced occurrence of acute diarrhoea in the probiotic group compared to nutrient group was not associated with any specific aetiology. No adverse event was observed in children of either probiotic or nutrient groups. The study suggests that daily intake of a probiotic drink can play a role in prevention of acute diarrhoea in young children in a community setting of a developing country.
Subject(s)
Diarrhea/prevention & control , Poverty Areas , Probiotics/therapeutic use , Child, Preschool , Diarrhea/epidemiology , Diarrhea/microbiology , Diarrhea/parasitology , Double-Blind Method , Female , Humans , India , Infant , Male , Nutritional Status , Urban Population/statistics & numerical dataABSTRACT
Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.
Subject(s)
Cholera Toxin/biosynthesis , Vibrio cholerae O1/classification , Vibrio cholerae O1/pathogenicity , Virulence Factors/biosynthesis , Animals , Blotting, Western , Cholera Toxin/toxicity , Culture Media/chemistry , Humans , Ileum/pathology , Rabbits , Virulence Factors/toxicityABSTRACT
Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of childhood diarrhoea in Bangladesh. Among the virulence factors of ETEC, toxins and colonization factors (CFs) play a major role in pathogenesis. Unlike Vibrio cholerae, the relationship between ETEC and ETEC-specific phages is poorly understood and the possible role of ETEC phages in the evolution of ETEC strains in the environment is yet to be established. This study was designed specifically to isolate phages that are specific for ETEC virulence factors. Among the 49 phages isolated from 12 different surface water samples, 13 were tested against 211 ETEC strains collected from clinical and environmental sources. One phage, designated IMM-001, showed a significant specificity towards CS7 CF as it attacked all the CS7-expressing ETEC. Electron microscopic analyses showed that the isolated phage possessed an isomeric hexagonal head and a long filamentous tail. An antibody blocking method and phage neutralization assay confirmed that CS7 pilus is required for the phage infection process, indicating the role of CS7 fimbrial protein as a potential receptor for IMM-001. In summary, this study showed the presence of a lytic phage in environmental water that is specific for the CS7 CF of ETEC.
Subject(s)
Coliphages/growth & development , Coliphages/isolation & purification , Enterotoxigenic Escherichia coli/metabolism , Enterotoxigenic Escherichia coli/virology , Escherichia coli Proteins/biosynthesis , Fimbriae Proteins/biosynthesis , Virulence Factors/biosynthesis , Coliphages/ultrastructure , Enterotoxigenic Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Humans , Microscopy, Electron, Transmission , Neutralization Tests , Receptors, Virus/biosynthesis , Virion/ultrastructure , Water MicrobiologyABSTRACT
During epidemics of cholera in two rural sites (Bakerganj and Mathbaria), a much higher proportion of patients came for treatment with severe dehydration than was seen in previous years. V. cholerae O1 isolated from these patients was found to be El Tor in its phenotype, but its cholera toxin (CT) was determined to be that of classical biotype. Whether the observed higher proportion of severe dehydration produced by the El Tor biotype was due to a shift from El Tor to classical CT or due to other factors is not clear. However, if cholera due to strains with increased severity spread to other areas where treatment facilities are limited, there are likely to be many more cholera deaths.
Subject(s)
Cholera/complications , Cholera/epidemiology , Asia/epidemiology , Cholera Toxin/metabolism , Disease Outbreaks , Humans , Retrospective Studies , Time Factors , Vibrio cholerae/classification , Vibrio cholerae/metabolismABSTRACT
A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men aged between 16 and 50 years from Kolkata, India. A dose of 5 x 10(9)CFU (n=186) or a placebo (n=116) containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal antibody response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively. In a subgroup, anti-CT antibody rose (> or =2-folds) in 23/30 (77%) and 6/19 (32%) in vaccine and placebo recipients, respectively. These studies demonstrate that VA1.3 at a dose of 5 x 10(9) is safe and immunogenic in adults from a cholera endemic region.