cholera

Recent Advances in Cholera : 

Recent Advances in Cholera Ashima Jain PG 2nd yr Dept. of Microbiology

Contents : 

Contents Historical aspect Taxonomy Epidemiology Virulence factors & Pathogenesis Clinical manifestations Diagnosis Typing methods Prevention Treatment

Historical aspect : 

Historical aspect V. cholerae first described and named by PACINI in 1854 who observed curved bacilli on denuded epithelium and in intestinal contents of Cholera patients in Florence. ROBERT KOCH : Isolated the organism in 1883 in Egypt and called it Kommabazilen based on its characteristic shape. JOHN SNOW : Suggested the disease to be water borne

Taxonomy : 

Taxonomy Family VIBRIONACEAE Vibrio Aeromonas Earlier Classification Photobacterium Plesiomonas Recent Modifications Aeromonas Aeromonadaceae Plesiomonas Enterobacteriaceae

Continued… : 

Continued… Family Vibrionaceae Genera: Allomonas Vibrio Shewanella Grimontia Listonella Photobacterium Salinivibrio Enterovibrio (Bergey’s Manual of Systematic Bacteriology.Release1.0., Springer–Verlag, 2001) V. anguillarum (fish pathogen) Listonella anguillarum V. damsela Photobacterium damselae (only pathogenic member of this genus)

Natural Habitat : 

Natural Habitat Vibrio spp. (including V. cholerae) grow in estuarine and marine environments worldwide Can survive and replicate in contaminated waters with increased salinity and at temperatures of 16-40o C Asymptomatically infected humans : reservoir in endemic regions Resistance Destroyed at 55ºC in 15 min Dried on linen : survive for 1-3 days On cover slips : 3 hrs Clean tap water : 30 days On fruits: 1-5 days at RT 1 week in refrigerator Gastric acidity acts as an important barrier against infection but may survive for 24 hrs in achlorhydric gastric juice

Classification of Vibrio cholerae : 

Classification of Vibrio cholerae Classical El Tor Each O1 biotype can have 3 serotypes V. Cholerae sp. is classified on basis of somatic O-Ag : 206 serogroups

Epidemiology : 

Epidemiology

Slide 10: 

Cholera in India: an analysis of reports, 1997–2006 S Kanungo et al. Bulletin of the World Health Organization 2010;88:185-191

Slide 11: 

There are an estimated 3–5 million cholera cases and 100 000–120 000 deaths due to cholera every year Recent Cholera Outbreaks Zimbabwe : August 2008, affecting all provinces in the country Iraq : August 2008 India : August 2007, Orissa

Cholera Pandemics : 

Cholera Pandemics

Continued… : 

Continued… 1st six pandemics Caused by classical strain Originated from India Very severe cases with high mortality rates 7th pandemic Caused by El Tor Originated from outside Indian subcontinent (Sulawesi, Indonesia in 1961) Severity less, with many mild/ asymptomatic case, low mortality and high carrier rate

The 8th Pandemic ? : 

The 8th Pandemic ? In 1992 December, a new epidemic began in the coastal areas of the Bay of Bengal It was not related to the 138 known serotypes Thus the term Bengal was given as a synonym of the O139 serotype Spread eastwards to South East Asia Spread westwards to Pakistan, Afghanistan, China and some parts of Europe Imported cases have also been reported from US and UK

Origin of O139 : 

Origin of O139 Virulence factors CT & TCP of O139 are indistinguishable from O1 El Tor The sequenced wb* regions of O1 & O139 have gmhD gene at left junction The sequenced right junction of O1 wbe cluster has a 30-bp overlap with that of O139 wbf right junction The intervening regions are divergent in the two serogroups except for the presence of an insertion sequence IS1358 The entire region shown here is 41,221 bp in length, of which the O139-specific wbf region is 35,807 bp in length, starting from wbfA to wbfX.

Differences between O1 & O139 : 

Differences between O1 & O139 Deletion of wbe (rfb) region (22 kb) of O1 Replacement by 35 kb wbf region encoding O139 O1 O139 At genetic level

Atypical El Tor Strains : 

Atypical El Tor Strains Mix of both classical and El Tor traits Matlab variants : Isolated between 1991-94 in Matlab, Bangladesh Mozambique Variants : Cholera outbreak in 2004 in Mozambique, East Africa Recently, have also been isolated in Vietnam (1995-2004) Altered El Tor : Isolated in Bangladesh after 2001 In Vietnam (2007-2008) Hybrid El Tor : Asian and African countries (1991-2004) other than India, Bangladesh and Mozambique

The changing epidemiology of Cholera : 

The changing epidemiology of Cholera 1992 – 1994 O139 strain dominated 1994-1996 New variant of O1 in India, northern and central Bangladesh In southern coastal regions , O139 continued Aug1996-Sept 1997 New O139 variant in India Both O1 & O139 cases in Bangladesh (late 1995 through 1996) O139 outbreak in Dhaka,2002

Modes of Transmission : 

Modes of Transmission Vibrio cholerae is spread mainly via the faeco-oral route. Drinking water that has been contaminated at its source, during storage or usage Contaminated foods, vegetables that have been fertilised with human excreta (nightsoil) or "freshened" with contaminated water Soiled hands can also contaminate clean drinking water and food Seafood , particularly shellfish taken from contaminated water and eaten raw or insufficiently cooked

Virulence Factors : 

Virulence Factors Toxins : Cholera toxin Zot toxin Ace toxin Hemolysin/cytolysin Shiga-like toxin ST New Cholera Toxin Sodium channel Inhibitor Colonization Factors : Toxin Coregulated Pilus Accessory colonization factor MFRHA (D-mannose, L-fucose resistant HA) MSHA (Mannose sensitive HA) Core encoded Pilin Soluble HA/ Protease OMP Motility LPS Polysaccharide capsule Neuraminidase

Cholera Toxin : 

Cholera Toxin Genes encoding Cholera Toxin (ctxAB) reside in the genome of a lysogenic bacteriophage ctx Ø Mol wt 84000 kDa Consists of five identical B subunits and a single A subunit (AB5) A subunit is proteolytically cleaved into A1 and A2 Neither of the subunits have any secretogenic activity by themselves

Slide 22: 

[Galβ1-3GalNAcβ1(NeuAcα2-3)-Glc-ceramide]

Alternate mechanisms of action : 

Alternate mechanisms of action Prostaglandins : Cholera patients in the active secretory disease stage have elevated jejunal concentrations of prostaglandin E2 (PGE2) Addition of CT increases both cAMP and PGE in rabbit loops and in CHO cells, resulting in the release of arachidonic acid from membrane phospholipids Enteric Nervous System : CT binds to enterochromaffin cells which release serotonin which activates dendrite like structures located beneath the intestinal epithelium. This leads to the release of VIP, resulting in electrolyte and fluid secretion

Other Toxins produced by V. Cholerae : 

Other Toxins produced by V. Cholerae Zot : Toxin that increases the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction, or zonula occludens Zot might cause diarrhea by leakage of water and electrolytes into the lumen under the force of hydrostatic pressure Ace : Increases potential difference rather than tissue conductivity. Strains containing the cloned ace gene caused significant fluid accumulation in rabbit ligated ileal loops. The ace and zot genes are almost always found in strains containing ctx but are rarely found in strains lacking ctx.

Continued… : 

Continued… Hemolysin/cytolysin : The hemolysin/cytolysin is initially made as an 82-kDa protein and processed in two steps to a 65-kDa active cytolysin Genes encoding this hemolysin, hlyA, are present in classical, El Tor, and non-O1 strains of V. Cholerae with no correlation seen with the presence of ctx sequences The purified hemolysin is capable of causing fluid accumulation in ligated rabbit ileal loops The accumulated fluid produced in response to hemolysin is invariably bloody with mucous

Slide 26: 

Genes encoding the three distinct toxins are located in tandem on a dynamic region of the V. cholerae chromosome This region was recently shown to also contain a gene encoding an intestinal colonization factor, cep (core encoded pilin) . Thus, this region may be perceived as a ‘‘virulence cassette’’ of V. cholerae

Toxin Co-regulated Pilus (TCP) : 

Toxin Co-regulated Pilus (TCP) TCP is the only colonization factor of V. cholerae whose importance in human disease has been proven Expression of the pilus correlates with expression of CT Intestinal colonization by V. cholerae was found to be abolished when the gene encoding the TCP subunit (tcpA) was inactivated in a derivative of the classical Ogawa strain 395 TCP has been studied almost exclusively in V. cholerae O1 TCP is expressed by V. cholerae O139 when strains are grown under conditions that favor expression of TCP in El Tor V. cholerae O1 The tcpA gene sequence of V. cholerae O139 is identical to that of an El Tor strain

Clinical Manifestations : 

Clinical Manifestations Incubation period: 1-5 days High infectious dose: >108 CFU 103 -105 CFU with achlorhydria or hypochlorhydria (lack of or reduced stomach acid) Abrupt onset of vomiting and life-threatening watery diarrhea (15-20 liters/day) As more fluid is lost, feces-streaked stool changes to rice-water stools: Colorless Odorless No protein Speckled with mucus

Continued.. : 

Continued.. Cholera Gravis Loss of 1 liter of fluid/hour >10% of body weight Hypotension within 1 hour (usually 4-12 hours) Death if untreated within 2 hours (usually >18 hours) Complications Hypokalemia Cardiac arrhythmia & renal failure Hypovolemic shock Metabolic acidosis

Diagnosis : 

Diagnosis

General characteristics : 

General characteristics Gram negative, short, curved rods (comma shaped) Curvature lost on subculture Motile with polar flagella Mucous flakes stained films : ‘fish in stream’ appearance Acute cholera stool/young culture : actively motile vibrios ‘swarm of gnats’ Strongly aerobic (scant, slow growth anaerobically) Temp. range 16-40oC pH : 7.4 – 9.6 (optimum 8.2) Optimal growth on most non selective media containing 0.5 -1% NaCl (>= 6% inhibitory for V. cholerae)

Culture media for Vibrio : 

Culture media for Vibrio Holding/Transport Media: Venkatraman - Ramakrishnan medium Cary-Blair medium Autoclaved Sea Water Enrichment Media: Alkaline Peptone water Monsur’s taurocholate Tellurite Peptone Water Plating Media: Mac Conkey agar Blood agar Alkaline Bile Salt Agar (BSA) Monsur’s Gelatin Taurocholate trypticase Tellurite Agar (GTTA) Thiosulphate Citrate Bile salt Sucrose medium (TCBS)

Biochemical Reactions : 

Biochemical Reactions Oxidase +ve Catalase +ve Indole +ve Nitrate +ve Carbohydrate metabolism : fermentative producing acid without gas MR –ve Urease –ve Gelatin liquefaction +ve Lysine decarboxylase +ve Ornithine decarboxylase +ve Arginine dihydrolase –ve

Newer Diagnostic Techniques : 

Newer Diagnostic Techniques Antigen Detection : Rapid Immunochromatographic Dipstick Kit Crystal VC (Span Diagnostics, Surat) for determination of LPS Ag of both O1 and O139 using Monoclonal antibodies specific to O1 and O139 Monoclonal Ab based, coagglutination test against O1 Ag (Cholera Screen; New Horizons Diagnostics Corp., Columbia) A colloidal gold based colorimetric immunoassay, Cholera SMART kit, a modification of Cholera Screen Fluorescent Ab technique using O1 antisera used to identify V. Cholerae O1 in stool and water samples

Continued… : 

Continued… Molecular Techniques : PCR for ctx gene sequences DNA probes used to distinguish strains with genes encoding CT (ctx) from those that do not have them Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the ctxA gene. positive sample visible, white precipitate Mg2+ pyrophosphate isothermal conditions (60-64 °C) for 60 min The presence or absence of the white precipitate allows the easy assessment of samples with the naked eye

Toxin Assays : 

Toxin Assays ELISA using GM1 ganglioside receptor as capture molecule Bead ELISA using Polystyrene beads coated with anti-CT antibody as solid phase Cell culture assays using Chinese Hamster Ovary (CHO) cells Latex agglutination assay : less complicated & less time consuming than ELISA

Toxin identification: Immunochemistry : 

Toxin identification: Immunochemistry The Reversed Passive Latex Agglutination (RPLA) Toxin Detection Kit relies on Latex particles sensitized with antibodies to Cholera toxin. In the presence of the toxin, the latex particles agglutinate to form a clearly visible lattice structure. Sensitivity 97%, specificity 100%

Typing : 

Typing Serotyping Biotyping Phage typing Molecular typing

Serotyping : 

Serotyping The H flagellar antigen is common to all Vibrio sp. Hence, the O antigen forms the basis for most Vibrio typing schemes Sakazaki & Shimada scheme : sera raised against heat killed organisms Smith scheme : sera against live organisms Siebeling scheme : same as Sakazaki & Shimada

Biotyping : 

Biotyping

continued.. : 

continued.. Differences in single gene clusters : VSP-І & ІІ ( Vibrio Seventh Pandemic Island І & ІІ) RTX ( Repeat In Toxin) Other genes used : tcpA (Toxin Co-regulated Pilin A) ctxB (Cholera Toxin B) rstR (Repeat Sequence Transcriptional regulator) CTB epityping

Biotypes Phenotypes Genotypes : 

Biotypes Phenotypes Genotypes

Phage typing : 

Phage typing Used for differentating between El tor and classical biotypes Also been developed for O139 serogroup The international phage typing scheme of Basu and Mukerjee includes five phages (I, II, III, IV, and V) In 1993, Chattopadhyay et al proposed five new phages (N4,S5,S20,M4 & D10) to increase the discrimination. Use of lysogenic bacteriophage has been done to distinguish strains on the basis of chromosomal integration sites

Cholera Phage (ctxФ) in Toxigenic V. Cholerae : 

Cholera Phage (ctxФ) in Toxigenic V. Cholerae Core ctxAB gene encoding CT Consist of 5 other genes required for phage morphogenesis psh Core encoded pilin(cep) pIII ctx Accessory Cholera enterotoxin (Ace) Zonula Occludens (Zot) RS2 region Encodes proteins with roles in rstA (replication) rstB (Integration) rstR (regulation of site specific recombination of ctxφ on the V. cholerae chromosome)

Molecular typing : 

Molecular typing ctx gene variation : RFLP analysis using ctx (Gene encoding CT) gene probes detect sequence divergence in genes flanking the ctx locus to differentiate between strains Multilocus Enzyme Electrophoresis (MEE) : Variations in the electrophoretic mobility of several enzymes have been found in V. Cholerae strains which can divide them into multiple electrophoretic types (ETs) and distinguish the El Tor biotypes rRNA gene variation : Examination of RFLPs in rRNA genes has been reported to distinguish the 2 biotypes. rRNA RFLP analysis yielded greater diversity among the El Tor isolates studied than did MEE

Prevention : 

Prevention Hygienic disposal of human waste Adequate supply of water Good food hygiene Thoroughly cooking food Eating food while its hot Preventing cooked foods from contacting raw foods (including water or ice) Avoiding raw fruits or vegetables Washing hands after defecation & before cooking

Cholera Vaccines : 

Cholera Vaccines

Continued.. : 

Continued.. Cholera vaccination is NOT recommended, and vaccines currently available DO NOT help in controlling cholera because : Vaccines lack the required potency and have low rate of protective efficacy Vaccines provide immunity of limited duration only They do not reduce the rate of asymptomatic infections, and prompt a false sense of security Due to these limitations, in 1973 the 26th World Health Assembly abolished the requirement in the International Health Regulations for a certificate of vaccination against cholera.

Future Cholera Vaccines : 

Future Cholera Vaccines Attenuated Vaccines : A live attenuated single dose oral vaccine developed in Finlay Institute at Cuba, already tested in Phase 1 trials New attenuated strains of serogroup O139 for use as oral vaccines (CVD112 and Bengal 15) are also under development Use of attenuated strains of V. Cholerae as expression vectors to deliver antigens directly to mucosal surfaces is also being explored Some of the live attenuated V. Cholerae O1 and O139 vaccines have undergone removal of the entire toxin genetic element (the filamentous phage ctxφ ) including the attRS1 site (site of reintegration) which decreases the chances of the vaccine strain becoming toxigenic again

Continued.. : 

Continued.. Recombinant Vaccine : A live oral recombinant vaccine strain (Peru 15) already tested by Avant Immunotherapeutics (USA) and Biosidus S.A. (Argentina) in Phase 2 trials Others : An injectable O-Antigen conjugated vaccine in preclinical phase at Pasteur Institute in Paris A DNA vaccine to be given i/m, in preclinical phase at Putra University in Malaysia Live combination vaccines ( V. Cholerae O1 and O139; V. Cholerae O1 ElTor and Classical biotypes) are also being considered

Treatment : 

Treatment

Steps in the Management of Suspected Cholera : 

Steps in the Management of Suspected Cholera Step 1: Assess dehydration Step 2: Rehydrate the patient, and monitor frequently, and reassess hydration status Step 3: Maintain hydration: replace continuing fluid losses until diarrhea stops.

Need for Antibiotics : 

Need for Antibiotics Antibiotic management is not recommended for cholera patients, including severe cases. No antidiarrhoeal, anti-emetic, antispasmodic, or corticosteroid drugs should be used to treat cholera. Blood transfusion and plasma volume expanders are not necessary. However, they reduce by about 50% the duration of illness, diarrhea volume and the rehydration requirements.

Antibiotic treatment reduces the cost and effort required to deal with an outbreak. : 

Antibiotic treatment reduces the cost and effort required to deal with an outbreak.

Antibiotic Resistance : 

Antibiotic Resistance Antibiotic-resistant Vibrio cholerae 01 should be suspected if diarrhoea continues after 48 hours of antibiotic treatment. Antimicrobial resistance is usually encoded by conjugative plasmids Strains in East Africa, Bangladesh and India can be taken to be tetracycline resistant unless results of susceptibility testing are available Some isolates have also been resistant to Chloramphenicol, TMP-SMX, and Furazolidione

Patterns of Drug Resistance in O139 : 

Patterns of Drug Resistance in O139 In last 9 yrs, Strains remained susceptible to Ciprofloxacin, Tetracycline and Gentamycin Susceptibility to ampicillin, TMP-SMX, Chloramphenicol, and streptomycin varied In 1992-93, strains in both India and Bangladesh were Tetracycline- sensitive TMP-SMX, streptomycin - resistant In 1997, TMP-SMX & streptomycin - sensitive Since 1997,O139 strains in India showed increasing trend of resistance to Ampicillin, neomycin and susceptibility to Chloramphenicol and Streptomycin

Slide 59: 

Thank You