cholera 1
TRANSCRIPT
CHOLERA
Presented - BY URVASHI Moderator - DR RITU BSc.M.L.T. 3rd year
WHAT IS CHOLERA ? It is an acute diarrheal disease caused by Vibrio
cholerae. Mode of transmission: Water (infectious dose =
109), Food (infectious dose = 103) ,Person-to-person.
Incubation period: less than 24 hrs to 5 days. Secretory diarrhoea induced by enterotoxin
secreted by Vibrio cholerae.
Clinical severity varies from rapidly fatal disease to transient asymptomatic colonisation of intestine.
In its most severe form,disease is characterized by profuse, painless watery diarrhoea and effortless vomiting may lead to death in less than 24hrs.
massive loss of fluid and electrolytes. Cholera stool is typically colorless watery fluid
with flecks of mucus ‘rice water stools’.
V. CHOLERAE MORPHOLOGY Gram-negative, non sporing, non capsulated. Short, curved, cylindrical rod (1.5 x 0.2-0.4µm ) rounded
or slightly pointed ends. Typical comma shaped, but curvature lost on repeated
subculture. S shaped or spiral forms may be seen. Actively motile by a polar flagellum ‘DARTING
MOTILITY’. When examined under the microscope they suggest
‘swarm of gnats’.
.
CULTURAL CHARACTERISTICS Facultative anaerobe- strongly aerobic , growth being
scanty and slow anaerobically. Temperature range 16-40°C ( optimum 37°C). Growth better in alkaline medium ( optimum
pH 8.2). NaCl 0.5-1% - for optimal growth. ≥ 6%- inhibitory. Grows well on ordinary media.
TRANSPORT MEDIA VR- Venkatraman-Ramakrishnan medium. :- in
this medium vibrio do not multiply but remain viable for several weeks.
Cary-Blair medium :- it is suitable transport medium for salmonella and shigella as well as for vibrios.
Autoclaved sea water.
Enrichment media Alkaline peptone water. :- this is both
transport as well as transport media.
Monsur’s taurocholate tellurite peptone water pH(9.2).
SELECTIVE MEDIA TCBS(thiosuphate citrate bile sucrose agar)-
cholera vibrios produce large yellow,convex colonies.
Monsur’s tellurite taurocholate gelatin agar-colonies are small,translucent with greyish black centre and a turbid halo.
Alkaline bile salt agar-moist,translucent,1-2mm in dia. colonies,with a bluish tinge in transmitted light.(pH8.2).
VR- VENKATRAMAN-RAMAKRISHNAN MEDIUMSimple and in-expensive.vibrios do not multiply but remain viable
for several weeks. It’s constituents:-boric acid. KCl. sea salt/common salt. NaoH.pH 9.2. Cary Blair mediumSodium thioglycollate,NaCl,di sodium
hydrogen phosphate,agar,Calcium chloride.
pH-8.4.
APW-ALKALINE PEPTONE WATER
Simple and extensively used. It’s constituents:-peptone. NaCl.pH 8.6.growth occurs as fine surface pellicle in
about 6 hours.
Monsur’s tellurite taurocholate gelatin agar
Contain’s 1%each of trypticase and NaCl,sodium taurocholate,Na carbonate,agar,gelatin,potassium tellurite.
pH-8.4-9.2.
THIOSULPHATE CITRATE BILE SUCROSE AGAR(TCBS) It,s contituents are:-Yeast extract.Peptone.NaCl.Sucrose.Bromothymol blue(indicator).Agar.Sodium citrate,sodium thiosuphate.Ferric citrate.Ox bile and water.pH-8.6
CLASSIFICATION HEIBERG (1932) xlassified vibrios into 6 groups
based on the fermentati of mannos, sucrose and Arabinose.
Cholera vibrio belong to group 1. Serological classification was introduced clasified as
A vibby Gardner and Venkatraman(1935). Vibrio possesing common flagellar(H antigen) were
classified as group a vibrios, and rest as group B vibrios.
Has over 150 identified serotypes based on O-antigen.
Only O-1 and O-139 are toxigenic and cause cholera disease.
There are 2 categories of O-1 serotype-classical and El Tor.
GARDNER AND VENKATARAMAN’S CLASSIFICATION
VIBRIO
GP A GP B (common H Ag) (antigenically
heterogenous)
O1 Non O1(0-139) classical ElTor
Ogawa Inaba Hikojima
O subgroups, serogroups, serovrs
BIOTYPES
DIFFERENTIATION B/W CLASSICAL AND ELTOR VIBRIOS
Haemolysis - + Voges-proskauer - + Chick-RBC agglutination - + Polymyxin-B Sensitivity + -
ElTor phage 5
Susceptibility - +
CLASSICAL El Tor
DIAGNOSISNo clinical manifestations help distinguish
cholera from other causes of severe diarrhoea.
enterotoxigenic E.coli. viral gastroenteritis. bacterial food poisoning.
Ogawa AB Inaba AC Hikojima ABC
O antigen
LAB DIAGNOSISSAMPLES
Faecal specimen from acute cases should be collected in a screw capped container.
Moistened rectal swabs may be taken from convalescent cases.
Specimen is best collected by introducing into the rectum a lubricated catheter and letting liquid stool flow directly into a screw cap container.collection of stool from a pans is not recommended
If there is likely to be a delay of more than 6hrs.in specimen reaching the lab, preserve the specimen at 4°C or in appropriate holding medium.
Macroscopic examination:-in case of cholera ‘rice watery stool’ with mucus flakes.
Direct microscopy:- Wet preparation in saline/dark field.
Darting movement
Movements stop on adding antisera.
PROCESSING
PROCESSING
STOOL SAMPLE Direct plating APW(6-8hrs.inc)on B/A,M/A,TCBS Hanging drop plating on (darting motility) B/A,M/A, TCBS
overnight incubation at 37 °C
examine the colony characteristics
COLONY MORPHOLOGY MacConkey’s agar –colourless at first but
becomes reddish on prolonged incubation,due to late lactose fermentation.
TCBS-yellow colonies of vibrio cholerae,2-3mm in dia.
COLONY MORPHOLOGYBlood agar-colonies initially surrounded
by a zone of greening,which later becomes clear due to hemodigestion.
HEMODIGESTIONON BLOOD AGAR
Gram’s staining from the colony.
ABOVE-V.CHOLERAE ON BLOOD AGAR.BELOW-V.CHOLERAE ON MAC CONKEY AGAR..
TCBS-V.Cholerae colonies-yellow.
V.Parahaemolyticus-green.
Gram Stain shows Gram negative comma-
shaped bacteria.
BIOCHEMICAL REACTIONSOxidase - positiveCatalase - positive. Indole - positive.Nitrate reduction - positive.Decarboxylates lysine and ornithine.Gelatin-liquified ( funnel - shaped).Peptone water-fine surface pellicle.‘Cholera red reaction’- add a few drops
of conc. Sulphuric acid to the growth of vibrios in peptone water,a reddish pink colour develops due to formation of nitroso-indole.
SEROTYPING Colonies suggestive of vibrio should be tested with cholera O subgroup
1 serum.
If positive, serotyping using Ogawa and Inaba sera can be done.
Hikojima strains will agglutinate with both Ogawa and Inaba sera.
If agglutination is negative with one colony it is essential to repeat with atleast five more colonies because V. cholerae 01and non-01 vibrios may coexist in the same specimen.
STRING TEST
Loopful of growth is mixed with growth of 0.5% sodium deoxycholate on a slide.
If the test is positive,the suspension becomes mucoid and forms a “string”when loop is drawn slowly away.
Positive in V.cholerae.
CONTROL MEASURES
Hygienic disposal of human waste.Adequate supply of water.Good food hygiene
Thoroughly cooking food.Eating food while it’s hot.Preventing cooked foods from contacting raw foods (including water or ice).Avoiding raw fruits or vegetables.Washing hands properly.
TREATMENT *Even before identifying the cause of the
disease,rehydration therapy must begin immediately because death can occur within hours*
Oral or intravenous rehydration.(ORS) oral rehydration salts.
Antimicrobial therapy-doxycycline.