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.