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 0.2-0.4m ) 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 16-40C ( optimum 37C). 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. medium :- it is suitable transport medium for salmonella and shigella as well as for vibrios.

Cary-Blair

Autoclaved

sea water.

Enrichment media
Alkaline

peptone water. :- this is both transport as well as transport media. pH(9.2). taurocholate tellurite peptone water

Monsurs

SELECTIVE MEDIA
TCBS(thiosuphate

citrate bile sucrose agar)cholera vibrios produce large yellow,convex colonies. tellurite taurocholate gelatin agarcolonies are small,translucent with greyish black centre and a turbid halo.

Monsurs

Alkaline

bile salt agar-moist,translucent,1-2mm

in dia. colonies,with a bluish tinge in transmitted light.(pH8.2).

VR- VENKATRAMAN-RAMAKRISHNAN MEDIUM Simple and in-expensive. vibrios do not multiply but remain viable for several weeks. Its constituents:-boric acid. KCl. sea salt/common salt. NaoH. pH 9.2. Cary Blair medium Sodium thioglycollate,NaCl,di sodium hydrogen phosphate,agar,Calcium chloride. pH-8.4.

APW-ALKALINE PEPTONE WATER

Simple

and extensively used. Its constituents:-peptone. NaCl. pH 8.6. growth occurs as fine surface pellicle in about 6 hours. Monsurs tellurite taurocholate gelatin agar Contains 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 VENKATARAMANS

CLASSIFICATION

VIBRIO GP A GP B (common H Ag) (antigenically heterogenous)

O subgroups, serogroups, serovrs

O1

Non O1(0-139)

BIOTYPES

classical ElTor
Hikojima

Ogawa Inaba

DIFFERENTIATION B/W CLASSICAL AND ELTOR VIBRIOS

CLASSICAL El Tor

Haemolysis Voges-proskauer Chick-RBC agglutination Polymyxin-B Sensitivity ElTor phage 5

+ + + -

Susceptibility

DIAGNOSIS
No

clinical manifestations help distinguish cholera from other causes of severe diarrhoea. enterotoxigenic E.coli. viral gastroenteritis. bacterial food poisoning.

O antigen

Ogawa Inaba Hikojima

AB AC ABC

LAB DIAGNOSIS
SAMPLES 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 4C or in appropriate holding medium.

PROCESSING

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

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

MacConkeys 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 MORPHOLOGY
Blood

agar-colonies initially surrounded by a zone of greening,which later becomes clear due to hemodigestion.

HEMODIGESTION ON BLOOD AGAR

Grams

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 commashaped bacteria.

BIOCHEMICAL REACTIONS
Oxidase

- positive Catalase - 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 stringwhen 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 its 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. therapy-doxycycline.

Antimicrobial