Bacteriological diagnosis of enteric

fever consists of:1. Isolation of bacilli2. Demonstration of antibodies3. Demonstration of circulating

antigen4. Other laboratory test

ISOLATION OF BACILLISpecimens:Blood Urine Faeces Aspirated duodenal

fluid,etc..

BLOOD CULTUREPositive in,90%-1st week75%-2nd week60% -3rd week25%-till the subsidence of pyrexia

contd…• Blood collected is cultured on glucose

&taurocholate broth..• We must take care of contamination with

external environment• Dilute or add sodium polyanethol

sulphonate• Incubated at 37degrees• Castaneda’s method is also practiced

CLOT CULTURE• Aseptically collected blood is

allowed to clot• Serum-used in widal test• Blood clot-added to bile broth

containing streptokinase(causes lysis of blood clot)

FAECES CULTURE• Salmonella are shed in the faeces

through out the disease even in convalescence

• Antibiotic does not eliminate the bacilli from the GUT as it does 4m BLOOD

CULTURE• On McConkey agar & DCA media-Pale yellow colonies

• On wilson blair media• S.typhi-black colinies(with metallic sheen)• S.paratyphi-green colonies(due to

absense of hydrogen sulphide• Enrichment media are incubated and

then subcultured on selective media

On mac conkey agar

• S.typhi • S.paratyhi

URINE CULTURE• Not much more imp as blood &

faeces culture• Positive only in 2nd& 3 rd weekes• Urine sample is centrifuged

&inoculated into enrichment &selective media

Other specimens1. Bone marrow2. Bile 3. Rose spots discharge4. Pus5. Suppurative lesions6. Csf&sputum At autopsy-gallbladder,liver,spleen

& mesenteric lymphnodes

SLIDE AGGLUTINATION TEST

• A loopful of growth from a nutrient agar is emulsified in two drops of saline on a microscopic slide

CONTD..• 1st emulsion-control• To show the strain not

agglutable• 2nd emulsion-test• Polyvalent “o”&polyvalent

“H”are added • Positive-presence of

salmonella sps• Then monovalent anti sera

belongs to S.typhi are added• If the isolate is fresh

agglutination is done with “vi”antisera,alternatively the bacterial suspension is boiled for 20 mins which removes the vi antigens

¤ Non typhoid Salmonella sproducing acid and gas 4m carboydrates is tested for “O” &”H”antisera for groups A,B,C…..

¤ NATIONAL SALMONELLA REFERENCE CENTRE at the CRI

¤ INDIAN VETERINARY RESEARCH INSTITUTE,Izatnagar

SLIDE AGGLUTINATION

DEMONSTRATION OF ANTIBODIES

WIDALTEST

PROCEDURE: DREYER”S TUBE - H agglitination FELIXTUBE - O agglutination

28 test tubes are arranged in 4 rows each row containing 7 tubes and named as

1st row-To2nd row-Th3rd row-Ah4th row-Ao

Contd….• To &Th for the detection of S.typhi• Ao &Ah for the detection of

S.paratyphi• Of the 7 tubes one acts as control

&remaining tubes as test

Contd…. • A low dilution of patient serum is

1:20 to 1:640• All tubes in rack are incubated at

37 degrees for 24hrs&results are read after over night refrigeration at 4 degrees

Contd….• Positive H agglutination-formation of

loose,cotton wooly clumps• Positive O agglutination-disc like pattern

at the bottom of tube• In both supernatent fluid is clear• Paratyphoid O antigens are not employed

because theycross react with typhoid O antigen due to their haring of factor 12…

RESULT

INTERPRETATION1. Titre depends on stage of disease2. On immunistion with TAB vaccine

individuals may show high titres of antibody

3. In non immunised individuals high titre of H agglutinin indicates entericfever or latent infection

4. ANAMNESTIC REATION:5. In carriers

DEMONSTRATION OF ANTIGENS

• Ag present in blood and urine• PREPARATION OF ANTIGENS: S.aureus is stabilised with

fprmaldehyde &then coated with S.typhi antibody

• PROCEDURE: Patients serum is mixed with staphylococcal

cells Positive-1st week..• CIEP,ELISA

OTHER….1. TLC2. DIAZO TEST IN URINE• TLC-leucopenia with a relative

lymphocytosis is found• DIAZO TEST:• Positive -in b/w 5th &14th weeks

Duration of diseaseS

Specimen examination

%positivity

1st week Blood culture 90%

2nd week Blood culture,Faeces cultureWidal test

75%50%Low titre

3rd week Widal testBlood cultureFaeces culture

80-100%60%80%

CARRIERS

Convalescent carriers Healthy carriers Chronic

carriers

DIAGNOSIS…..• SPECIMENS:stool,urine,bile,duodenal drainage Antibody ti vi antigen in serum is clamied

indicate thecarrier stateConvalescent &healthy carriers shed bacilli in

faeces from 2 months to 1 yr…Where as chronic carriers shed bacilli in faeces

from 1yr to several years

• BACTERIOPHAGE TYPING:• BIO TYPING• PLASMID TYPING

• ANTOIBIOTICS:• chloramphenicol• ampicillin• furazolidone• cotrimoxazole• For multiresistent cases:• flouroquinolones• 3rd generation cephalosporins• Resistence has been devoloped to antibiotics• In salmonella gastro enterits antibiotics should

not be used

1. Maintaining highgenic conditions2. Carriers should not be engaged in

food preparation3. Vaccination

TAB vaccine• Heat killed,whole celled,• S.typhi-1,000 millions/ml• S.paratyphi A-750 millions/ml• S.paratyphi B-750 millions/ml• Dose schedule:• Protection:• Side effects:

TYPHOID VACCINE• Live oral,• Avirulent mutent strain• S.typhi lacking UDP-galactose-4-

epimerase has been used`as live oral vaccine

• Dose schedule:• Protection:

TYPHIM-Vi• Purified Vi antigen• Dose schedule :im • singledose• 25 micro grams