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WIDAL TEST: Widal test is a tube agglutination test employed in the serological diagnosis of enteric fever.

The test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist, born March 9, 1 !", #lgeria$ died %anuary 1&, 19"9, 'aris. Principle: 'atients( suffering from enteric fever )ould possess antibodies in their sera )hich can react and agglutinate serial doubling dilutions of *illed, colored +almonella antigens in a tube agglutination test. Requirements: Widal rac*, round,bottomed Feli- tubes, conical,bottomed .reyer(s tubes, )ater bath, doubly diluted patient serum in three,four ro)s, /illed colored suspensions of S.typhi 0 antigen, S.typhi 1 antigen, S.paratyphi #1 antigen and optionally S.paratyphi 21 antigen. Preparation of antigens: Salmonella typhi 931 strain is used to prepare S.typhi 0 and S.typhi 1 antigens. 0 antigens for S.paratyphi # and S.paratyphi 2 are not ta*en as they cross,react )ith S.typhi 0 antigen. 1 antigen suspension is prepared by treating overnight broth culture or saline suspension of +almonella )ith 3.14 formalin. For preparing 0 antigen suspension, +almonella are gro)n on phenol agar 516 337 to inhibit flagella. The gro)th is then emulsified in small volume of saline, mi-ed )ith "3 times its volume of alcohol, heated at &3o8 to 93o8 for :3 minutes and centrifuged. The antigens are treated )ith chloroform 5preservative7 and appropriate dyes are added for easy identification of antigens. Procedure: 'atient serum is doubly diluted by mi-ing and transferring from 1613 to 16!&3 in three, four ro)s. First ro) usually comprises of Feli- tubes, )here somatic S.typhi 0 antigen is added. For all the remaining ro)s, .reyer(s tubes are ta*en$ )here different flagellar 1 antigens are added. ;ach tube must contain 3.9ml of diluted serum. # test tube )ith only saline is *ept in each ro) as control. #ll the tubes 5including control7 in a ro) are mi-ed )ith 3.9ml of antigen suspension. The first ro) is treated )ith S.typhi

0 antigen, the second ro) )ith S.typhi 1 antigen, the third ro) )ith S.paratyphi #1 antigen and the fourth ro) )ith S.paratyphi 21 antigen. +ince infections by S.paratyphi 2 are rare, this antigen is usually omitted in the test. #fter all the tubes have been treated )ith specific antigen suspensions, the )idal rac* is placed in a thermostatically controlled )ater bath maintained at :<o8 for overnight incubation. #nother approach is to incubate the tubes at 93,99o8. Reading the results: The control tubes must be e-amined first, )here they should give no agglutination. The agglutination of 0 antigen appears as a =matt> or =carpet> at the bottom. #gglutination of 1 antigens appears loose, )ooly or cottony. The highest dilution of serum that produces a positive agglutination is ta*en as titre. The titres for all the antigens are noted. Slide widal test: # slide )idal test is more popular among diagnostic laboratories as it gives rapid results. ualitati!e test: 0ne drop each of undiluted patients( serum samples for the four antigens are placed on the circled card and one drop of each of the four +almonella antigens are added separately and gently rotated for one minute. #ppearance of agglutination gives ?ualitative results. To *no) the titre for each of the antigens, the test is repeated )ith dilutions of serum. uantitati!e test: 3 @l, &3 @l, "3 @l, 13 @l and 9 @l of patient(s serum each for the four antigens are placed on the circled card. To each series of serum specimen, one drop of specific antigen is added to each, mi-ed and rotated for one minute. #gglutination in each of these is noted. 3 @l corresponds to 1in "3 dilution, &3 @l to 1 in &3, "3 @l to 1 in 3, 13 @l to 1 in 1!3 and 9 @l correspond to 1 in :"3 titre. Interpretation of widal test:

Timing of test is important, as antibodies begin to arise during end of first )ee*. The titres increase during second, third and fourth )ee* after )hich it gradually declines. The test may be negative in early part of first )ee*. +ingle test is usually of not much value. # rise in titre bet)een t)o sera specimens is more meaningful than a single test. If the first sample is ta*en late in the disease, a rise in titre may not be demonstrable. Instead, there may be a fall in titre. 2aseline titre of the population must be *no)n before attaching significance to the titres. The antibody levels of individuals in a population of a given area give the baseline titre. # titre of 133 or more for 0 antigen is considered significant and a titre in e-cess of "33 for 1 antigens is considered significant. 'atients already treated )ith antibiotics may not sho) any rise in titre, instead there may be fall in titre. 'atients treated )ith antibiotics in the early stages may not give positive results. 'atients )ho have received vaccines against +almonella may give false positive reactions. This can be differentiated from true infection by repeating the test after a )ee*. True untreated infection results in rise in titre )hereas vaccinated individuals don(t demonstrate any rise in titre. Those individuals, )ho had suffered from enteric fever in the past, sometimes develop anti,+almonella antibodies during an unrelated or closely related infection. This is termed anamnestic response and can be differentiated from true infection by lac* of any rise in titre on repetition after a )ee*. #ntigen suspensions )ith fimbrial antigens may sometimes give false positive reactions due to sharing of fimbrial antigens by some ;nterobacteriaceae members. #ntigen suspension must be devoid of fimbrial antigens.