Widal Test

 Introduction

Widal Test is an agglutination test which detects the presence of serum agglutinins (H and O) in patients serum with typhoid and paratyphoid fever. When facilities for culturing are not available, the Widal test is the reliable and can be of value in the diagnosis of typhoid fevers in endemic areas. It was developed by Georges Ferdinand Widal in 1896

The patient’s serum is tested for O and H antibodies (agglutinins) against the following antigen suspensions (usually stained suspensions):

  • S. Typhi H antigen suspension, d
  • S. Typhi 0 antigen suspension, 9, 12
  • S. Paratyphi A 0 antigen suspension, 1, 2, 12
  • S. Paratyphi A H antigen suspension, a
  • S. Paratyphi B 0 antigen suspension, 1, 4, 5, 12
  • S. Paratyphi B H antigen suspension, b, phase 1
  • S. Paratyphi C 0 antigen suspension, 6, 7
  • S. Paratyphi C H antigen suspension, c, phase 1
Salmonella antibody starts appearing in serum at the end of first week and rise sharply during the 3rd week of endemic fever. In acute typhoid fever, O agglutinins can usually be detected 6–8 days after the onset of fever and H agglutinins after 10–12 days. It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a rising antibody titre. Salmonella antigen suspensions can be used as slide and tube techniques.
Principle of Widal Test
Bacterial suspension which carry antigen will agglutinate on exposure to antibodies to Salmonella organisms. Patients’ suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial doubling dilutions of killed, coloured Salmonella antigens in a agglutination test.
The main principle of widal test is that if homologous antibody is present in patients serum, it will react with respective antigen in the reagent and gives visible clumping on the test card and agglutination in the tube. The antigens used in the test are “H” and “O” antigens of Salmonella Typhi and “H” antigen of S. Paratyphi. The paratyphoid “O” antigen are not employed as they cross react with typhoid “O” antigen due to the sharing of factor 12. “O” antigen is a somatic antigen and “H” antigen is flagellar antigen.

Preparation of Widal Antigens
  1. H suspension of bacteria is prepared by adding 0.1 per cent formalin to a 24 hours broth culture or saline suspension of an agar culture.
  2. For preparation of O suspensions of bacteria, the organisms is cultured on phenol agar (1:800) to inhibit flagella. Standard smooth strains of the organism are used; S Typhi 901, O and H strains are employed for this purpose.
  3. The growth is then emulsified in small volume of saline, mixed with 20 times its volume of alcohol, heated at 40° C to 50° C for 30 minutes and centrifuged.
  4. The antigens are treated with chloroform (preservative) and appropriate dyes are added for easy identification of antigens.
Procedure of Widal Test
SLIDE TEST
  1. Place one drop of positive control on one reaction circles of the slide.
  2. Pipette one drop of Isotonic saline on the next reaction cirlcle. (-ve Control).
  3. Pipette one drop of the patient serum tobe tested onto the remaining four reaction circles.
  4. Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction circles. (PC & NC).
  5. Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction circles.
  6. Mix contents of each circle uniformly over the entire circle with separate mixing sticks.
  7. Rock the slide, gently back and forth and observe for agglutination macroscopically within one minute.
SEMI-QUANTITATIVE METHOD
  1. Pipette one drop of isotonic saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul of the test sample on the remaining circles.
  2. Add to each reaction circle, a drop of the antigen which showed agglutination with the test sample in the screening method.
  3. Using separate mixing sticks, mix the contents of each circle uniformly over the reaction circles.
  4. Rock the slide gently back and forth, observe for agglutination macroscopically within one minute.
STANDARD TUBE TEST METHOD (TITRATION)
In Widal Test, two types of tubes were originally used:
(1) Dreyer’s tube (narrow tube with conical bottom) for H agglutination and
(2) Felix tube (short round-bottomed tube) for O agglutination.
Now a days 3 x 0.5 ml Kahn tubes are used for both types of agglutination.

Take 4 sets of 8 Kahn tubes/test tubes and label them 1 to 8 for O, H, AH and BH antibody detection.
  1. Pipette into the tube No.1 of all sets 1.9 ml of isotonic saline.
  2. To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
  3. To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested and mix well.
  4. Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
  5. Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix well. Continue this serial dilution till tube no.7 in each set.
  6. Discard 1.0 ml of the diluted serum from tube No.7 of each set.
  7. Tube No.8 in all the sets, serves as a saline control. Now the dilution of the serum sample achieved in each set is as follows: Tube No. : 1 2 3 4 5 6 7 8 (control) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280.
  8. To all the tubes (1 to 8) of each set add one drop of the respective WIDALTEST antigen suspension (O, H, AH and BH) from the reagent vials and mix well.
  9. Cover the tubes and incubate at 37° C overnight (approximately 18 hours).
  10. Dislodge the sedimented button gently and observe for agglutination
Interpretation of Widal Test- Tube Method
The titre of the patient serum using Widal test antigen suspensions is the highest dilution of the serum sample that gives a visible agglutination.
The sample which shows the titre of 1:100 or more for O agglutinations and 1:200 or more for H agglutination should be considered as clinically significant (active infection). Demonstration of 4-fold rise between the two is diagnostic. H agglutination is more reliable than O agglutinin.
Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre remains steady till 4th week after which it declines.

Limitations of Widal Test
  1. The Widal test is time consuming (to find antibody titre) and often times when diagnosis is reached it is too late to start an antibiotic regimen.
  2. The Widal test should be interpreted in the light of baseline titers in a healthy local population.
  3. The Widal test may be falsely positive in patients who have had previous vaccination or infection with S. Typhi.
  4. Besides cross-reactivity with other Salmonella species, the test cannot distinguish between a current infection and a previous infection or vaccination against typhoid.
  5. Widal titers have also been reported in association with the dysgammaglobulinaemia of chronic active hepatitis and other autoimmune diseases.
False positive Widal test results are also known to occur in typhus, acute falciparum malaria
(particularly in children), chronic liver disease associated with raised globulin levels and disorders such as rheumatoid arthritis, myelomatosis and nephrotic syndrome. False negative results may be associated with early treatment, with “hidden organisms” in bone and joints, and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic.

False negative Widal tests may be due to antibody responses being blocked by early antimicrobial treatment or following a typhoid relapse. Severe hypoproteinaemia may also prevent a rise in 0 and H antibody titres. The antibody levels found in a healthy population however, may vary from time to time and in different areas, making it difficult to establish a cut off level of baseline antibody in a defined area and community. In low typhoid endemic areas, weak and delayed O and H antibody responses limit the usefulness of the Widal test. Variations also exist between laboratories in the performance and reading of Widal tests which compromise further the reliability of the test.
The World Health Organization (WHO) has said that due to the various factors that can influence the results of a Widal test, it is best not to rely too much on this test.


Comments

Post a Comment

Popular posts from this blog

Gram Stain

Image
What is Gram Staining? Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process. How Does Gram Staining Work? Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker pept...
Read more

White blood cells

Image
Granulocytes  Are phagocytes, which have the ability to ingest viruses, bacteria and other parasites. They have visible granules or grains in their cytoplasm and have large elongated or lobed nuclei. The  diameter of cell measures approximately from (12 – 20) μ, and their nucleoli cannot be seen. They account for approximately 60% of our WBCs. The sub-types of granulocytes are: neutrophil, basophil and eosinophil.    Neutrophils   are a part of the innate immune system and an essential line of defense against bacteria. The shape of  nucleus is like a “U” or a curled rod prior to segmentation. They are also known as “band neutrophils”. The diameter usually ranges between (10–18)μ. The cytoplasm is moderate to abundant with a few non-specific granules. Neutrophils account  for  approximately  (1%–3%) of the peripheral WBCs. The diameter of a segmented neutrophil cell  usually ranges between (9–16)μ. They have a multi-lobed nucleus (three o...
Read more

Laboratory Disinfectants.

Image
This post describes the use of disinfectants for routine laboratory decontamination of surfaces and equipment. Definitions:   Disinfectants : Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on the objects. Microorganisms : A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms. Antiseptic : Any substance that inhibits the growth and reproduction of microorganisms. Generally includes only those that are used on living objects (as opposed to disinfectants) and aren’t transported by the lymphatic system to destroy bacteria in the body (as opposed to antibiotics ). Decontamination – A process that removes the total burden of all classes of microorganisms, usually using chemicals, heat, and/or pressure.  Disinfection – A process that reduces microbial burden on a surface or object.  Inactivation – The pro...
Read more

Laboratory diagnosis of urinary tract infection

Image
Definition Urinary tract infection (UTI) is the active infection in any part of urinary tract beyond distal urethra which is normally bacteriologically sterile. Causative Agents A large number of organisms gaining access to urinary tract are capable of causing UTI (Table 1). These organisms may reach the urinary tract through ascending route or by haematogenous route. Table 1 : Causative agents of UTI Factors Predisposing to UTI Various bacterial attributes and host factors favour urinary tract infections. Mechanical factors are important. Anything that disrupts normal urine flow or complete emptying of the bladder, or facilitates access of organisms to bladder, will predispose an individual to UTI. Clinical Specimen: Collection and Transportation Mid-stream sample of urine is the ideal specimen for the diagnosis of UTI. First part of the urine washes away the surface commensals from the distal urethra and hence the midstream specimen indicates actual bacteriological pi...
Read more

Morphological classification of medical important bacteria

Image
Morphological classification of medical important bacteria Morphologically bacteria can resemble: Cocci (Singular: coccus) Bacilli / rods (Singular: rod, bacillus) Vibrios (Singular: vibrio) Spirilla (Singular: spirillum) Spirochaetes (Singular: spirochaet Cocci : These are round or oval bacteria measuring about 0.5–1.0 m in diameter. When multiplying, cocci may form pairs, chains, or irregular groups:   cocci in pairs are called diplococci, e.g. meningococci and gonococci. cocci in chains are called streptococci, e.g. Streptococcus pyogenes. cocci in irregular groups are called staphylococci, e.g. Staphylococcus aureus. Gram reaction : Staphylococci and streptococci are Gram positive, whereas diplococci can be Gram positive or Gram negative. Rods (bacilli): These are stick-like bacteria with rounded, tapered (fusiform), square, or swollen ends. They measure 1–10 µ m in length by 0.3–1.0 µ m in width. The short rods with rounded ends are often called coccobacilli. When mul...
Read more

Autoimmune disease

Image
  What is an autoimmune disease? An autoimmune disease is a condition in which your immune system mistakenly attacks your body tissues and cells as foreign and launches an inappropriate immune response. The immune system normally guards against germs like bacteria and viruses. When it senses these foreign invaders, it sends out an army of fighter cells to attack them. Normally, the immune system can tell the difference between foreign cells and your own cells. In an autoimmune disease, the immune system mistakes part of your body, like your joints or skin, as foreign. It releases proteins called autoantibodies that attack healthy cells. Some autoimmune diseases target only one organ. Type 1 diabetes damages the pancreas. Other diseases, like systemic lupus erythematosus (SLE), affect the whole body. Why does the immune syste attack the body? Doctors don’t know exactly what causes the immune-system misfire. Yet some people are more likely to get an autoimmune disease than others. Ac...
Read more

Coagulase test

Image
Coagulase Test- Principle, Procedure, Types, Result, Uses Coagulase Test Definition A coagulase test is a biochemical test that is used to differentiate Staphylococcus aureus from other Staphylococci species like S. epidermidis and S. saprophyticus on the basis of the ability to produce the coagulase enzyme. The coagulase test is an important test that differentiates the species of the genus Staphylococci into two groups; Coagulase positive Staphylococci and Coagulase Negative Staphylococci. The coagulase enzyme acts as a virulence factor in some organisms as it interacts with the fibrinogen present on the host’s cell surface. Organisms with coagulase usually have a protective barrier around themselves, increasing their pathogenicity and resistance against the immune system. Coagulase is of two types; free coagulase and bound coagulase, each of which is detected by different methods. The bound coagulase is called the clumping factor and is detected rapidly by a slide test. The free c...
Read more