Weil Felix Test for Rickettsial Diseases: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Weil Felix Test for Rickettsial Diseases: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Introduction of Weil Felix Test for Rickettsial Diseases

Weil  Felix test is a serological assay that is used for the diagnosis of rickettsial infection by indicating immunoglobulins/antibodies in the serum. In this test,  OX -19, OX -2 strain of Proteus vulgaris, and OX-K of P. mirabilis are used instead of specific rickettsial pathogens as antigens. This test was developed from the observation that certain strains of Proteus species isolated from the patient’s urine with epidemic typhus were agglutinated by the sera of the typhus patients. This assay has been used for presumptive serological evidence of rickettsial disease.

Principle of Weil Felix Test 

Weil Felix test is an agglutination test for heterophilic antibodies (antobodies produced in certain clinical condition have the ability  to cross react with more than one antigen). It is based on the principle that many patients infected with one of the  Rickettsia produce antibodies that can be agglutinate certain strains of bacteria of genus Proteus because of the presence of a common alkali stable carbohydrate antigen. Rickettsia prowazaki and R. moonseri share alkali stable carbohydrate with Proteus vulgaris  OX 19 while  Rickettsia tsutsugamushi  with P. mirabilis OX- K  and R. rickettsia and R. conori with P. vulgaris OX2.

Requirements for Weil Felix Test 

  1. Small test tubes, tubes racks, and pipettes
  2. Physiological saline (0.85% NaCI), and P. vulgaris OX-19, OX-2 and P. mirabilis OX- K antigens.
  • Specimen- Serum

Procedure of Weil Felix Test for Rickettsial Diseases

  1. Add 2.7 ml of physiological saline in test tube.
  2. Add 0.3 ml of serum to the test tube which makes the master serum dilution as 1:10.
  3. Arrange 3 rows of the test, each row containing  7 test tubes.
  4. Add 1.0 ml of saline to all the tubes.
  5. Add 1.0 ml of diluted serum  from master dilution into the first tubes in all the three rows of the tubes.
  6. Make doubling dilution in the first row from the first tube (serum dilution 1: 20) and discard  1 ml from the 6th tube (serum dilutions  1:640). Note: Leave the 7th tube as control without serum.
  7. Similarly, make doubling dilutions in the second and third row tubes.
  8. Add 0.5 ml of concentrated Proteus  vulgaris  OX-19 to all 7 tubes in the  first row tubes.
  9. Add  0.5 ml  of concentrated  P. vulgaris OX-2 to all tubes in the seconds row tubes.
  10. Add 0.5 ml drop of concentrated P. mirabilis OX -K  to all 7 tubes in the third  row of tubes.
  11. Incubate the racks in water  bath at 37°C for 2 hours, followed by reincubating  at 4°C for  18 hours.

Quality Control

7th tube in each row acts as a negative control. The same dilutions of a known positive serum are tested with Proteus  OX 19, OX2 and OX-K antigen.


Observe the tubes after overnight incubation for agglutination and note the results. The results are studied by viewing the tubes under good light against a dark background with the help of a magnifying lens. Complete agglutination is shown by complete clearing of the supernatant and formation of white flocculent masses in the bottom of tubes. The last tubes showing is considered as the endpoint. The reciprocal of the dilution is considered as the  tire (e.g. If the dilution of the last tube showing agglutinations is 1:640, then the titer is 640)

Results –  Interpretation of Weil Felix Test for Rickettsial Diseases

A serum  titer of 1:80 and above is considered significant titer but four-fold or greater increase of antibody between acute  and convalescent sera is considered diagnostic.

  1. Agglutination with Proteus OX 19 and OX 2 is suggestive of spotted fever.
  2. Agglutination  with Proteus 0X2 is suggestive of murine typhus.
  3. Agglutination  with Proteus 0XK is suggestive of murine typhus.


  1. Weil- Felix false positive reaction may occur in urinary tract infection with Proteus, in leptospirosis, in Borrelia disease and even  in severe liver disease.
  2. The antigens are stored at 4°C prior to use.
  3. Proper precautions should be considered for the standardization of antigens. It should not be standardized against sera from rabbits immunized with a homologous strain of Proteus species, but with sera derived from patients infected with rickettsiae.
  4. The test is not helpful for the detection of antibodies in rickettsial pox, trench fever or Q fever as these persons do not develop Proteus agglutinins.
  5. Streptococcus MG agglutination test for the diagnosis of atypical pneumonia, Paul -Bunnel test for the diagnosis of Epstein Bar virus infection and, cold agglutination test for the diagnosis of primary atypical pneumonia are the examples of detecting heterophilic antibodies.

Further Reading

  1. Collins CH, Lyne PM and Grange JM, Microbiological Methods. Butterworth, London, 94-96.
  2. Koneman EW, Allen SD, Janda WM, Sschrekenbergu PC and Winn Jr. WC color Atlas and Textbook of Diagnostic Microbiology. 5th  Edition. Lippincott Williams and Wilkins. 1997: pp 1395
  3. Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Editions. 2001, pp 902
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