mr-said warsame nur

1. LECTURE -3-AGGLUTINATION TEST
FOR
FEBRILE DISEASE
M r. S a i d W a r s a m e N u r

2. Review: lecture 1 and 2
 Plasma = water + proteins + dissolved substances. It
is 90-92 percent water
 The clear liquid that can be separated from clotted
blood is called serum
 Lab diagnosis of infectious diseases
Isolation and identification of causative agent
Detection of specific Ab in sera of infected
 Serological Reactions
Primary
Secondary
Tertiary

3. Review lecture 1 and 2
• Specificity: ability of a test to identify correctly
those who do not have the disease
• Sensitivity: Ability of a test to identify correctly
those who have the disease
• Quantitative test:
– It measures the amount of Ag or Ab.
• Qualitative test :
– It detects the presence or absence of Ag or Ab.

4. Review lecture 1 and 2
• Dirty glasswares easily affect
serological test.
• Incubator and water bath are usually
used in serologic tests.
• Rotating machines are required to
facilitate antigen antibody reactions.

5. Review lecture 1 and 2
• Specimens that are used for serologic test
include: serum, plasma & cerebrospinal
fluid.
• Serum or plasma sample could be
obtained from venous blood, which can be
performe by the laboratory personnel
however.
• Cerebrospinal fluid should be collected by
a physician or a trained.

6. Review lecture 1 and 2
• The last tube that shows visible
immunologic reaction is known as end
point of the test,
• the dilution of the antiserum at the end
point is known as the titer.
• The reciprocal of the greatest reacting
dilution of the serum is considered as the
measure of titer or the concentration of
the antibody.

7. Review lecture 1 and 2
Syphilis is a sexually transmitted infection
caused by the bacterium Treponema pallidum
subspecies pallidum.
Stages of Syphilis
Primary syphilis
Secondary syphilis
Latent syphilis
Late (Tertiary) syphilis

8. Review lecture 1 and 2
The serologic methods for syphilis measure
the presence of two types of antibodies:
Treponemal
FTA-ABS
MHA-TP.
Non treponemal
VDRL
RPR

9. Learning Objective
• At the end of this lecture, the learner
should be able to:
–Explain the etiology and way of
transmission of febrile disease
–Practice widal and Weil-felix tests

10. Introduction
• Febrile agglutinins are antibodies that clump
upon exposure to their antigen, causing fever
• The 2 most clinically important antibodies are
called IgM and IgG
• IgM is produced within days of infection as part of the
primary immune response. Elevated levels of IgM
indicate a ‘new’ infection
• IgG is produced several weeks after infection as part of
the secondary immune response. Elevated levels of IgG
indicate a chronic or past infection

11. Introduction
• When any pathogenic microorganism
invades the human body, the natural
response is the production of antibodies.
• Some of the causative agents of febrile
diseases are:
– salmonella species
– rickettsial
– brucella abortus.

12. Typhoid and Paratyphoid Fever
• Salmonella of medically important
species are:
– S.typhi (typhoid fever)
–S.paratyphi A and B (paratyphoid
fever).
• Typhoid and paratyphoid fever is
transmitted through ingestion of
contaminated food or water.

13. Incubation of S.typhi and
Identification of salmonella
• incubation period ranges from 7 to 14
days. In 5% to10% of untreated patients
relapse may occur
• Salmonella species can be identified
based on their antigenic structure they
possess.

14. three different antigenic
structures of salmonella
• O- antigen (somatic antigen)-It is
lipopolysaccharide of the outer
membrane
• H-antigen (flagellar antigen)-H-antigen
is protein, which makes the
perithrchous flagella.
• Vi- Antigen-This is the antigen that
determines the virulence, the ability to
cause disease, of the organism.

15. Preparation of antigen suspension
• Salmonella antigen suspension
is available commercially and
it’s also possible to prepare in
the laboratory.

16. Widal test
• Widal test is a serological test, which is
commonly used to diagnose typhoid
and paratyphoid fever.
• The patient’s serum is tested for O and
H antibodies

17. Rapid slide (Screening) test
1-Clean the glass slides supplied in the kit
well and wipe it free of water.
2- Place one drop of undiluted test serum
in each of the first circle (1to4) and one
drop of positive control serum in each of
the last two circles.

18. Rapid slide (Screening) test
3-Place one drop of antigen O, H, A (H)
and B (H) in circle 1,2,3, &4 respectively and
O antigen in circle five and antigen in
circle 6
4-Mix the contents of each circle with
separate applicator stick and spread to
fill the whole area of the individual circle.

19. Rapid slide (Screening) test
5-Rotate the slide for one minute and
observe for agglutination.
• If agglutination is visible, quantitative
estimation of the titer of the
appropriate antibodies should be done

20. Tube agglutination method
1-Take a set of 8 clean dry test tubes for
each serum to be tested.
2-Place 1.9ml of saline in tube 1 and 1 ml of
saline in other tuber (2-8)
3-Transfer 0.1 ml of undiluted serum to
tube 1. Mix thoroughly. The resultant
dilution of serum is 1:20.

21. Tube agglutination method
5-Add one drop of appropriate antigen in
each (use only that antigen suspension
which has given a positive reaction in the
screening test).
• Note: Each antigen (O, H, AH, BH) will
require a series of 8 tubes for determine
the titer of their corresponding
antibodies.

22. Tube agglutination method
4-Further dilutions are done in the following
a) Transfer 1ml of the diluted serum from
tube 1 and place in tube 2 this leads to 1:40
dilutions in tube2
b) Repeat the transfer process for tube 7
after mixing.
c) Leave 1 ml of saline in tube 8 at the
‘saline control’
• Note. Tube 1 has a serum dilution of 1:20, 1:40
(2), 1:80 (3), and 1:160 (4), 1:320 (5),
1:640 (6) 1:1280 (7).

23. Tube agglutination method
6-Mix well and incubate overnight (16-18
hrs) at 37C0.
7-Examine agglutination macroscopically.
8-Two tubes for positive control O and it
antigen should be included.

24. Interpretation
1- Only a titer above 1:80 should be
considered as significant.
2-A rise in titer (done each week) is
considered to be definite evidence of
infection. A single test result is considered
of diagnostic value only when it is
usually high(above 160).

25. Interpretation
3-Antibiotic treatment in typhoid fever often
prevents a rise in titer,
4- A negative test does not rule out the possibility
of infection because of the tine when the blood
sample was taken in relation to the stage of the
disease
5-Positive results should always be
interpreted with reference to clinical
findings.

26. Any question