BRUCELLA

AGGLUTINATION
TEST
PRESENTED BY:
NEETU AMATYA
B.Sc.MLT 3rd YEAR
JFIHS/LACHS
 CONTENTS
 INTRODUCTION TO BRUCELLA

 BRUCELLOSIS

 BRUCELLA AGGLUTINATION TEST

A.QUALITATIVE SLIDE AGGLUTINATION TEST (SCREENING TEST)

B.SEMI-QUANTITAIVE SLIDE AGGLUTINATION TEST( TITRATION)

C. SEMI-QUANTITATIVE TUBE AGGLUTINATION TEST(TITRATION)

 LIMITATIONS OF PROCEDURE
 INTRODUCTION TO BRUCELLA

• Brucellae are small, facultative, intracellular, nonmotile, non-

capsulated ,aerobic, gram-negative coccobacilli or short rods.

CAUSATIVE AGENT:
• Brucella abortus
• Brucella melitensis
• Brucella suis
• Brucella canis
 BRUCELLOSIS
• Brucellosis is a zoonotic
disease,primarily affecting
goats, sheeps, cattle,
buffaloes, pigs .

• Transmitted to human by
contact with infected animal
or through their products
(through raw milk, fresh
cheese, cream or other milk
products), enter damaged skin
or the eye, or are inhaled in
aerosols.
• Human infection may be of three types:

1. Latent infection with only serological but no clinical

evidence;
2. Acute or subacute brucellosis; and
3. Chronic brucellosis.
Clinical Description
Council of State and
Territorial Epidemiologists
(CSTE)1 2010 Case Definition

• An illness characterized by acute or

insidious onset of fever and one or
more of the following: night sweats,
arthralgia, headache, fatigue,
anorexia, myalgia, weight loss,
arthritis/spondylitis, meningitis, or
focal organ involvement
(endocarditis, orchitis/epididymitis,
hepatomegaly, splenomegaly).
 Seroconversion during brucellosis

• Brucella-specific agglutination tests involve direct agglutination of

bacterial antigens by specific antibodies. Agglutination tests detect
antibodies of IgM, IgG, and IgA classes.

• IgM antibodies are predominant in acute infection but decline within

weeks. Relapses are accompanied by transient elevations of IgG and IgA
antibodies but not IgM.
BRUCELLA AGGLUTINATION TEST

A. QUALITATIVE SLIDE AGGLUTINATION TEST

(SCREENING TEST)

B. SEMI-QUANTITAIVE SLIDE AGGLUTINATION

TEST( TITRATION)

C. SEMI-QUANTITATIVE TUBE AGGLUTINATION

TEST(TITRATION)
A. QUALITATIVE SLIDE AGGLUTINATION TEST
(SCREENING TEST)

Pipette/drop onto Cell 1 Cell 2 Cell 3

separate cells of the
slide(1drop+50μl)
1. PC 1 drop _ _

NC _ 1 drop _
SPECIMEN _ _ 1 drop
2. AG next to control & 1 drop 1 drop 1 drop
control
3. Mix with separate, disposable sticks and spread the fluid over the entire area of the particular
cell.
4. Tilt slide back and forth for 1 min or place on automated rotator at 100rpm.

5.At the end of rotation read results under bright artificial light.
 Interpretation:

Positive: Distinct agglutination within

1 min after rotation.

Negative: No agglutination

NOTE:
Positive sera (visible clumping) may be
titrated by tube agglutination.
Note:
• This is a useful rapid screening test to detect a reactive
serum which requires titrating.

• It should not replace the standard tube agglutination

test nor be used to determine the Brucella antibody
titre.

• To avoid a false negative result due to the prozone

phenomenon both neat and diluted serum samples should
be tested.
B. SEMI-QUANTITAIVE SLIDE AGGLUTINATION
TEST( TITRATION)

Prepare serum dilutions in saline( 9gm Nacl/l) a/c to the following scheme:

SLIDE 1 2 3 4 5 6

Dilution 1/2 1/4 1/8 1/16 1/32 1/64

Saline 100μl 100μl 100μl 100μl 100μl 100μl

Serum 100μl

Mix + 100μl 100μl 100μl 100μl 100μl 100μl

transfer discard
Continue test as described under A, employing each dilution as specimen.
Examine macroscopically for the presence / absence of distinct agglutination within 1 min after
  Interpretation

• The result will be read at the highest dilution still

showing an agglutination.
C. SEMI-QUANTITATIVE TUBE AGGLUTINATION
TEST(TITRATION)

• The standard agglutination test (SAT) is confirmatory serological test

to diagnose brucellosis.

• SAT is the reference method, of which BMAT (Brucella

microagglutination test) is a modified format.

• This is a tube agglutinationtest in which equal volumes of serial dilutions

of the patient's serum and the standardised antigen (a killed suspension
of a standard strain of Br. abortus) are mixed and incubated at 37 °C
for 24 hours or 50 °C for 18 hours.
• A titre of 160 or more is considered significant.

• Most patients with acute brucellosis develop titres of

640 or more by 3-4 weeks of illness.

• Titres tend to decline after the acute phase of the

illness.
1.Prepare for each antigen to be tested a series 6 tubes and 2 tubes for the controls

2. Prepare a row of serum dilutions in saline (9gm Nacl/l) a/c to following scheme:

TUBE 1 2 3 4 5 6

DILUTION 1/20 1/40 1/80 1/160 1/320 1/640

SALINE 1.9 ml 1.0ml 1.0 ml 1.0ml 1.0ml 1.0ml

SERUM 100μl _ _ _ _ _

MIX + 1 ml 1ml 1ml 1ml 1ml 1ml

TRANSFER

3. Add to specimen dilutions and controls AG 1drop each

4. Close tubes with stoppers or sealing tape and mix thoroughly

5. Incubate for 24 hrs at 37 °C

6. Examine macroscopically the pattern of agglutination and compare it with the results of the control
tubes.
  Interpretation

RESULT:
• A titer of 1 : 160 or greater in the
SAT is considered diagnostic if
this result fits the clinical and
epidemiologic findings
• Acute brucellosis: titres of 640 or
more by 3-4 weeks of illness

PC: Partial or complete agglutination

NC: No visible clumping
LIMITATIONS OF PROCEDURE
• False negative results may be obtained in the early phase
of disease, prozone phenomenon and antibiotic
treatment. Sera from low or non-immune responders will
also produce false negative results.

• Serological cross-reactions with brucella have been

reported in cases of infection or vaccination with some
strains of V. cholera, Pasteurella, Proteus OX19 and Y.
enterolitica, serotype 9.
• Several sources of error have to be guarded against. Sera often
contain `blocking' or 'non-agglutinating' antibodies.

• The blocking effect may sometimes be removed by prior heating of the

serum at 55 °C for 30 minutes or by using 4% saline as the diluent for
the test.

• The most reliable method for obviating the blocking effect and
detecting the ‘incomplete' antibodies is the antiglobulin (Coombs) test.
THANKYOU
REFERENCES
• https://www.cdc.gov/brucellosis/pdf/brucellosi-reference-guide.pdf
• Bailey & Scott’s Diagnostic Microbiology, 13th E D I T I O N
• District Laboratory Practice in Tropical Countries, Part Second Edition , Monica
Cheesbrough
• Ananthanarayan & Panikers Text Book of Microbiology, 7 th Edition
• Internet sources for images