Febrile Agglutinins
1
Febrile Agglutinins


Agglutinating antibodies that arise from
infections with some microorganisms that
induce fever
Among bacteria that induce febrile
agglutinins are:



2
Rickettsia (typhus )
Brucella (Brucellosis)
Salmonella (typhoid fever)
Typhus
3
Typhus
• Any of several similar diseases caused by
louse-borne bacteria.
• The causative organism Rickettsia is an
obligate parasite and cannot live long
outside living cells.
• Rickettsia is endemic in rodent hosts,
including mice and rats, and spreads to
humans through mites, fleas and body
4
lice.
Rickettsial Diseases



5
Rickettsias as a group have a worldwide
distribution
Rickettsias are associated with variety of
different vectors and hosts
Most types of rickettsiosis are geographic areaspecific
Rickettsial Diseases

The rickettsiae that are pathogens of humans
are subdivided into three major groups
based on clinical characteristics of disease:



6
spotted fever group (R. rickettsii )
typhus group (Rickettsia prowazekii )
and scrub typhus group
Rickettsial Diseases



7
Rickettsial diseases are clinically non-specific
with many overlapping signs and symptoms
Rickettsiosis in humans may be:
Subclinical
Mild, self-limited
Severe, life-threatening
Confirmation of rickettsiosis cases requires
consideration of clinical, and
epidemiological, and laboratory data
Weil-Felix Agglutination Test




Developed in 1916
Confirm diagnosis for rickettsial infection
Nonspecific rickettsial test based on cross-reacting
antibodies
Antibodies are produced that cross-react with
polysaccharide “O” antigens of some Proteus species




8
P. vulgaris, OX-19 strain
P. vulgaris, OX-2 strain
P. mirabilis, OX-K strain
By the Weil-Felix test, agglutinating antibodies
are detectable after 5 to 10 days following the
onset of symptoms
Weil-Felix Agglutination Test

There are two protocols for the Weil-Felix
agglutination reaction:


9
Rapid slide test
Tube method (confirmation method)
Routine Laboratory Tests
Neutropenia in the acute phase
 Thrombocytopenia
 Hypoproteinemia, hypoalbuminemia
 Elevated ALT, AST, and alkaline
phosphatase
 CPK and LDH often elevated in acute
infection

10
Brucellosis
11
Brucella spp.



Gram negative, coccobacilli bacteria
Facultative, intracellular organism
Environmental persistence
–
–

Multiple species
•
12
Temp, pH, humidity
Frozen and aborted materials (fetuses or placentas)
Three species (B melitensis, B abortus, B suis)
are important human pathogens; B canis is of
lesser importance
Transmission to Humans

Conjunctiva or broken skin contacting infected
tissues
–

Ingestion
–
–


13
Raw milk & unpasteurized dairy products
Rarely through undercooked meat
Inhalation of infectious aerosols
–

Blood, urine, vaginal discharges, aborted fetuses, placentas
Pens, stables, slaughter houses
Person-to-person transmission is very rare
Incubation varies
–
7-21 days to several months
14
Diagnosis in Humans

Isolation of organism
–

Serum agglutination test
–
–


15
Blood, bone marrow, other tissues
Fourfold or greater rise in titer
Samples 2 weeks apart
Immunofluorescence of organism in clinical
specimen
PCR
Diagnosis
 Depends on the presence of clinical features
and +ve blood or tissue culture and/or
detection of raised brucella agglutinins in the
blood.
 Culture: +ve in about 50 -70% of cases.
 Standard agglutination Test:
• a titre of 1/160 in non endemic areas
• & 1/320 in endemic areas are significant.
16
Typhoid Fever
17
Definition
• Typhoid fever is an acute infectious disease of digestive
tract caused by typhoid bacillus
•
Causative organism: Typhoid bacillus
•
genus salmonella group D
•
Pathogenicity: endotoxin
•
Resistance: Stable in environment, sensitive to heat, acid,
common disinfectants
18
Salmonella
19
•
Salmonella is a Gram-negative facultative
rod-shaped bacterium
•
Salmonella are the cause of two diseases called
•
salmonellosis: enteric fever (typhoid), resulting from
bacterial invasion of the bloodstream,
•
and acute gastroenteritis, resulting from a food borne
infection/intoxication
Antigenicity
• Salmonellae possess three major
antigens:
• H or flagellar antigen (heat-labile
proteins );
• O or somatic antigen (heat stable );
• and Vi antigen (possessed by only a few
serotypes).
20
Exercise 5
Widal Test
21
Widal Test



22
To detect the febrile agglutinins to
Salmonella species
The “O” and “H” antigens are affixed to
particles
The antigens are adsorbed to differently
colored latex particles
Widal test

WIDAL test can be performed by two methods
1.
2.

23
Slide Agglutination
Tube agglutination
Tube agglutination is more accurate in
comparison to slide agglutination you can go
upto 1:1280 titre but in case of slide you are
restricted to 1:320 titre only but it is more rapid
result comes within 5 min
Widal test – Slide Method




24
Add the following volumes of serum to a slide
80 µl, 40 µl , 20 µl , 10 µl , and 5 µl
Mix the reagent containing the Ags well and
add one drop to each drop of serum
Mix well
Rotate for one minute and then read the
result
Widal test – Tube Method
1.
2.
3.
4.
25
Make a series of serum dilutions for each antigen to
be tested. Include tubes with 0.5 ml saline for
control of each antigen to be used.
Use perfectly clean and dry test tubes and prepare
dilutions beginning with 1:10 and doubling through
1:320 or so.
Add 0.1 ml of serum to 0.9 ml of physiological saline
and then dilute serially by mixing 0.5 ml diluted
serum with 0.5 ml saline and discarding 0.5 ml from
the last tube.
From specimen submitted to detect possible rise in
titre, prepare a series of 10 dilutions, ending with
1:5120.
Widal test – Tube Method
26
Preparation of antigen
 Use the killed standardized suspension of S.typhi O ,
S.typhi H ,S.paratyphi AH and S.paratyphi AO. These are
commercially available and their preparation should be
undertaken as per the instructions of the manufacturers
Procedure of test
 Shake the antigen suspension to ensure even
distribution.
 Add 0.5 ml to each serum dilution and to saline for
controls.
o
 Incubate the test overnight at 37 C and let tubes stand at
room temperature for 15-20 minutes before reading.
Reading the result


27
In all cases, look first at the control tubes and proceed only if
they show satisfactory results. There should be no appreciable
sedimentation of the bacteria. Although a fine button may have
been deposited at the bottom of the tube, no discernible
clearing should have occurred. The antigen suspension should
be evenly distributed as a rule.
Pick up the individual tubes of each row of the patient’s
specimens and look first at the supernatant. When a complete
agglutination occurs, practically all the bacteria are removed
from the supernatant which appears absolutely clear. When the
reaction is negative the suspension should look as turbid as the
antigen control is. The in-between reactions can be categorized
into.+++ ,++ ,+
Reading the result





28
Examine the tubes with a hand lens against
a dark background
End point carpet formation
Granular agglutination
Floccular agglutination
Also look at the supernatant fluid above the
aggultinate
29
Carpet formation
Larger flocules
30
Widal /reporting




31
Highest dilution showing agglutination
If no agglutination report as
Salmonella typhi O titer < 1 in 20
Salmonella tyhphi H titer < 1in 20
Widal interpretation



32
A 2-4 four fold rise in titer from the serum
tested during the first week and the 2-4 week
very significant
High titer > 1:160 against O antigen suggest
infection
High titer > 1:160 against H antigen (develop
later in infection but persist for long periods)
suggest past infection
Limitations




Titers against O and H antigens may be
raised in the following diseases/conditions
Other salmonellosis
Chronic liver disease
Immunological disorders
–
–
–
33
rheumatic fever
Nephrotic syndrome
Ulcerative colitis