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Serology in Infectious Diseases
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Serology Principles and Interpretation
in Infectious diseases Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
Beginning of Serology
• Serology as a science began in 1901. Austrian American immunologist Karl Landsteiner (1868-1943) identified groups of red blood cells as A, B, and O. From that discovery came the recognition that cells of all types, including blood cells, cells of the body, and microorganisms carry proteins and other molecules on their surface that are recognized by cells of the immune system.
Dr.T.V.Rao MD 2
Karl Landsteiner (1868-1943)
• An Austrian physician
by training,
Landsteiner played an
integral part in the
identification of blood
groups. He
demonstrated the
catastrophic effect of
transfusing with the
wrong type of blood,
Dr.T.V.Rao MD 3
Purpose of Serological Tests
• Serological tests may be performed for
diagnostic purposes when an infection is
suspected, in rheumatic illnesses, and in
many other situations, such as checking
an individual's blood type. Serology blood
tests help to diagnose patients with certain
immune deficiencies associated with the
lack of antibodies, such as X-linked
agammaglobulinemia.
Dr.T.V.Rao MD 4
Serology • The branch of
laboratory medicine
that studies blood
serum for evidence of
infection and other
parameters by
evaluating antigen-
antibody reactions in
vitro
Dr.T.V.Rao MD 5
Serology
• Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum
We can detect antigens too
Dr.T.V.Rao MD 6
Serology prerogative of
Microbiology
• It is rather curious that, although serum for
a multitude of constituents in biochemistry
and haematological laboratories, the term
serology has come to imply almost
exclusively the detection of antibodies in
serum for antibodies in infectious
diseases, and terminology has become
prerogative of microbiologists.
Dr.T.V.Rao MD 7
Immunology/ Serology?
Precipitation Reactions • Capillary tube precipitation (Ring Test)
• Ouchterlony Double Diffusion (Immunodiffusion)
• Radialimmunodiffusion (RID)
• Immunoelectrophoresis (IEP)
• Rocket Electroimmunodiffusion (EID)
• Counterimmunoelectrophoresis (CIEP)
The above tests have moved to Biochemistry
Dr.T.V.Rao MD 8
Terms used in evaluating test
methodology
• Sensitivity –Analytical Sensitivity – ability of a
test to detect very small amounts of a substance
–Clinical Sensitivity – ability of test to give positive result if patient has the disease (no false negative results)
Dr.T.V.Rao MD 9
Specificity
• Analytical Specificity – ability of test to
detect substance without interference from
cross-reacting substances
• Clinical Specificity – ability of test to give
negative result if patient does not have
disease (no false positive results)
Dr.T.V.Rao MD 10
Affinity
• Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site.
• Affinity is the equilibrium constant that describes the antigen-antibody reaction
Dr.T.V.Rao MD 11
Affinity
• Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.
• It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site .
Dr.T.V.Rao MD 12
Avidity • Avidity is a measure of
the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies
• Avidity is influenced by both the valence of the antibody and the valence of the antigen.
• Avidity is more than the sum of the individual affinities.
Dr.T.V.Rao MD 13
Dr.T.V.Rao MD 14
Dilution
• Estimating the
antibody by
determining the
greatest degree to
which the serum may
be diluted without
losing the power to
given an observable
effect in a mixture
with specific antigen
Dr.T.V.Rao MD 15
Titer
• Different dilutions of
serum are tested in
mixture with a
constant amount of
antigen and greatest
reacting dilution is
taken as the measure
or Titer
Dr.T.V.Rao MD 16
Expression of Titers
• Expressed in term of the
was in which they are
made
• Dilution 1 in 8 is a
dilution made by mixing
one volume of serum
with seven volumes of
diluents (Normal Saline )
• Incorrect to express
dilution as 1/8
Dr.T.V.Rao MD 17
Common methods in creating
dilutions
Dr.T.V.Rao MD 18
Sero Conversion
• Seroconversion is the
development of
detectable specific
antibodies to
microorganisms in the
blood serum as a
result of infection or
immunization.
Dr.T.V.Rao MD 19
Sero reversion
• Seroreversion is the
opposite of
seroconversion. This
is when the tests can
no longer detect
antibodies or antigens
in a patient’s serum
Dr.T.V.Rao MD 20
Testing paired Samples
• Testing for infectious
diseases is performed on
acute and convalescent
specimens (about 2
weeks apart) Paired
sample.
• Must see 4-fold or 2-
tube rise in titre to be
clinically significant
Dr.T.V.Rao MD 21
Majority Diagnostic tests are
Serological tests
• There are several
serology techniques
that can be used
depending on the
antibodies being
studied. These
include: ELISA,
agglutination,
precipitation,
complement-fixation,
and fluorescent
antibodies. Dr.T.V.Rao MD 22
Antigen and Antibody
reactions can be identified by
different methods
Dr.T.V.Rao MD 23
Precipitation
• Principle – Soluble antigen + antibody (in proper proportions) –>
visible precipitate
– Lattice formation (antigen binds with Fab sites of 2 antibodies)
• Examples – Double diffusion (Ouchterlony)
– Single diffusion (radial Immunodiffusion)
– Imunoelectrphoresis
– Immunofixation
Dr.T.V.Rao MD 24
Agglutination
• Principle
– Particulate antigen + antibody –> clumping
– Lattice formation (antigen binds with Fab sites of 2
antibodies forming bridges between antigens)
• Examples
– Direct agglutination (Blood Bank)
– Passive Hemagglutination (treat RBC's with tannic
acid to allow adsorption of protein antigens)
– Passive latex agglutination (antigen attached to latex
particle)
Dr.T.V.Rao MD 25
Neutralization reactions
• Similar in principle and interpretation of results
• Antibody-binding
• Hemagglutination inhibition (serum antibody reacts with known nonparticulate antigen –> binding occurs)
• Neutralization (antibody neutralizes toxin)
• After binding, antibody is not available to react in indicator system
• Results:
• NO agglutination or NO haemolysis = positive reaction
• Agglutination or haemolysis = negative reaction (antibody not bound in origin
Dr.T.V.Rao MD 26
• Generally, positive control samples used in inhibition or neutralization tests show no reaction and negative control samples show a reaction (opposite of results in direct agglutination testing)
• Example of inhibition: Hemagglutination inhibition test for rubella
• Example of neutralization: antistreptolysin O test (ASO)
Neutralization reactions
Dr.T.V.Rao MD 27
Complement fixation (CF)
• Antibody and antigen allowed to combine in presence of complement
• If complement is fixed by specific antigen-antibody reaction, it will be unable to combine with indicator system
• Precautions
• Serum must be heat-activated
• Stored serum becomes anti-complementary
• Extensive QC/standardization required
• Only use for IgM antibodies
Dr.T.V.Rao MD 28
Imunoelectrphoresis (IEP)
Qualitative • A serum sample is
electrophoresed through an agar medium.
• A trough is cut in the agar and filled with Ab.
• A precipitin arc is then formed.
• Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation.
Dr.T.V.Rao MD 29
Serology can be done on various
specimens
• Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva, which have (roughly) similar properties to serum.
• Serological tests may also be used forensically, generally to link a perpetrator to a piece of evidence (e.g., linking a rapist to a semen sample).
Dr.T.V.Rao MD 30
Enzyme immunoassay
(EIA/ELISA) • Sandwich technique”
• Monoclonal or polyclonal antibody adsorbed on solid surface (bead or microliter plate)
• Add patient serum; if antigen is present in serum, it binds to antibody coated bead or plate
• Add excess labelled antibody (antibody conjugate); forms antigen-antibody-labelled antibody “sandwich” (antibody in conjugate is directed against another epitope of antigen being tested)
• Add substrate, incubate, and read absorbance
• Washing required between each step
• Absorbance is directly proportional to antigen concentration
Dr.T.V.Rao MD 31
ELISA methods takes over
• Enzyme-linked immunosorbent assay, also
called ELISA, enzyme immunoassay or EIA, is
a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample. The ELISA
has been used as a diagnostic tool in medicine
• Because the ELISA can be performed to
evaluate either the presence of antigen or the
presence of antibody in a sample
Dr.T.V.Rao MD 32
ELISA Most popular technological
advance in Laboratory Medicine
• ELISA methods can
detect any infectious
disease provided if
we have antibodies
and antigen to any
infection, enzyme or
any substance
Dr.T.V.Rao MD 33
Serology applications
in.. • HIV testing
• Serum HCG
(pregnancy)
• Tests for hepatitis
antigens and
antibodies
• Antibodies to bacteria
• Hepatitis Serology
Dr.T.V.Rao MD 34
Nephelometry
• Procedure
– Serum substance reacts with specific antisera and
forms insoluble complexes
– Light is passed through suspension
– Scattered (reflected) light is proportional to number of
insoluble complexes; compare to standards
• Examples
– Complement component concentration
– Antibody concentration (IgG, IgM, IgA, etc.)
• Immunofluorescence
Dr.T.V.Rao MD 35
Immunofluorescence
• Direct – add fluorescein-labelled antibody to
patient tissue, wash, and examine under
fluorescent microscope
• Indirect – add patient serum to tissue containing
known antigen, wash, add labelled antiglobulin,
wash, and examine under fluorescent
microscope
• Examples
– Testing for Antinuclear Antibodies (ANA)
– Fluorescent Treponemal Antibody Test (FTA-Abs)
Dr.T.V.Rao MD 36
Fluorescence polarization
immunoassay (FPIA) • Principle
• Add reagent antibody and fluorescent-tagged antigen to patient serum
• Positive test – Antigen present in patient serum binds to reagent leaving most
tagged antigen unbound
– Unbound labelled antigens rotate quickly reducing amount of polarized light produced
• Negative test – If no antigen present in patient serum, tagged antigen binds to
reagent antibody
– Tagged antigen-antibody complexes rotate slowly giving off increased polarized light
Dr.T.V.Rao MD 37
Flow cytometry
• Method of choice for T- and B-cell analysis (lymphocyte phenotyping)
• Principle
• Incubate specimen with 1 or 2 monoclonal antibodies tagged with fluorochrome
• Single cells pass through incident light of instrument (laser) which excites fluororochrome and results in emitted light of different wavelength
• Intensity of fluorescence measured to detect cells possessing surface markers for the specific monoclonal antibodies that were employed
• Forward light scatter indicates cell size or volume
• 90° side-scattered light indicates granula
Dr.T.V.Rao MD 38
Common uses Flow cytometry
• DNA analysis
• Reticulocyte counts
• Leukaemia/lymphoma
classification
• CD 4 cell estimations
in AIDS/HIV patients.
Dr.T.V.Rao MD 39
Other Applications of
agglutination tests in Serology
i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.
Dr.T.V.Rao MD 40
Georges-Fernand-Isidor Widal
• Widal in 1896, and
Widal & Sicard in
1896 described the
Widal reaction, and
this test has proved
of value in cases
where positive
cultures have been
unobtainable
Dr.T.V.Rao MD 41
Widal test a Popular test in
diagnosis of Typhoid Fever
• The Widal test is a
presumptive
serological test for
Enteric fever or
Undulant fever. In
case of Salmonella
infections, it is a
demonstration of
agglutinating antibodies against
antigens O-somatic and
H-flagellar in the blood. Dr.T.V.Rao MD 42
Widal test is century old ,
Is it loosing importance ?
• In this reaction antibodies react with antigens on
the surface of particulate objects and cause the
objects to clump together, or agglutinate. These
reactions were the earliest to be adapted to
diagnostic laboratory. Widal test is used for
diagnosis of typhoid fever. This test, developed
by Georges Fernand I. Widal (French physician)
in 1896, is now supplemented by more
sophisticated procedures.
Dr.T.V.Rao MD 43
Widal test – A standard tube
agglutination test
• Test can be performed by the tube dilution
technique which permits, the assay of antibody
titre. In this, a constant amount of the antigen is
added to a series of tubes containing serum
dilutions. After mixing, the tubes are incubated at
a particular temperature and the highest dilution
of serum showing visible agglutination is
determined.
Dr.T.V.Rao MD 44
Agglutination how it appear after
reactivity
• O agglutination is granular
• H agglutination is loose and floccular
Dr.T.V.Rao MD 45
Dr.T.V.Rao MD 46
Principle of the Test
• A classic example of the agglutination
reaction is seen in the widal test for
diagnosis of typhoid fever. In this test the
antibody content of the patient's serum, is
measured by adding a constant amount of
antigen (Salmonella typhi) to the serially
diluted serum.
Dr.T.V.Rao MD 47
Reading the Widal Test
• Read the results by viewing the tubes under good light against the dark background with x2 magnifying lens
• Do not shake tubes before reading the results
• Read titers as greatest dilutions giving visible agglutinations.
• Limiting agglutination is 1in 200 the titer is 200 not to be reported as 1/200.
Dr.T.V.Rao MD 48
Interpretation of Widal test
• Test results need to
be interpreted
carefully in the light of
past history of enteric
fever, typhoid
vaccination, general
level of antibodies in
the populations in
endemic areas of the
world.
Dr.T.V.Rao MD 49
Testing in Typhoid carriers
• Many known carriers of typhoid bacilli
possess antibody against the Vi
(virulence) antigen of S. typhi. This is a
surface antigen easily lost during
cultivation(Vi tires seem to correlate
better with the carrier state than do O
or H titres). For this reason, Felix et al.
suggested the use of Vi agglutination
for detection of carriers.
Dr.T.V.Rao MD 50
Importance of Vi antibodies
• Many known carriers of typhoid bacilli
possess antibody against the Vi
(virulence) antigen of S. typhi. This is a
surface antigen easily lost during
cultivation(Vi tires seem to correlate
better with the carrier state than do O
or H titres). For this reason, Felix et al.
suggested the use of Vi agglutination
for detection of carriers.
Dr.T.V.Rao MD 51
Prozone phenomenon in
Agglutination tests
Prozone effect - Occasionally, it is observed that
when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs.
The lack of agglutination at high concentrations of antibodies is called the prozone effect. Lack of agglutination in the prozone is due to antibody excess resulting in very small complexes that do not clump to form visible agglutination
Dr.T.V.Rao MD 52
Causes Of False-positive Widal
Agglutination Tests
• Previous immunization with Salmonella
antigen.
• Cross-reaction with non – typhoidal
Salmonella.
• Variability and poorly standardized
commercial antigen preparation.
• Infection with malaria
• other Enterobacteriaceae charring the
same s-LPS . Dr.T.V.Rao MD 53
Causes of Negative Widal
Agglutination Test
• The carrier state
• An inadequate inoculum of bacterial
antigen in the host to induce antibody
production
• Technical difficulty or errors in the
performance of the test.
• Previous antibiotic treatment
• Variability in the preparation of
commercial antigens. Dr.T.V.Rao MD 54
Declining importance of Widal
test
• The value of the salmonella agglutination
tests has declined as the incidence of
typhoid fever has decreased, at least in
the developed world, the general use of
vaccines has increased, and ever
increasing -numbers of antigenically
related serotypes of Salmonella have been
recognised.
Dr.T.V.Rao MD 55
Serology - Importance of repeated
tests Criteria for diagnosing Primary Infection
• 4 fold or more increase in titer of IgG or total antibody between acute and convalescent sera
• Presence of IgM
• Seroconversion
• A single high titer of IgG (or total antibody) - very unreliable
Criteria for diagnosing Reinfection
• Four fold or more increase in titer of IgG or total antibody between acute and convalescent sera
• Absence or slight increase in IgM Dr.T.V.Rao MD 56
Typical Serological Profile After Acute
Infection
Note that during Reinfections, IgM may be absent or present at a low level transiently Dr.T.V.Rao MD 57
Antigen – Antibody reactions
presenting with precipitation
Dr.T.V.Rao MD 58
Precipitation Curve
Dr.T.V.Rao MD 59
Precipitation Curve
Dr.T.V.Rao MD 60
Measurement of Precipitation by
Light
• Antigen-antibody complexes, when formed
at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy
appearance.
• Turbidimetry measures the turbidity or
cloudiness of a solution by measuring
amount of light directly passing through a
solution. • Nephelometry indirect measurement, measures amount
of light scattered by the antigen-antibody complexes. Dr.T.V.Rao MD 61
Screening Tests for Syphilis
• Serologic methods are divided into two
classes. One class, the nontreponemal
tests, detects antibodies to lipoidal
antigens present in either the host or T.
pallidum; examples are the Venereal
Disease Research Laboratory and rapid
plasma reagin and tests.
Dr.T.V.Rao MD 62
Serological Diagnosis Of
Syphilis
I. Specific Anti-
Treponemal
Antibody
II. Anti – Treponemal
Antibody
III. Reagin Antibody
(VDRL and RPR)
Associated with higher
false positives
Dr.T.V.Rao MD 63
Indication for testing for Syphilis
Pregnant women
sexual contacts or partners of patients diagnosed with syphilis
children born to mothers with syphilis
patients with HIV infection
Dr.T.V.Rao MD 64
Tests For Reagin Antibody
• A large numbers of tests for Reagin: • VDRL (Venereal Diseases Reference
Laboratory).
• RPR (Rapid Plasma Reagin)
• ART (Automated Reagin Test)
Good sensitive screening
Titer falls rapidly with treatment
• Reagin titer falls with treatment.
Dr.T.V.Rao MD 65
VDRL – A standard test for
Syphilis
• NONTREPONEMAL ANTIGEN TESTS. Nontreponemal antigen tests are used as screeners. They measure the presence of reagin, which is an antibody formed in reaction to syphilis. In the venereal disease research laboratory (VDRL) test, a sample of the patient's blood is mixed with cardiolipin and cholesterol. If the mixture forms clumps or masses of matter, the test is considered reactive or positive. The serum sample can be diluted several times to determine the concentration of reagin in the patient's blood.
Dr.T.V.Rao MD 66
Screening tests should be
reported with cautions
• Reactivity in these tests generally
indicates host tissue damage that may not
be specific for syphilis. Because these
tests are easy and inexpensive to perform,
they are commonly used for screening,
and with proper clinical signs they are
suggestive of syphilis. The other class of
test, the Treponemal tests, uses specific
Treponemal antigens.
Dr.T.V.Rao MD 67
Combination of testes are
desirable
• Syphilis
serodiagnosis relies
on a combination of
nonspecific screening
tests (antilipoidal
antibodies) and
Treponema pallidum-
specific tests (anti-T.
pallidum antibodies).
Dr.T.V.Rao MD 68
Measurement of Precipitation by
Light
• Antigen-antibody complexes, when formed
at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy
appearance.
• Turbidimetry measures the turbidity or
cloudiness of a solution by measuring
amount of light directly passing through a
solution. • Nephelometry indirect measurement, measures amount of light
scattered by the antigen-antibody complexes. Dr.T.V.Rao MD 69
Confirmation is warranted
• Confirmation of infection requires a reactive
Treponemal test. Examples of the Treponemal
tests are the microhemagglutination assay for
antibodies to T. pallidum and the fluorescent
Treponemal antibody absorption test. These
tests are more expensive and complicated to
perform than the nontreponemal tests. On the
horizon are a number of direct antigen, enzyme-
linked immunosorbent assay, and PCR
technique
Dr.T.V.Rao MD 70
Non reactive and Reactive VDRL
Tests
Dr.T.V.Rao MD 71
Rapid plasma reagin
• The rapid plasma reagin (RPR) test works on the same principle as the VDRL. It is available as a kit. The patient's serum is mixed with cardiolipin on a plastic-coated card that can be examined with the naked eye.
Dr.T.V.Rao MD 72
Agglutination++ RPR Agglutination+
Dr.T.V.Rao MD 73
Agglutination+: ve RPR Agglutination : -
Dr.T.V.Rao MD 74
Biological false positives
• Biological False Positive Antibody (BFP)
Reagin Antibody: associated with other
diseases (BFP)
A. Acute: • Pneumonia
• Vaccination with live attenuated viruses.
• Malaria
• Pregnancy
B. Chronic: • Leprosy – the only infection
• Reagin titer falls rapidly with treatment
Dr.T.V.Rao MD 75
Serological Diagnosis Of
Syphilis
Test for specific Anti - Treponemal Antibody
1. Absorbed fluorescent Treponemal
antibody (FTA - ABs)
2. Treponema Pallidum Immobilization Test
(TPI) A. Most sensitive
B. Utilize living Treponema maintained by passage
in rabbits testes.
C. Expensive
D. Potentially hazardous.
E. Not done in the present contest as Technically demanding Dr.T.V.Rao MD 76
Doing a quantization test RPR
Dr.T.V.Rao MD 77
Other Serological Methods in
Diagnosis Of Syphilis
Treponema pallidum haemagglutination
(TPHA) test. A. Sheep, chicken or turkey RBCs. Sensitized by
attaching killed Treponema pallidum.
B. Agglutinate by presence of antibody
C. Less sensitive than FTA – Abs
D. Less reliable in the diagnosis of primary syphilis.
E. Sometimes false positive
Dr.T.V.Rao MD 78
Treponema Palladium
Haemagglutination test
TPHA
Dr.T.V.Rao MD 79
Other Serological Tests for
Syphilis
• Anti – Treponemal Antibody
• Anti-Treponemal ABs group detected by
Reiter Protein Complement Fixation Test
(RPCFT)
A. Appears later than specific ABs
B. Some syphilis patient do not produce the
form of ABs
C. Used is limited.
Dr.T.V.Rao MD 80
Detection by FTA-ABS IgG and
IgM
• In the FTA-ABS tests,
the patient's blood
serum is mixed with a
preparation that
prevents interference
from antibodies to
other Treponemal
infections.
Dr.T.V.Rao MD 81
FTA abs IgG and IgM detection continues to be
a confirmatory test in diagnosis of Syphilis
• The test serum is added to a slide
containing T. pallidum. In a positive
reaction, syphilitic antibodies in the blood
coat the spirochetes on the slide. The slide
is then stained with fluorescein, which
causes the coated spirochetes to fluoresce
when the slide is viewed under ultraviolet
(UV) light..
Dr.T.V.Rao MD 82
Principle of Fluorescent Method
Dr.T.V.Rao MD 83
Active Treponema Pallidum
Infection
1. Positive Specific Tests e.g. TPHA
2. Positive ( ≥1/ 8) of non-specific test
(VDRL)
• TPI-T (Treponema Pallidum
Immobilization Test)
• FTA –T (Fluorescent Treponema Test)
• Sometimes needed for confirmation.
Dr.T.V.Rao MD 84
Emerging Methods in Diagnosis
of Syphilis
• Currently, ELISA,
Western blot, and
PCR testing are being
studied as additional
diagnostic tests,
particularly for
congenital syphilis
and Neurosyphilis.
Dr.T.V.Rao MD 85
SPINAL FLUID TESTS in
Syphilis.
. Testing of cerebrospinal fluid (CSF) is an
important part of patient monitoring as well
as a diagnostic test. The VDRL and FTA-
ABS tests can be performed on CSF as
well as on blood. An abnormally high white
cell count and elevated protein levels in
the CSF, together with positive VDRL
results, suggest a possible diagnosis of
Neurosyphilis.
Dr.T.V.Rao MD 86
CSF testing is indicated only in…
• CSF testing is not
used for routine
screening. It is used
most frequently for
infants with congenital
syphilis, HIV-positive
patients, and patients
of any age who are
not responding to
penicillin treatment.
Dr.T.V.Rao MD 87
Biological false reactive VDRL test among the
HIV infected patients
• Fewer reports on the biological false positive VDRL in HIV individuals are documented. In this work, the author studied the rate of biological false reactive VDRL among the HIV-infected patients. Of interest, in this study, the rate is significantly lower (by Fishers exact test) than a recent previous report among prostitutes in India (10/94, about 10.6 %). In the general population, the biological false positive VDRL generally returns to negative within 14 weeks, without other clinical significance.
• Dr.T.V.Rao MD 88
Rickettsia and Serology
• Rickettsia is a genus of motile, Gram-negative, non-spore forming, highly pleomorphic bacteria that can present as cocci (0.1 μm in diameter), rods (1–4 μm long) or thread-like (10 μm long). Obligate intracellular parasites
• Because of this, Rickettsia cannot live in artificial nutrient environments and are grown either in tissue or embryo cultures (typically, chicken embryos are used).
• Still we have to dependent on Weil Felix test
Dr.T.V.Rao MD 89
Weil and Felix contribute for
testing
• In 1915, Weil and Felix showed that serum
of patients infected with any member of
the typhus group of diseases contains
agglutinins for one or more strains of O X
Proteus. In cases of typhus fever the
reaction usually appears before the sixth
day and reaches its height in the second
week.
Dr.T.V.Rao MD 90
Weil-Felix reaction – A
Heterophile agglutination Test
• A Weil-Felix reaction is a type of
agglutination test in which patients serum
is tested for agglutinins to O antigen of
certain non-motile Proteus and rickettsial
strains(OX19, OX2, OXk)
• OX19, OX2 are strains of Proteus vulgaris.
OXk is the strain of Proteus mirabilis.
Dr.T.V.Rao MD 91
Weil-Felix a Heterophile
agglutination test • The agglutination reactions,
based on antigens common to both organisms, determine the presence and type of rickettsial infection
• Because Rickettsiae are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification
• Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris.
Dr.T.V.Rao MD 92
Interpretations in Weil-Felix
reaction
• Sera from endemic typhus agglutinate OX19,
OX2.
Tick borne spotted fever agglutinate OX19, OX2.
• Scrub Typhus agglutinate OXk strain
• Test is negative in rickettsialpox, trench fever
and Q-fever.
False positive reaction may occur in urinary or
other Proteus infections
Test may be negative in 50 percent scrub typhus
Dr.T.V.Rao MD 93
Weil-Felix test
indicated in when patients present
with rashes • Test for diagnosis of
typhus and certain other rickettsial diseases. The blood serum of a patient with suspected rickettsial disease is tested against certain strains of (OX-2, OX-19, OX-K)..
Dr.T.V.Rao MD
Weil Felix test and Concentration
Camps
Dr.T.V.Rao MD 95
Weil-Felix test positivity saves
from Nazis • In Poland, during World War II, where a pair of
quick-thinking doctors used a little-known organism to keep the Nazis at bay. The microorganisms is Proteus OX19. . Its one remarkable feature is that human antibodies for Proteus OX19 cross-react with the antibodies for Ricksettia – the bacterium responsible for the deadly disease typhus. Blood from a patient infected with Proteus Ox19 will give a false-positive in the most common typhus screening method, the Weil-Felix test.
Dr.T.V.Rao MD 96
How they made Weil-Felix test
Positive
• While the Polish doctors could, and did,
inject a number of other people with
Proteus to induce positive Weil-Felix
results, an on-site Nazi medical team
could well have proved their undoing.
Fortunately, ingenuity and a good dose of
hospitality and alcohol prevented them
from being uncovered.
(From the British Medical Journal )
Dr.T.V.Rao MD 97
Other Emerging
Serological Tests
Dr.T.V.Rao MD 98
Co-agglutination
• Co agglutination is similar to the latex agglutination technique for detecting antigen (described above). Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions
Dr.T.V.Rao MD 99
Co agglutination Test
Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.
Dr.T.V.Rao MD 100
Chemiluminescence
• Chemiluminescen
ce is the
emission of light
with limited
emission of heat
(luminescence),
as the result of a
chemical
reaction. Dr.T.V.Rao MD 101
Chemiluminescent
Immunoenzymatic Assay
• Process for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescent labelling substance activated or excited to chemiluminescence by an analytical reagent. By means of a serological reaction, initially an antigen/antibody complex is formed which is treated with a chemiluminescent conjugate containing chemiluminescent triphenylmethane dyes and the chemiluminescence of the chemiluminescent complex formed is measured.
Dr.T.V.Rao MD 102
Recent testing Advances
• The ToRC IgG kit simultaneously detects
IgG class antibodies to Toxoplasmosis
gondii, rubella and cytomegalovirus
(CMV).
• The HSV-1 and HSV-2 IgG kit utilises
type-specific proteins to simultaneously
detect and differentiate IgG class
antibodies to the two most common
herpes subtypes, HSV-1 and HSV-2. Dr.T.V.Rao MD 103
False Positive Serological Tests
1. Cross reacting antibody
2. Cross reactivation of latent organism (Influenza
Virus A infection activate CMV IgM –
production
3. Presence of Rheumatoid factors
RF = IgM
RF + IgG = Complexed
= False positive organism-
specific IgM Antibody
Dr.T.V.Rao MD 104
False Negative Serologic Test
1. Immune system not intact
2. Delay in Antibody response (Lyme
disease - Legionnaire’s Disease)
3. Competition for Antigen binding site of
antibody)
IgM binds to the Antigen IgG site
IgG binds to the Antigen IgM site
4. Prozone Phenomena
Dr.T.V.Rao MD 105
Usefulness of Serological
Results
• How useful a serological result is depends on
the individual virus.
• For example, for viruses such as rubella and
hepatitis A, the onset of clinical symptoms
coincide with the development of antibodies.
The detection of IgM or rising titers of IgG in
the serum of the patient would indicate active
disease.
Dr.T.V.Rao MD 106
Rota Virus - whether serology
useful ?
• However, many viruses
often produce clinical
disease before the
appearance of antibodies
such as respiratory and
diarrheal viruses. So in this
case, any serological
diagnosis would be
retrospective and therefore
will not be that useful.
• Acute presence of Antigen
is much useful in Diagnosis
Dr.T.V.Rao MD 107
Antibody detection is definitive
Diagnosis
• There are also viruses
which produce clinical
disease months or years
after seroconversion e.g.
HIV and rabies. In the
case of these viruses,
the mere presence of
antibody is sufficient to
make a definitive
diagnosis.
Dr.T.V.Rao MD 108
Problems with Serology
• Long period of time required for diagnosis for paired
acute and convalescent sera.
• Mild local infections such as HSV genitalis may not
produce a detectable humoral immune response.
• Extensive antigenic cross-reactivity between related
viruses e.g. HSV and VZV, Japanese B encephalitis and
Dengue, may lead to false positive results.
Dr.T.V.Rao MD 109
Problems with Serology
Other Health conditions interfere
• Immunocompromised patients often give a
reduced or absent humoral immune response.
• Patients with infectious mononucleosis and those
with connective tissue diseases such as SLE may
react non-specifically giving a false positive result.
• Patients given blood or blood products may give a
false positive result due to the transfer of antibody
Dr.T.V.Rao MD 110
If we are a busy lab….
Dr.T.V.Rao MD 111
Why Automate?
• Reduce variability and improve quality
• Reduce labor and test costs
• Improve workflow in the laboratory
• Avoid potential ergonomic issues
Dr.T.V.Rao MD 112
Automations
• One of the first successful attempts to automate
• antibody tests was made by Weitz (1967) at the Lister Institute, London. The apparatus developed by Weitz (Fig. 3) allowed the performance of up to 12 titrations in a single operation, with even less manipulation than that required for a single test done by a more conventional technique.
Dr.T.V.Rao MD 113
Definitions (1)
• Quality Control - QC refers to the measures that must be included
during each assay run to verify that the test is working properly.
• Quality Assurance - QA is defined as the overall program that
ensures that the final results reported by the laboratory are correct.
• “The aim of quality control is simply to ensure that the results
generated by the test are correct. However, quality assurance is
concerned with much more: that the right test is carried out on the right
specimen, and that the right result and right interpretation is delivered
to the right person at the right time”
Dr.T.V.Rao MD 114
Definitions (2)
• Quality Assessment - quality assessment (also known as
proficiency testing) is a means to determine the quality of
the results generated by the laboratory. Quality assessment
is a challenge to the effectiveness of the QA and QC
programs.
• Quality Assessment may be external or internal, examples
of external programs include NEQAS, HKMTA, and Q-
probes.
Dr.T.V.Rao MD 115
Variables that affect the quality
of results
• The educational background and training of the
laboratory personnel
• The condition of the specimens
• The controls used in the test runs
• Reagents
• Equipment
• The interpretation of the results
• The transcription of results
• The reporting of results
Dr.T.V.Rao MD 116
Errors in measurement
• True value - this is an ideal concept which cannot be
achieved.
• Accepted true value - the value approximating the
true value, the difference between the two values is
negligible.
• Error - the discrepancy between the result of a
measurement and the true (or accepted true value).
Dr.T.V.Rao MD 117
Random Error
• An error which varies in an unpredictable manner, in magnitude
and sign, when a large number of measurements of the same
quantity are made under effectively identical conditions.
• Random errors create a characteristic spread of results for any test
method and cannot be accounted for by applying corrections.
Random errors are difficult to eliminate but repetition reduces the
influences of random errors.
• Examples of random errors include errors in pipetting and changes
in incubation period. Random errors can be minimized by training,
supervision and adherence to standard operating procedures.
Dr.T.V.Rao MD 118
Random Errors
x
x x
x x
True x x x x
Value x x x
x x x
x
x
x
Dr.T.V.Rao MD 119
Systematic Error
• An error which, in the course of a number of measurements of the same value of a given quantity, remains constant when measurements are made under the same conditions, or varies according to a definite law when conditions change.
• Systematic errors create a characteristic bias in the test results and can be accounted for by applying a correction.
• Systematic errors may be induced by factors such as variations in incubation temperature, blockage of plate washer, change in the reagent batch or modifications in testing method.
Dr.T.V.Rao MD 120
Systematic Errors
x
x x x x x x x
True x
Value
Dr.T.V.Rao MD 121
Internal Quality Control Program for
Serological Testing
An internal quality control program depend on the use of
internal quality control (IQC) specimens, Shewhart Control
Charts, and the use of statistical methods for interpretation.
Internal Quality Control Specimens
IQC specimens comprises either (1) in-house patient sera
(single or pooled clinical samples), or (2) international serum
standards with values within each clinically significant ranges.
Dr.T.V.Rao MD 122
Dr.T.V.Rao MD 123
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