1. Diagnosis of Infectious Diseases
Dr. G. V. Mali
Bharati Vidyapeeth’sMBSK Kanya
Mahavidyalaya, Kadegaon. Dist. Sangli 415304.
Maharashtra ( India)

2. Laboratory diagnosis of infectious diseases
1- Microscopic examination of the clinical specimen
2- Isolation of the culture & its identification based on
biochemical reactions
3- Serological identification / Immunological
identification of Ags or Abs.
4- Nucleic acid based / Molecular biology techniques
( Among these 1 & 2 are conventional methods )

3.  Limitations of the conventional methods –
 1. Lengthy, time consuming and tedious.
 2. Culturing of certain organisms like viruses, fungi &
parasites may not be possible
 3. Associated with risk.
 4. Impossible in all laboratories, requires
sophisticated labs. e. g Mycobacteria
 5. Culture may be negative due to prior antimicrobial
therapy.

4. Serological identification of Ags / Abs
OR
Immunological assays
 Important Advantages -
 They provide early diagnosis
 Important for uncultivable organisms in the lab.
 Useful for differential diagnosis of certain
diseases e.g. Typhoid fever
 Useful to measure the antibody level (titer).

5. Conventional Serological Methods
1. Precipitation
2. Agglutination
3. Haemagglutination
4. Haemagglutination inhibition
5. Complement Fixation Test
6. Fluorescent Antibody Test

6. 1. Precipitation:
Reaction between soluble antigen and antibody =
Insoluble PPT
- If ppt sediments – Precipitation
- If remains suspended as floccules – flocculation
 Carried out either in liquid media / in gels
e.g. agar, agarose, polyacrylamide
 Can be Qualitative or quantitative
 Sensitive, can detect as little as 1μg of protein
- Applications
 Slide test ( Qualitative ) – VDRL test for syphilis
 Tube test ( Quantitative )– Kahn test for syphilis

7. - Precipitation in gel is called immunodiffusion
. Used for detection of fungal antigens
- Immunoelectrophoresis –
- Combination of ectrophoresis & Immunodiffusion
- Here, process of immunodiffusion is enhanced
electrically.
- Two Common ways of Immunoelectrophoresis –
Counter immunoelectrophoresis –
For detection of HBs surface Ags, specific
bacterial & Cryptococcal Ags.
Rocket immunoelectrophoresis –
Quantitative estimation of Ags

8. 2. Agglutination
 Reaction of antibodies with
particulate or insoluble antigens in
presence of an electrolyte at
suitable pH & temp.
= formation of visible clumps
 Applications-
 Slide test- primary diagnosis of
typhoid
 Tube test- Widal test used for
diagnosis of typhoid fever
 Tube test for Brucellosis
 Weil felix test for typhus fever
 Passive agglutination : Agglutination
 Agglutination of soluble Ags by
coating them on inert particles like
latex beads or carbon particles
 E.g. RPR test ( Rapid plasma
reagin test ) to detect cardiolipin
antibodies in sera of syphilis
patients.

10. Passive Agglutination

11. 3. Haemagglutination
 Agglutination of RBCs
 Useful for diagnosis of viral
infections
e.g. influenza, mumps &
measles.
 Haemagglutination
inhibition test :
 To detect Abs in serum
against haemagglutinating
viruses .
 Positive Test :
 Virus + RBCs + test serum
= No hemagglutination
 Negative Test :
 Virus + RBCs + test serum
= Hemagglutination

12. 4. Complement Fixation Test
 Complement – Complex system of
some serum proteins , activated by
Ag-Ab complexes
• Ability to fix on Ag-Ab complex ,
if Ab is involved
• In presence of appropriate Ab, ‘C’
causes lysis of RBCs, bacteria
• Two steps –
• 1.Complement Fixation Step
• Inactivated serum of patient + Ag + C
= incubation at 37o C for 1 hr.
2. Indicator Step
Addition of sheep RBCs & antisheep
RBC antibodies )
 No hemolysis = Positive test
 Hemolysis = Negative Test
 E.g. Wasserman test – for syphilis,
Also used for viral, fungal,
rickettsial, chlamydial & protozoal

13. 5. Fluorescent Antibody Test
 Use antibodies labeled with fluorescent dyes.
 e.g. fluorescein isothiocynate (Green fluorescence ),
Rhodamine B ( Orange red )
 Two types –
1. Direct FAT : Used to identify specific microorganisms
(antigens).
 Specimen ( Ag) is fixed on slide + labelled Abs = examined
under fluorescent microscope =
 If fluorescene = + ve test.
E.g. Diagnosis of Ags on group A streptococci,
enteropathogenic E.coli, H.influenzae type b, rabies etc.
2. Indirect FAT : Used to detect Abs in serum.
 Known Ag fixed on slide + test serum + labelled
antiimmunoglobulin = observation under fluro microscope
* If fluorescence = +ve test ( Abs are present )
* Used to detect Treponemal Abs for syphilis diagnosis.

14. Direct and Indirect FAT

15. New immunological methods /Immunoassays
1. Enzyme linked immunosorbant assay ( ELISA )
2 . Radioimmunoassay
1.ELISA –
 Uses antibodies linked to an enzyme,
 E.g. horseradish peroxidase or alkaline phosphatase.
 Antigen – antibody reactions are detected by enzyme
activity.
 The specific Antigen is added to the test well.
 An antibody linked to the enzyme is added to it.
 It will bind if the antigen specific for it is present.
 To determine whether the enzyme-linked antibody is
bound in the well, substrate for the enzyme is added.
 If the enzyme linked antibody is present, the substrate is
converted to a product that causes a color change.

16. Three ways of performing ELISA –
ANTI-HUMAN
1.Indirect – IMMUNOGLOBULIN
WITH DETECTOR
 Wells coated with known Ags
 Test serum
 Add conjugate of anti-antibody linked
with enzyme ( HRPO)
 Detection of enzyme by addition of enzyme
substrate ( ortho- phenylene –dihydro
dichloride solution )
SAMPLE
 Development of yellow orange colour
Ab
 Positive test
* Colour produced will be proportional
ANTIGEN
to the conc. of Abs.
* Used for the detection of Abs SOLID PHASE
against HIV 1 & HIV 2, rubella
virus.

17. 2. Competitive ELISA
 Used for the detection of Abs in test sample
 Competition between Abs & labelled known Abs.
 If Abs are present in test serum, labelled Abs will not
bind & will not produce a colour = Positive test
 If Abs are absent in test serum, labelled Abs will bind
& produce colour = Negative test

18. 3. Sandwich ELISA :
 Detection of Ags and not for Abs
 Two types – Direct sandwich and Indirect Sandwich
 Direct Sandwich / Single Ab :
 Abs are coated on solid surface
 Test sample (Ag)
 Enzyme linked known Ab
 Indirect Sanwich / Double Ab :
 Abs are coated on solid surface
 Test sample (Ag)
 Second Ab ( known )
 Enzyme linked anti-antibody.

20. ELISA kits are available for both clinical
diagnostics and home use. These tests are used
for everything from screening blood for anti-HIV
antibodies to home pregnancy tests.

21. 2. Radioimmunoassay ( RIA )
 Steps are very similar with ELISA
 In RIA, instead of enzyme linked Abs ,
radiolabelled Abs i.e. antiglobulin
labelled with a radioactive compound is
added.
 The amount of radioactivity in wells
provides an estimate of the titer of
target antibody.

22. Immunoblotting : e.g. Western blot
analysis
 Technique of separation & detection of Ags.
 Ags (e.g. HIV Ags in serum) are first
separated by polyacrylamide gel
electrophoresis
 Separation takes place on the basis of size
 Separated molecules are transferred to
another matrix e.g. nitrocellulose membrane.
 Enzyme labelled Abs against the molecules of
interest is added and then sbstrate is added
for visualization.
 Used to confirm the presence of specific Ags
of HIV 1 & HIV 2.

23. Nucleic Acid Based Methods / Molecular
Biology Techniques
 It involves the study of relevant DNA
sequence by nucleic acid techniques.
 The most common methods are –
A- Polymerase chain reaction (PCR):
B- Restriction fragment length polymorphisms
( RFLP)
C- Genetic probes (DNA or RNA probes):

24. 1.Polymerase Chain Reaction
 Amplification of a short sequence of target DNA or
RNA which is then detected by a labeled probe.
 Highly sensitive – detects infectious agents in host
tissues and vectors, even when a small number of
host cells are infected.
 PCR can target and amplify a gene sequence that is
integrated into the DNA of infected host cells.
 It can also target and amplify un-integrated viral
gene sequences.
 Very useful in the diagnosis of chronic-persistent
infections, such as retroviruses (bovine leukemia
virus, caprine arthritis /encephalitis virus, etc.).

25. Steps in PCR
 Cells separated and lysed.
 Each cycle of PCR consists of three cycles:
1.Denaturation of target DNA at 950Cto separate 2
strands.
2.Annealing step - in which the reaction mix is cooled to
550 C to allow the primers to anneal to target sequence
 Primers are small segments of DNA , no more than
20-30 nucleotides long.
 Primers are complementary to segments of opposite
strands of the target sequence.
3.Extension reaction in which primers initiate DNA
synthesis ( at 720C) using a DNA polymerase.
• These three steps constitute a thermal cycle
• Only the segments of target DNA between the primers
will be replicated.
 Each PCR cycle results in a doubling of target sequences.
 One cycle takes approximately 60-90 seconds.

28.  Specific primers are designed for identification of
different classes of pathogens.
 The best example is the use of sequences of the 16s
rRNA gene which is an evolutionarily conserved gene
in bacterial species.
 Using such primers, one can determine the presence
of any bacteria from the sample.
 Positive PCR result needs to be further characterized
by hybridization with species-specific probes, analysis
by restriction enzyme digestion, or by sequencing.

29.  Classical PCR methods are now replaced with real-
time PCR assays.
 They can be used to quantify the DNA or RNA content
in a given sample.
 In contrast to conventional PCR, real-time PCR
requires less manipulation
 Is more rapid .
 Is highly sensitive and specific.
 Provides quantitative information.

30. 2.Restriction fragment length polymorphisms
 Fact - the genomes of even closely related pathogens
are identified by variation in sequence.
 Steps - Isolation of target pathogen,
- Extracting DNA or RNA (with subsequent
reverse transcription to DNA)
- Digesting the nucleic acid with one of a
panel of restriction enzymes.
 Separation of the individual fragments of DNA by gel
electrophoresis
 Visualization by staining with ethidium bromide.
 Ideally each strain will reveal a unique pattern, or
fingerprint.
 A good example of the application - differentiation of
rabies virus biotypes from dog.

31. RFLP
GGATCC
CCTAGG

32.  A modification to the basic RFLP technique -
 Incorporation of PCR as a preliminary step to amplify
a specific region of the genome.
 Amplified DNA serves as the template DNA for the
RFLP technique.
 This new combination (PCR-RFLP) offers - greater
sensitivity for the identification of pathogens
 Especially useful when the pathogen are in small
numbers or is difficult to culture.
 Both RFLP and PCR-RFLP are immensely useful for the
genotyping of strains of Cryptosporidium
 Examples in which the RFLP / PCR - RFLP techniques
are useful to differentiate between the genotypes –
the fungus Candida, - the bacterium Helicobacter
pylori .

33. 3.Diagnosis by DNA probes and DNA
Microarray technology
 DNA probes are specific short sequence of single-
stranded DNA or RNA which are labeled and bind with
specific complementary strand of nucleic acid of
organism in question
 Used in the detection of a segment of DNA sequence
(gene) in unknown organism.
 Used for the rapid identification of bacteria in
specimens e.g. Hepatitis B virus, EB Virus, N.
gonorrhoe.
 In conventional DNA probing, the unknown DNA (or
RNA), is immobilized on a solid surface e.g. a filter.
 The known DNA ( labeled probe )is in the liquid phase

34.  The target can be nucleic acids extracted from clinical
material or cultured cells
 It is then either - (a) added to filters (a dot or slot blot)
or
(b) transferred to a filter
after gel electrophoresis.
 If the amount of pathogen in a clinical sample is too
low for detection , one can amplify the nucleic acid by
PCR
 In order to visualize a probe bound to its target, the
probe can be labelled with a radioactive nucleotide

35. In situ
Hybridization

36. Microarray technology
 In microarray diagnosis, the known DNA ( large
oligonucleotides or complementary DNA) are
immobilized on a glass slide, and the unknown DNA
( labeled probe ) is in the liquid phase.
 A microarray is so-called because it consists of
20,000 or more different known DNAs, each DNA
being spotted onto glass slides, to form the array.
 Each spot is only around 10 μm in diameter. DNAs
complementary to the selected genes of pathogens
can be used to make the arrays .

37.  In microarray probing , the probe is made from the
sample
 Nucleic acid is extracted from a sample and an PCR is
performed using random oligonucleotide primers.
 Part of all the nucleic acids in the sample (– both of
host and pathogen origin ) are amplified.
 These PCR products are labelled with a fluorescent dye
and applied to the microarray.
 Under optimized conditions , only the DNA derived from
the pathogen will bind to the DNA on the glass slide.
 If one is interested in detecting only a particular
pathogen or group of related pathogens, then
pathogen-specific oligonucleotides can be used to
amplify these within the sample for probe production.

38. Molecular diagnosis
 Merits  Demerits
 Reduce reliance on  Technically
culture demanding
 Faster  Relatively
 More sensitive expensive
 More definitive  Can be too
 More discriminating sensitive
 Techniques
 Provides no
adaptable to all information if
pathogens results are
negative

39. Future of Molecular Diagnostic
Techniques
 Rapid diagnosis will result in decreased cost.
 Example: Mycobacteria - quick diagnosis - no need
for expensive laboratory isolation.
 Increased specificity and sensitivity of molecular
testing will become the standard of practice in
immunology and microbiology.
 Testing will become more rapid as assays are
automated which will also bring down the costs.

40.  THANK YOU