Music 9 - 4th quarter - Vocal Music of the Romantic Period.pptx
Diagnosis of infectious diseases by dr.g.v.mali
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).
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.
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.
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.
19.
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.
26.
27.
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.
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
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.