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Diagnosis of infectious diseases by dr.g.v.mali

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Diagnosis of infectious diseases by dr.g.v.mali

  1. 1. Diagnosis of Infectious Diseases Dr. G. V. Mali Bharati Vidyapeeth’sMBSK KanyaMahavidyalaya, Kadegaon. Dist. Sangli 415304. Maharashtra ( India)
  2. 2. Laboratory diagnosis of infectious diseases1- Microscopic examination of the clinical specimen2- Isolation of the culture & its identification based on biochemical reactions3- Serological identification / Immunological identification of Ags or Abs.4- Nucleic acid based / Molecular biology techniques( Among these 1 & 2 are conventional methods )
  3. 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. 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. 5. Conventional Serological Methods1. Precipitation2. Agglutination3. Haemagglutination4. Haemagglutination inhibition5. Complement Fixation Test6. Fluorescent Antibody Test
  6. 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 gelse.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. 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, specificbacterial & Cryptococcal Ags.Rocket immunoelectrophoresis – Quantitative estimation of Ags
  8. 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.
  9. 9. Passive Agglutination
  10. 10. 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
  11. 11. 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
  12. 12. 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.
  13. 13. Direct and Indirect FAT
  14. 14. New immunological methods /Immunoassays 1. Enzyme linked immunosorbant assay ( ELISA ) 2 . Radioimmunoassay1.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.
  15. 15. Three ways of performing ELISA – ANTI-HUMAN1.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.
  16. 16. 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
  17. 17. 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.
  18. 18. ELISA kits are available for both clinicaldiagnostics and home use. These tests are usedfor everything from screening blood for anti-HIV antibodies to home pregnancy tests.
  19. 19. 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.
  20. 20. 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.
  21. 21. 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):
  22. 22. 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.).
  23. 23. 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.
  24. 24.  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.
  25. 25.  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.
  26. 26. 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.
  27. 27. RFLPGGATCCCCTAGG
  28. 28.  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 .
  29. 29. 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
  30. 30.  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
  31. 31. In situHybridization
  32. 32. 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 .
  33. 33.  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.
  34. 34. 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
  35. 35. 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.
  36. 36.  THANK YOU

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