Diagnosis of Infectious Diseases Dr. G. V. Mali Bharati Vidyapeeth’sMBSK KanyaMahavidyalaya, Kadegaon. Dist. Sangli 415304. Maharashtra ( India)
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 )
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.
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).
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
- 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
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.
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
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
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.
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.
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.
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
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.
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.
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.
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.
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):
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.).
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.
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.
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.
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.
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 .
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
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
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 .
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.
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
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.