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Diagnosis of HIV and HCV
- 1. IMMUNE & MOLECULAR DIAGNOSTICS CONVENTIONAL& MOLECULARDIAGNOSISOF HIVAND HCV A PRESENTATION DONE BY TAMILSELVAN.D BMS14330 IV YEAR M.SC BIOMEDICAL SCIENCE
- 2. The Human Immunodeficiency Virus HIV is a retro virus infection with which leads to a condition called Acquired ImmunoDeficiency Syndrome (AIDS) It is spherical shaped virus with a diameter of 132-146nm The virus is transmitted mainly sexual contact and also by means of Blood transfusion. It is transmitted from an infected mother to a child by placenta and breast feeding AIDS is a non-communicable disease that cause the depletion of Helper T Lymphocytes (CD4 T cells) which play an essential role in the activation of immune system It is considered as a life threatening disease as the immunodeficiency caused by this will lead to other infections such as Tuberculosis
- 3. The most common symptoms of HIV infection are Fever Night sweat Joint pain Sore throat Loss of weight and CD4 count of less than 200 cells / microliter of blood
- 4. Diagnosis of HIV Conventional methods ELISA Western Blot Molecular methods HIV quantitative nucleic acid assay RNA load assay
- 5. Conventional methods ELISA The Enzyme Linked Sorbet Assay for the diagnosis of HIV infection is the commonly used screening test Diagnosis of HIV by ELISA involves the detection of Antibodies in the serum that are specific to the viral antigens Protein 24(P24) which makes up the viral core, Glycoproteins 160 & 120 This involves coating the antigens on the wells of 96 well plate When serum is added to the well the primary antibody (if present) that is specific to the antigens interact to form an Antigen-Antibody complex It is followed by the addition of secondary antibody which is conjugated with an enzyme most commonly HRP or ALP
- 6. The plate is then washed in order to remove the unbound secondary antibodies Then a chromogenic substrate is added which produces the colour
- 7. Western Blot It is the widely used confirmatory test for HIV diagnosis This is an electrophoretic technique which also detects the antibodies specific to viral antigens The serum sample is subjected to SDS-PAGE that separates the serum proteins based on molecular weight This separated proteins are transferred into a nitrocellulose or PVDF membrane using western blotting When the antigens are added to the membrane agglutination happens if the antibodies specific to those antigens are present
- 8. molecular methods Qualitative Nucleic Acid Assays This method involves the detection of HIV pro-viral DNA and HIV RNA from the blood sample The detection of pro-viral DNA and RNA involves PCR and RT-PCR techniques respectively To detect the pro-viral DNA the total DNA from blood sample is isolated and subjected for PCR with the primers specific for the pro-viral DNA Whereas to detect the HIV RNA total RNA is isolated using Guanidium thiocyanate extraction method. The isolated RNA is then subjected to RT-PCR
- 9. Quantitative Nucleic Acid Assays The most commonly employed assay for the quantification of HIV RNA is Cobas Amplicor HIV load assay The main specimen for this assay is plasma how ever other specimen such as CSF, Saliva can also be used As the RNA is an unstable molecule it is mandatory that the sample to be tested is stored at -70˚C prior to testing This method involves the following steps
- 10. Automated isolation of total RNA from the sample RT-PCR to amplify the HIV RNA Followed by the quantification of HIV RNA using fluorescent dye labelled oligonucleotide probes by including a Quantitation standard of known copy number Initially this test is done manually and was capable of detecting only about 400 copies of HIV RNA from per 1ml of sample But with advancements in the technologies and usage of automated techniques the limit has been increased to one million copies/ml of sample
- 11. Advantages of molecular methods over conventional methods Conventional methods can not determine the stage of the disease There are possibilities of getting false positive results in conventional methods due to manual errors Molecular methods are more sensitive because it identifies a target molecule that is present in a very minute quantity
- 12. The Hepatitis c virus HCV is a small enveloped ssRNA virus belonging to the viral family Flaviviridae It is also a spherical shaped virus with a diameter of about 55-65nm It is responsible for hepatitis and some times hepatic carcinoma HCV is transmitted by blood contact via transfusion, IV drug administration, poorly sterilized medical equipment and also by lactation There is no effective vaccine for this virus till date
- 13. Hepatitis C is almost asymptomatic Complications such as fever, dark urine, abdominal pain and yellow coloration of skin and eyes are rarely reported
- 14. Diagnosis of HcV Conventional methods ELISA Western Blotting Point of care Rapid assay Molecular methods NAT assay Qualitative assay Viral Genotyping
- 15. conventional methods ELISA In HCV diagnosis ELISA is used to detect the presence of antibodies against the core, non structural 3, non structural 4 and non structural 5 antigens in the serum Western blotting This assay is often termed as Recombinant Immunoblot Assay (RIBA) This is based on the interactions of antibodies blotted on a membrane with recombinant HCV antigens Usually three antigens are used for this and if the antibodies are present for at least two antigens the test is considered positive
- 16. Point of care rapid assay This is a kit based method that can diagnose the HCV infection very rapidly In this method whole blood sample is used only a few drops are needed so the blood obtained by finger prick is more than enough A few drops of blood is added to the test device and the device is then dipped into a buffer solution containing the HCV antigens This is followed by the incubation at room temperature for 20-40 minutes The result is observed either as reactive or non reactive
- 18. molecular methods NAT assay It is nothing but a RT-PCR amplification of HCV RNA For this method liver biopsy is done From the liver tissue total RNA is isolated Then it is subjected for RT-PCR by using specific primers Quantitative assay It is similar to the Cobas Amplicor method of quantifying HIV RNA The detection limit of this method is 10 IU -10 million IU of HCV RNA /ml of sample
- 19. Viral Genotyping Till date there are seven genotypes and 80 subtypes of HCV Viral genotyping is done by sequencing of the cDNA fragments obtained by reverse transcription of the specific regions viral RNA Sanger Coulson method of gene sequencing is most commonly used to sequence the cDNA fragments
- 20. Limitations of conventional methods The conventional methods are very sensitive with a sensitivity range of 95%. But it has a certain limitations as follows They could not differentiate whether the infection is current or resolved These tests cannot be used in immunocompramized people and people who undergo hemodialysis It can not detect the infection in a early stage