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Laboratory diagnosis of HIV infection.


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laboratory diagnosis of HIV infection.
different tests performed.
strategies in performing tests.

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Laboratory diagnosis of HIV infection.

  1. 1. LABORATORY DIAGNOSIS OF HIVTarun Prudvi B MBBS 2nd Professional
  2. 2. HIV AIDS has turned out to be Social Problem. Global Problem. So precise diagnostic methods and strategies are designed now.
  3. 3. HIV A pretest and post-test counseling is Growing importance Counseling gives the confidence!
  4. 4. PURPOSE OF TESTING FOR HIV • A matter of great importance as in Blood donors to prevent infected blood being transfused. • To diagnose the patients infected with HIV virus. • For surveillance purpose. • Persons with high risk behavior. • In Pregnant women to prevent Mother to Child transmission. • Patient's presenting with opportunistic infections. • For persons who need to undergo a surgery. Tarun Prudvi BHIV
  5. 5. TYPES OF TESTS Specific tests and Nonspecific tests. In India, National AIDS Control Organisation (NACO) to ensure quality and uniformity has provided guidelines for conducting serological tests and their interpretation. Tarun Prudvi BHIV
  6. 6. Antigen detection Specific HIV Tests Antibody detection Cultivation of Virus P24 Ag detection Screening- ELISA; Sandwich ELISA, Indirect ELISA, Competitive ELISA. RAPID TESTS; Cassette ELISA, Particle agglutination, Dot Blot. Confirmatory/Supplementary- Immuno Blot, Western Blot, RIPA, IFA HIV
  7. 7. The HIV virus has several antigens GP120, GP41, p24, p31, p18, p15. P24 is the earliest to appear in blood. (Major core antigen); In cases of small viral load, like needle prick injury it takes much longer time and appearance of p24, viremia and IgM coincides with seroconversion illness. P24 antigen will be reactive only after 48 hrs. SPECIFIC TESTS / Antigenic detection 1 HIV
  8. 8. Free p24 antigen disappears later and appears during the end stage while remaining absent through out the long asymptomatic stage. However, antibody bound p24 is demonstrable after dissociation. The p24 capture essay that uses anti-p24 antibody can be used to demonstrate this. It is positive in 30% cases. It is positive in first few weeks after infection and in terminal phase it is uniformly positive. It can be used for screening blood donors. SPECIFIC TESTS / Antigenic detection 2 2 4 8 12 A B C p24 Ig M IgG Temporal sequence in appearance of p24, IgM and IgG antibodies © Tarun Prudvi B p24 can be detected in early phase of infection, it disappears later through out the asymptomatic stage. IgM can be detected 4-6 weeks, followed by IgG. HIV
  9. 9. In the HIV viral infection the antibodies are not readily detected in the serum immediately after infection. It needs at least 2-8 weeks to several months to be detected. During which the antibodies cannot be identified even though the individual is highly infectious- the “WINDOW PERIOD”. ( In window period the infection can be detected by p24 assay.) Once they appear they increase in titre. And IgM disappears following the appearance of IgG. So, antibody testing will have to be done after 2-6 months to ascertain whether the infection has occurred or not. SPECIFIC TESTS / Antibody detection 1 HIV
  10. 10. SPECIFIC TESTS / Antibody detection 2 Serological tests of HIV are of two types: screening test and Confirmatory/supplementary tests. Screening Tests: ELISA, Rapid tests, simple tests They are not always positive may give false positive results. All positive results must be rechecked before sample is confirmed positive. HIV Screening test High degree of Sensitivity Few false negatives Confirmatory test High degree of specificity Few / No false positive results.
  11. 11. ELISA- Indirect ELISA, Sandwich ELISA, Competitive ELISA A common method used in Blood banks in mass screening of Human blood. Useful in large scale screening. SPECIFIC TESTS / Antibody detection 3 This is inexpensive, but false positive results may occur. Tarun Prudvi BHIV
  12. 12. 1st generation All antigens used to bind HIV antibodies, indirect immunoassay format employs labeled antihuman IgG for detection of IgG antibodies. 2nd generation Synthetic peptide or recombinant protein antigens alone are used to bind HIV antibodies. Design of the specific antigenic epitopes improves sensitivity for HIV-1 group O and HIV-2 3rd generation Synthetic peptide or recombinant protein antigens are used to bind HIV antibodies in an immunometric antigen sandwich format. This allows detection of IgM and IgG antibodies. Lower sample dilutions and the ability to detect IgM antibodies (which are expressed before IgG antibodies) increase sensitivity during early seroconversion. 4th generation Synthetic peptide or recombinant protein antigens are used in the same antigen sandwich format as 3rd generation assays to detect IgM and IgG antibodies, and monoclonal antibodies are also included to detect p24 antigen. Inclusion of p24 antigen capture allows detection of HIV-1 infection before seroconversion. Evolution of HIV Immunoassay Technology HIV
  14. 14. A growing importance Results can be issued within < 20 minutes. Limited protocols, and less demanding technical skills. But needs confirmation with ELISA / Western Blot testing. Can differentiate HIV 1 and HIV 2. RAPID TESTS Cassette ELISA, Immune chromatography, Coated particle agglutination tests, Dot Blot test SIMPLE TESTS They are also based on ELISA principle. Takes 1- 2 hrs. HIV
  15. 15. In patients in Labor whose Immune status is not known, Resource poor establishments. But needs confirmation with ELISA / WB DOT BLOT TEST
  16. 16. It is important to confirm all screening tests with Confirmatory tests, or we brand some one without infection as infected, Confirmatory tests differentiates false reactive tests and identifies truly infected or not. CONFIRMATORY/ SUPPLEMENTARY TESTS 1 / Western blot test 1. HIV proteins are separated on polyacrylamide gel by electrophoresis 2. Blotted on to strips of nitrocellulose paper. 3. These strips are reacted with the test sera 4. The anti-human globulins are added with attached enzyme and suitable substrate added, produces colour HIV
  17. 17. CONFIRMATORY/ SUPPLEMENTARY TESTS/ Western Blot 2 The antibodies in the serum should react with at least two of gp160/120, gp41, p24 antigens. If does not meet requirements, marked as indeterminate. / INTERPRETATION
  18. 18. SPECIFIC TESTS / Antibody detection 3 False positive results: A positive HIV test may be obtained in the absence of HIV antibodies in blood. • Influenza immunisation may temporarily cause false positive result • Autoimmune disorders RA /SLE • Presence of autoantibodies against lymphocytes • Sera stored for longer durations All positive results must be conformed by other method. Other tests in Antibody detection RIFA IFA Immuno Blot HIV
  19. 19. Recommended Laboratory HIV Testing Algorithm for Serum or Plasma Specimens HIV
  20. 20. Reporting of all Positive results is a great concern, Avoid casual reporting
  21. 21. PCR Viral Load Tests CD4 Count Prognostic Tests HIV
  22. 22. Nucleic acid test (NAT) Nucleic-acid-based tests amplify and detect one or more of several target sequences located in specific HIV genes In the RT-PCR test, viral RNA is extracted from the patient’s plasma and is treated with reverse transcriptase (RT) to convert the viral RNA into cDNA. The polymerase chain reaction(PCR)process is then applied. After PCR is complete, the resulting DNA products are hybridized to specific oligonucleotides bound to the vessel wall, and are then made visible with a probe bound to an enzyme. HIV
  23. 23. Once infected the virus is present in circulation and in fluids. During asymptomatic phase the viral titres are low and are antibody bound. Their titres remain high in early infection and in the end stage. Thus, infected person is infectious through out the life, infectivity being high in early and end stages. It can be isolated by co-cultivation of patients lymphocytes and uninfected lymphocytes and the reverse transcriptase activity can be observed. However, it is not used as a routine diagnostic test. Viral isolation HIV
  24. 24. Viral Load tests HIV-1 RNA Polymerase chain reaction (PCR) Multiplies amount of HIV RNA in a blood sample through use of an enzyme. The resultant chemical reaction marks the virus and the markers are measured to calculate the amount of HIV-1 RNA Branched chain DNA (bDNA) Uses a substance that produces light when it combines with HIV particles. The amount of light is measured and converted to a viral count Nucleic acid sequence-based amplification (NASBA) Continues amplification of nucleic acid sequences Advantage is that it is more sensitive and gives faster results HIV
  25. 25. Steep rise in plasma HIV-1 RNA levels that reach a peak of between 105 and 106 copies/ml approx. 2 weeks after infection. Once the host defences are mobilized against the virus, there is a slow decline to a steady-state viral load, or set point, of between 104 and 105 copies/ml at approx. 4 months post infection. Viral load in ADULTS Viral load in infected infants may rise above one million copies/ml within weeks of infection and remain at this level during first year of life. The reported levels of HIV-1 RNA viral loads in infants during primary and early infection appear to be higher than those seen in adults. A reduction in viral load produced by antiretroviral therapy is a reliable marker of reduced risk of disease progression HIV
  26. 26. When the counts drops < 20% we have to watch for onset of opportunistic infections and malignancy. Most widely used predictor of HIV progression. Risk of progression to an AIDS defining illness, opportunistic infections or malignancy is high, when the counts drop below 200/mcl. CD4 Counts Detected by Flowcytometry HIV < 200 : Die 80% within 4 yrs. 200 - 400 : Die 50% within 4 yrs.
  27. 27. Positive HIV NAT results at any age should be confirmed by repeat testing as soon as possible on a new sample. Two independent positive test results definitively diagnose paediatric HIV infection in HIV-exposed infants and subsequent testing is not necessary. HIV nucleic acid testing (NAT) to detect HIV RNA or DNA should be performed for early diagnosis of paediatric HIV infection at the following ages • Within 48 hours of birth • At 2 weeks of life • At 4 to 6 weeks of life • At 4 to 6 months of life EVALUATION IN PAEDIATRIC AGE GROUP HIV Two negative HIV NAT results, one obtained >4 weeks of age and one obtained >4 months of age, definitively exclude paediatric HIV infection in HIV-exposed infants.
  28. 28. State of infection p24 Anti HIV IgG Anti HIV IgM Western Blot pattern Early infection - - - - Acute (seroconversion illness) + to - - To + + Partial: p24 and gp120 Carrier asymptomatic - + - Full pattern PGL + + - LOSS OF P24/ P55 AIDS + ABSENCE OF P24; LOSS OF OTHER REACTIVITIES Evolution of serological markers in HIV infection
  29. 29. Other tests /findings Total Leukocyte count and lymphocyte count <2000 cells per T cell counts; CD4+T cell count <200 cells per Thrombocytopenia Raised IgG & IgM Levels. Mantoux –ve (diminished CMI in the fourth stage) IgG IgM ThrombocytopeniaCD4+ T cell decreased CMI
  30. 30. Hb To assess Anaemic conditions in AZT (azidothymidine; Zidovudine) treatment. Bone marrow suppression. Serum alanine or aspartate amino transferase Assess possible hepatitis coinfection And monitoring liver toxicity Serum creatinine / BUN Renal function assessment / renal toxicity Serum glucose Hyperglycaemia and DM in cases of PI based regimen (Atazanavir, Indinavir) OTHER TESTS Tarun Prudvi BHIV
  31. 31. Post test counseling a Must in all Positive results
  32. 32. HIV infections cause production of antibodies and viral products which can be used to make diagnosis Elisa and rapid tests are commonly used for diagnosis for HIV CD4 counts can be used as a guide for initiating prophylactic medications and ART to provide information on the efficacy of ARV’S and to monitor HIV progression. Viral Load tests can be used for early detection of new HIV infections, for determination of response to therapy and sometimes for when to initiate therapy. SUMMARY Tarun Prudvi BTarun Prudvi BHIV
  33. 33. AIDS is Caused by fascinating Virus to - Scientists. Friendly in approach to - Risk group. Dangerous to Life to - Infected.