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Diagnosis of

Diagnosis of

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Diagnosis of

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  1. Diagnosis of AIDS Dr.T.V.Rao, MD.

  2. AIDS A Global Concern TODAY THERE IS NO REGION OF THE WORLD UNTOUCHED BY PANDEMIC OF AIDS

  3. Majority of persons infected with HIV present with AIDS soon or later.

  4. Why AIDS is more important TODAY • Apart from Men, there is a increasing burden of Infections in Women. • Implication on Mother to Child Transmission • Female Adolescents are infected 3 - 6 times higher than male counterparts. AIDS has turned out to be Social Problem.

  5. Implication of AIDS • Being diagnosed as having HIV/AIDS has life time repercussions. • Every body understands AIDS as a life threatening condition. • A casual way of dealing as being HIV + will have Moral, Legal, and Social consequences.

  6. Prompt and Precise Diagnosis of AIDS make great impact on Humanity

  7. Importance of precise Diagnosis of AIDS • All patients should be informed about consequences of Testing for HIV infection, • Legality of respective National Laws should be followed. • A pretest and posttest counseling is Growing importance, even in Developing countries. • Today’s debate on testing for HIV status as matter of Health protocol ?

  8. Counseling Improves the confidence

  9. Purpose of Testing for HIV Infection • 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.

  10. Diagnosis of HIV/ AIDS Basic Tests. 1.We can detect Antigen or Antibody 2.We can detect Antigen and Antibody. 3.Majority of Laboratories depend on Commercial Kits. 4.Devloped Nations going for Molecular and more precise Methods.

  11. Screening Tests for HIV/AIDS Detection • A screening test posses high Sensitivity • We rarely miss the Diagnosis in Infected patients. • Done as mass screening procedure. • Economical. • Developing countries depend on these tests even for Diagnosis.

  12. Confirmatory Tests in AIDS • 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.

  13. Screening test High degree of Sensitivity Few false negatives Confirmatory test High degree of specificity Few / No false positive results. Differences of Screening and Confirmatory Tests.

  14. Why patients should we tested withScreening and Confirmatory tests. • Before you declare a person infected with HIV / AIDS you should perform both methods. • Faulty testing methods can lead to catastrophic consequences, and legal litigations.

  15. Choosing Screening Testing methods • Our aim to obtain 100 % specific results ? • But impossible. • As few may be infected and do not have sufficient antibodies to detect in the testing methods. • Depend on time tested methods. • Explore the reputation of the suppliers.

  16. ELISA METHOD • Universally accepted test ,most popular even in the developing nations. • Useful in large scale screening. • A common method used in Blood banks in mass screening of Human blood.

  17. ELISA method – A ELISA plate

  18. Sequence of Events in ELISA test

  19. Different Methods of ELISA testing.

  20. Why ELISA is very trust worthy. • Easy to perform • Free from Radiation. • Relatively specific • Sensitive. • With scientific advances we have advanced to new generation of testing methods • Today we depend on 3rd and 4th generation ELISA methodologies.

  21. Significance of ELISA • Most reliable screening test for HIV infection. • 50% of the infected will show positive reactivity in < 22 days. • 95% show reactivity < 6 weeks • Sensitivity > 99.9 % • But needs confirmation with Western Blot.

  22. Advances in ELISA Methods. 1st and 2nd generation ELISA methods have become obsolete we have to switch to • Antigen sandwich ELISA protocols ( called as 3rd generation ELISA ). • Many Nations have switched to 3rd generation methods.

  23. What is 3rd Generation ELISA • Helps in detection of early infections, • Can detect all classes of antibodies including Ig M • All isotypes of antibodies can be captured. • But proving to be expensive to developing Nations.

  24. Need for 4th Generation ELISAMethods The quest for better Diagnosis of AIDS continues

  25. What is 4th generation ELISA. • A new generation Method in ELISA • Can detect both Antigen and Antibody in the same run ( in the same ELISA Plate ) • Helpful to make early diagnosis at least in few cases.

  26. Determination of P 24 Antigen by ELISA • Helps in the early detection of HIV infection. • Screening of Blood for HIV infection along with detection for antibody detection. • Early diagnosis of HIV infection in resource poor nations. • Monitoring Anti-retroviral therapy. But not a replacement for RNA quantization. • Even P 24 Antigens are subject of false positive results.

  27. Limitation of Screening Tests. • Imperfect Sensitivity, • Sensitivity to detect HIV2 ? • Failing to detect HIV variants. • Other technical errors.

  28. Confirmatory Serological Tests All the Screening tests need to be confirmed before being declared as HIV infected.

  29. Confirmatory Serological Tests. • HIV Western Blots. • Line Immunoassay ( LIA ) • Immuno fluorescent Assay ( IFA ) Utility is limited due technical and economical reasons

  30. WESTEN BLOT TEST • A Gold standard confirmatory test. • When tested combined with ELISA specificity is > 99.9 %.

  31. Relation of HIV structure and Western Blot configuration

  32. Principle of Western Blot • HIV viral antigens are separated as; gp160,gp120,p66,p55,p51,gp41,p31,p24, p17, and p15. Interpretation depends on presence / absence of reactivity to specific antigens.

  33. Interpretation of Western Blot. • The antibodies in the serum should react with at least two of following antigens, gp 160/120 If does not meet gp 41 requirements, marked p 24 as indeterminate.

  34. Negative Western Blot. • Absence of any reactivity to the bands is declared as Negative. False positives are rare. All doubtful results should be reported as Negative.

  35. Profile of Western Blot Test

  36. Limitation of Western Blot Test • If not designed for HIV 2 inclusion, we miss HIV2 infections, • Can give Indeterminate results in Pregnancy After administration of Tetanus Toxoid. Autoimmune conditions.

  37. Rapid tests in the Diagnosis of AIDS • 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.

  38. Rapid Testing for AIDS with Dot Methods

  39. Dot / Rapid Methods Useful • In patients in Labor whose Immune status is not known, • Resource poor establishments. • But needs confirmation with ELISA / WB

  40. Other supporting tests in AIDS Several tests will help in supporting to identify the progression of Disease.

  41. Flowcytometry for Estimation of CD 4 lymphocytes

  42. Use of CD4 counts in Progress of AIDS • CD4 count measurements help for staging the diseases. • Flow cytometry has become the prominent tool in estimations of CD4. • Specimens should be promptly processed.

  43. Uses of Absolute CD4 Lymphocyte counts. • 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.

  44. Uses of CD4 Lymphocyte percentage. • Estimation of percentages is more useful than CD4 counts. • When the counts drops < 20% we have to watch for onset of opportunistic infections and malignancy.

  45. Molecular Methodsin Diagnosis of AIDS

  46. Molecular Methods in Diagnosis of HIV / AIDS. 1. The reverse transcription polymerase chain reaction. 2. Nucleic acid sequence amplification. ( NASBA ) 3. Branched chain DNA bDNA.

  47. DNA –PCR ( RT-PCR) popular among Molecular Method

  48. Polymerase Chain Reaction.-RT- PCR • RT-PCR Need the use of Reverse transcriptase enzyme that converts RNA to DNA. • The testing is based on artificially replicating Nucleic acid sequence of the target ( viral sequence ) so that Millions to Billions are made available for detection.

  49. HIV Viral Load tests. • The tests measure the amount of actively replicating HIV virus. • Correlates with response to antiretroviral drugs • Most helpful in diagnosis of HIV before Sero conversion. • False positives should be possible with low viremia < 500 copies.