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

14. Why patients should we tested with Screening 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 ELISA Methods
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/ If does not meet
gp requirements, marked
p 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 Methods in
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.

50. 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.

51. Alternative Testing Media. From other Specimens.
Saliva Detection of Ig G
Urine Detection of Ig G
Both ELISA and Western Blot
can be performed.

52. Reporting of all Positive results is a great concern, Avoid casual reporting

53. Post test counseling a Must in all Positive results

54. Precautions in HIV Testing.
Casual testing to be curtailed.
To do with a purpose.
HIV positive result has Moral,social,and Economic consequences.
Post test counseling a must in all HIV+ individuals.

55. AIDS is Caused by fascinating Virus to - Scientists,
Friendly in approach to - Lovers.
Dangerous to Life to - Infected.
Dr.T.V.Rao,MD

56. STOP AIDS. Dr.T.V.Rao MD. Professor of Microbiology
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