Diagnosis of

AIDSDr.T.V.Rao, MD.

AIDS A Global Concern

TODAY THERE IS NO REGION OF THE WORLD UNTOUCHED BY PANDEMIC OF

AIDS

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

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.

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.

Prompt and Precise Diagnosis of AIDS make great impact on Humanity

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 ?

Counseling Improves the confidence

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.

Diagnosis of HIV/ AIDS

Basic Tests. 1.We can detect Antigen or Antibody2.We can detect Antigen and Antibody.3.Majority of Laboratories depend on

Commercial Kits.4.Devloped Nations going for Molecular and

more precise Methods.

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.

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.

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.

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.

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.

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.

ELISA method – A ELISA plate

Sequence of Events in ELISA test

Different Methods of ELISA testing.

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.

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.

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.

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.

Need for 4th Generation ELISA

Methods

The quest for better Diagnosis of AIDS

continues

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.

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.

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

Confirmatory Serological Tests

All the Screening tests need to be confirmed before being declared as

HIV infected.

Confirmatory Serological Tests. HIV Western Blots. Line Immunoassay ( LIA ) Immuno fluorescent Assay ( IFA )

Utility is limited due technical and economical reasons

WESTEN BLOT TEST A Gold standard confirmatory test. When tested combined with ELISA

specificity is > 99.9 %.

Relation of HIV structure and Western Blot configuration

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.

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.

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.

Profile of Western Blot Test

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.

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.

Rapid Testing for AIDS with Dot Methods

Dot / Rapid Methods Useful

In patients in Labor whose Immune status is not known,

Resource poor establishments. But needs confirmation with ELISA / WB

Other supporting tests in AIDS

Several tests will help in supporting to identify the progression of

Disease.

Flowcytometry for Estimation of CD 4 lymphocytes

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.

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.

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.

Molecular Methodsin

Diagnosis of AIDS

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.

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

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.

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.

Alternative Testing Media.From other Specimens.

Saliva Detection of Ig G Urine Detection of Ig G

Both ELISA and Western Blot

can be performed.

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

Post test counseling a Must in all Positive results

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.

AIDS is

Caused by fascinating Virus to - Scientists,

Friendly in approach to - Lovers.

Dangerous to Life to - Infected.

Dr.T.V.Rao,MD

STOP AIDS. Dr.T.V.Rao MD. Professor of Microbiology e mail tvraodoctor2000@yahoo.co.in