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AIDSA Global ConcernTODAY THERE IS NO REGION OF THEWORLD UNTOUCHED BY PANDEMIC OFAIDS
Majority of persons infected with HIVpresent with AIDS soon or later.
Why AIDS is more importantTODAY Apart from Men, there is a increasing burden ofInfections in Women. Implication on Mother to Child Transmission Female Adolescents are infected 3 - 6times higher than male counterparts.AIDS has turned out to be SocialProblem.
Implication of AIDS Being diagnosed as having HIV/AIDS haslife time repercussions. Every body understands AIDS as a lifethreatening condition. A casual way of dealing as being HIV + willhave Moral, Legal, and Socialconsequences.
Prompt and Precise Diagnosis ofAIDS make great impact on Humanity
Importance of precise Diagnosis ofAIDS All patients should be informed aboutconsequences of Testing for HIV infection, Legality of respective National Laws should befollowed. A pretest and posttest counseling is Growingimportance, even in Developing countries. Today’s debate on testing for HIV status asmatter of Health protocol ?
Counseling Improves the confidence
Purpose of Testing for HIV Infection A matter of great importance as in Blood donorsto 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 Childtransmission. Patients presenting with opportunistic infections.
Diagnosis ofHIV/ AIDSBasic Tests.1.We can detect Antigen or Antibody2.We can detect Antigen and Antibody.3.Majority of Laboratories depend onCommercial Kits.4.Devloped Nations going for Molecular andmore precise Methods.
Screening Tests for HIV/AIDSDetection 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 evenfor Diagnosis.
Confirmatory Tests in AIDS It is important to confirm all screening testswith Confirmatory tests, or we brand someone without infection as infected, Confirmatory tests differentiates falsereactive tests. and identifies truly infectedor not.
Differences of Screening andConfirmatory Tests.Screening testHigh degree ofSensitivityFew false negativesConfirmatory testHigh degree ofspecificityFew / No false positiveresults.
Why patients should we tested withScreening and Confirmatory tests. Before you declare a person infected withHIV / AIDS you should perform bothmethods. Faulty testing methods can lead tocatastrophic consequences, and legallitigations.
Choosing Screening Testing methods Our aim to obtain 100 % specific results ? But impossible. As few may be infected and do not havesufficient antibodies to detect in the testingmethods. Depend on time tested methods. Explore the reputation of the suppliers.
ELISA METHOD Universally accepted test ,most populareven in the developing nations. Useful in large scale screening. A common method used in Blood banks inmass 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 tonew generation of testing methods Today we depend on 3rd and 4th generationELISA methodologies.
Significance of ELISA Most reliable screening test for HIVinfection. 50% of the infected will show positivereactivity 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 havebecome obsolete we have to switch to Antigen sandwich ELISA protocols ( calledas 3rd generation ELISA ). Many Nations have switched to 3rdgeneration methods.
What is 3rd Generation ELISA Helps in detection of early infections, Can detect all classes of antibodiesincluding Ig M All isotypes of antibodies can be captured. But proving to be expensive to developingNations.
Need for 4thGeneration ELISAMethodsThe quest for better Diagnosis ofAIDScontinues
What is 4th generation ELISA. A new generation Method in ELISA Can detect both Antigen and Antibody inthe same run ( in the same ELISA Plate ) Helpful to make early diagnosis at least infew cases.
Determination of P 24 Antigen byELISA Helps in the early detection of HIV infection. Screening of Blood for HIV infection along withdetection for antibody detection. Early diagnosis of HIV infection in resource poornations. Monitoring Anti-retroviral therapy. But not areplacement for RNA quantization. Even P 24 Antigens are subject of false positiveresults.
Limitation of Screening Tests. Imperfect Sensitivity, Sensitivity to detect HIV2 ? Failing to detect HIV variants. Other technical errors.
ConfirmatorySerological TestsAll the Screening tests need to beconfirmed before being declared asHIV infected.
Confirmatory Serological Tests. HIV Western Blots. Line Immunoassay ( LIA ) Immuno fluorescent Assay ( IFA )Utility is limited due technical andeconomical reasons
WESTEN BLOT TEST A Gold standard confirmatory test. When tested combined with ELISAspecificity is > 99.9 %.
Relation of HIV structure and WesternBlot 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 reactwith at least two of following antigens,gp 160/120 If does not meetgp 41 requirements, markedp 24 as indeterminate.
Negative Western Blot. Absence of any reactivity to thebands is declared as Negative.False positives are rare.All doubtful results should bereported as Negative.
Profile of Western Blot Test
Limitation of Western Blot Test If not designed for HIV 2 inclusion, we miss HIV2infections, Can give Indeterminate results inPregnancyAfter administration of TetanusToxoid.Autoimmune conditions.
Rapid tests in the Diagnosis of AIDS A growing importance Results can be issued within < 20 minutes. Limited protocols, and less demandingtechnical skills. But needs confirmation with ELISA /Western Blot testing. Can differentiate HIV 1 and HIV 2.
Rapid Testing for AIDS with DotMethods
Dot / Rapid MethodsUseful In patients in Labor whose Immune statusis not known, Resource poor establishments. But needs confirmation with ELISA / WB
Other supportingtests in AIDSSeveral tests will help in supportingto identify the progression ofDisease.
Flowcytometry for Estimation ofCD 4 lymphocytes
Use of CD4 counts in Progress ofAIDS CD4 count measurements help for stagingthe diseases. Flow cytometry has become the prominenttool in estimations of CD4. Specimens should be promptly processed.
Uses of Absolute CD4 Lymphocytecounts. Most widely used predictor of HIVprogression. Risk of progression to an AIDS definingillness, opportunistic infections ormalignancy is high, when the counts dropbelow 200/mcl.
Uses of CD4 Lymphocyte percentage. Estimation of percentages is more usefulthan CD4 counts. When the counts drops < 20% we have towatch for onset of opportunistic infectionsand malignancy.
Molecular MethodsinDiagnosis of AIDS
Molecular Methods in Diagnosis ofHIV / AIDS.1. The reverse transcription polymerasechain reaction.2. Nucleic acid sequenceamplification. ( NASBA )3. Branched chain DNA bDNA.
DNA –PCR ( RT-PCR) popularamong Molecular Method
Polymerase Chain Reaction.-RT- PCR RT-PCR Need the use of Reverse transcriptaseenzyme that converts RNA to DNA. The testing is based on artificially replicatingNucleic acid sequenceof the target ( viral sequence ) so thatMillions to Billions are made availablefor detection.
HIV Viral Load tests. The tests measure the amount of activelyreplicating HIV virus. Correlates with response to antiretroviraldrugs Most helpful in diagnosis of HIV beforeSero conversion. False positives should be possible with lowviremia < 500 copies.
Alternative Testing Media.From other Specimens. Saliva Detection of Ig G Urine Detection of Ig GBoth ELISA and Western Blotcan be performed.
Reporting of all Positive results is agreat concern, Avoid casual reporting
Post test counseling a Must in allPositive results
Precautions in HIV Testing. Casual testing to be curtailed. To do with a purpose. HIV positive result has Moral,social,andEconomic consequences. Post test counseling a must in all HIV+individuals.