1. D.Vijay kumar Dist.Sales Manager Abbott Hyderabad.
HIV TESTING PROTOCOLS
D.Vijay kumar
Dist.Sales Manager
Abbott
Hyderabad.

2. * HIV testing – voluntary Mandatory testing – counterproductive
Informed consent prior to testing is essential to deal with issues like-
Confidentiality
Discrimination
Victimization
Psychological harm
* HIV testing – voluntary
Mandatory testing – counterproductive

3. Purpose of HIV testing

4. Transfusion safety
Epidemiological - Sentinel surveillance
Diagnostic purpose
Voluntary testing
Research

5. PPTCT Pregnant HIV infected women can-
make informed decisions about dealing with pregnancy
receive appropriate and timely interventions to decrease MTCT
ensure safe delivery
secure early access to HIV care and treatment
educated in prevention of HIV transmission
receive follow-up health care for self and child
Pregnant HIV non infected women can be educated and counselled to remain uninfected

6. Serological Profile of HIV infection

7. Tests for detecting HIV INFECTION
Detection of specific antibodies
screening tests: 100% sensitive
supplemental/ confirmatory tests-
sens. > 99.8%
spec. > 98.5%
Detection of specific antigens

8. Accuracy with which a test can confirm the presence of an infection.
SELECTION OF TESTS : BASED ON SENSITIVITY ,SPECIFICITY,EFFICIENCY,PPV & NPV
Sensitivity –
Accuracy with which a test can confirm the presence of an infection.
Test with high sensitivity – few false negatives TP
Sensitivity = x 100
TP + FN

9. SPECIFICITY-
Accuracy with which the test can confirm the absence of an infection
test with high specificity – few false positives
used for diagnosing infection in an individual TN
Specificity = X 100
TN + FP

10. Efficiency - ability of a test to correctly identify -
all positives as positives
all negatives as negatives
TP + TN
Efficiency = X 100
TP + FN + TN + FP

11. Predictive values
PPV – identifies ACTUALLY infected individuals TP
X 100
TP + FP
NPV – identifies ACTUAL non infected TN
TN + FN

12. ELISA Most common screening test
Indirect solid phase enzyme linked immunosorbent assay/ EIA
Used in blood banks/ tertiary labs
Fourth generation ELISA decreases window period
DISADVANTAGES
Many false positives
Few false negatives
Time consuming
Needs infrastructure & tech. expertise

13. PRINCIPLE OF INDIRECT ELISA
Substrate
Enzyme conjugated
Anti-HIV antibody in specimen
Antigen
On solid phase

14. ELISA cont. False positives: auto immune diseases multiple pregnancies
hematologic malignancies
primary biliary cirrhosis
alcoholic hepatitis
CRF
False negatives
window period
immunosuppressive therapy
malignant disorders
late stage disease
technical errors

15. Rapid tests Dot blot assay- immuno concentration method
Retroquic -line assay
Tridot - dot assay
Immunocomb assay – dipstick/comb
ELISA based
HIV comb
coomb AIDS
Immunochromatography – lateral flow assays
Determine,Unigold,Hemastrip
Particle aggluttination – Capillus,Serodia
ELISA based - EIA

16. Comb AIDS NON-REACTIVE INVALID TEST Dot immunoassay for HIV 1&2 using
whole blood,serum or plasma.
Comb with 8 teeth- Megenta red spot
Synthetic& recombinant peptides used
Two spots-- Control spot & test spot.
NON-REACTIVE
REACTIVE
For HIV-1 &2
INVALID TEST

17. TRIDOT
HIV 1 – gp 41, gp 120.
HIV 2 – gp 36.
Highly specific. C 1 2

18. RETROQUIC HIV

19. HIV EIA Comb

20. INTERPRETATION OF RESULTS
HIV EIA Comb
Rapid Visual EIA Test
Detection of Antibodies to HIV-1 (including
subgroups O & C) and HIV-2
Sensitivity (100%)
Specificity (99.9%)
INTERPRETATION OF RESULTS
NON-REACTIVE
HIV-1
HIV-2
HIV-1&2
REACTIVE
INVALID TEST

21. SUPPLEMENTAL TESTS 1.Rapid tests. 2.Western blot. 3.Immunoblot.
4.Line immunoassay.
WB/IB/LIA: highly specific but Expensive Labour intensive Needs expertise Equivocal/indeterminate results.

22. Western Blot for HIV Delineates the antibody profile of reactive serum
Used to grade intensity of ab response
Qualitatively
Quantitatively
Procedure based on principle of ELISA
WB Rotator platform
WB strips

23. Western blot for HIV antibody
gp 36
Control band
p 24
gp 31
gp 41
gp 120 & 160

24. TESTS DONE IN VCCTC, PPTCT & BLOOD BANK
Test – I: HIV Comb / Comb Aids – RS
Test – II: Tridot / Retroquic
Test – III: EIA Comb
FOR SURVEILLANCE:
BLOOD BANK:
Only Test: Microlisa HIV

25. STRATEGIES/ALGORITHMS OF HIV TESTING
1.Screening ELISA/Rapid tests – used in strategy I, II, III.
2.Supplemental tests – E/R & Western Blot.
Strategy I (for transfusion/transplantation safety)
one test kit required AI
A A positive negative

26. Strategy/Algorithm II A (for surveillance) 2 test kits required A1
A A Report negative A2
A1 + A A1+ A Report positive Report negative

27. Strategy/Algorithm II B (Diagnosis of an individual with AIDS indicator disease symptoms) test kits required A1
A A report negative A2
A1+ A A A report positive A3
A1+ A2 – A A1+ A2 - A indeterminate report negative

28. Strategy/Algorithm III To detect HIV infection in asymptomatic individuals (VCTCs, PPTCTs) test kits required A1
A A Report negative A2
A1+ A A1+ A2 – A3
A1+ A2+ A A1+ A2+ A A Report positive indeterminate
A1+ A2- A A1+ A2- A Indeterminate report negative

29. INDETERMINATE STATUS: Repeat test after 14-28 days
INDETERMINATE STATUS: Repeat test after days Results continue to be indeterminate – WB/PCR refer to NRL.
EQUIVOCAL WB: Rpt. WB after 2 weeks weeks weeks one year Correlate with high risk behaviour & clinical parameters.

30. TESTS TO DETECT ANTIGENS
P24 Antigen: Uncomplexed in serum, plasma, CSF, cell culture Indicates Active infection especially in newborn Resolves equivocal WB Window period CNS disease Immune collapse Monitoring response to ART.
Method: EIA
Disadvantages: Expensive Limited sensitivity Failure to detect HIV Failure to detect Ag when complexed with Antibody

31. Limitations of antigen detection methods
Not reliable
Expensive
Limited sensitivity-69% in patients with AIDS,15% in neonates
Detection not possible in patients with high anti p24 antibody
Cannot be used as a screening test

32. PCR Highly specific test-more than 95%
Highly sensitive-infants over 1 month
Detects proviral DNA .
Detects both latent viral infection and active viral transcriptipn.
Detects viral load.
Detects both HIV1 & HIV2.

33. PCR AS VIRAL ASSAYS IN INFANTS
Counselling for infant feeding & therapeutic intervention.
First done at 6 weeks.
Not Breast-fed, to say not infected: 2 negative test after 1 month (include 1 at 4 months)
Not done as part of PPTCT in India.
If symptoms occur at < 18 months: go for viral assays.

34. Diagnosing HIV infection in an infant born to HIV positive mother
Protocol 1
Asymptomatic
18 months-ELISA
Non-reactive Reactive
Conditionally consider confirm with a retest
Non-infected
Re-test NR
HIV Unifected

35. PROTOCOL 2 SYMPTOMATIC Early symptoms AIDS
Earlier than 12 mon defined symptoms
Viral assay at 6 weeks same as for early
Whenever symptoms occur symptoms
Retest 1 month later
If both are reactive consider
reactive
ELISA at 12 months
Non reactive Reactive
Follow protocol conditionally consider HIV +ve
Retest at 18 months retest at 18 months
Non reactive reactive
HIV UNIFECTED HIV INFECTED

36. HIV TESTING POLICY IN PPTCT
PARENT: Informed consent of the patient Pre n post test counselling Routine testing with three rapid tests first: highly sensitive test-NR-reported with exception of WP Indeterminate: 1st test reactive, 2nd/3rd NR repeat test after days WB/PCR: For persistent indeterminate cases.
INFANT OF HIV + MOTHER, ASYMPTOMATIC: ELISA at 18 months—NR—Retest—NR—uninfected Reactive—consider infected, confirm with retest.
INFANT OF HIV + MOTHER, SYMPTOMATIC:
ELISA at 12 months—NR—retest NR—uninfected.
Reactive—retest R at 18 months—infected.