HIV TESTING PROTOCOLS
D.Vijay kumar
Dist.Sales Manager
Abbott
Hyderabad.
Informed consent prior to testing is essential to
deal with issues likeConfidentiality
Discrimination
Victimization
Psychological harm
* HIV testing – voluntary
Mandatory testing – counterproductive
Purpose of HIV testing
 Transfusion safety
 Epidemiological - Sentinel surveillance
 Diagnostic purpose
 Voluntary testing
 Research
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
Serological Profile of HIV infection
Tests for detecting HIV INFECTION
 Detection of specific antibodies
screening tests: 100% sensitive
supplemental/ confirmatory testssens. > 99.8%
spec. > 98.5%
 Detection of specific antigens
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
SPECIFICITYAccuracy 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
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
Predictive values
PPV – identifies ACTUALLY infected
individuals
TP
X 100
TP + FP
NPV – identifies ACTUAL non infected
TN
X 100
TN + FN
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
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DISADVANTAGES
Many false positives
Few false negatives
Time consuming
Needs infrastructure & tech. expertise
PRINCIPLE OF INDIRECT ELISA
Substrate
Enzyme conjugated
Anti-HIV antibody in specime
Antigen
On solid phase
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
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
Comb AIDS
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
TRIDOT
 HIV 1 – gp 41, gp 120.
 HIV 2 – gp 36.
 Highly specific.
C
1
2
RETROQUIC HIV
HIV EIA Comb
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
REACTIVE
HIV-1
HIV-2
HIV-1&2
INVALID TEST
SUPPLEMENTAL TESTS

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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.
Western Blot for HIV
 Delineates the antibody
profile of reactive serum
WB Rotator platform
 Used to grade intensity of ab
response
– Qualitatively
– Quantitatively
 Procedure based on principle
of ELISA
WB strips
Western blot for HIV antibody
gp 36
Control band
p 24
gp 31
gp 41
gp 120 & 160
TESTS DONE IN VCCTC, PPTCT & BLOOD BANK
VCCTC & PPTCT:
Test – I:
HIV Comb / Comb Aids – RS
Test – II:Tridot / Retroquic
Test – III:
EIA Comb
FOR SURVEILLANCE:
Test – I:
HIV Comb / Comb Aids – RS
Test – II:Tridot / Retroquic
BLOOD BANK:
Only Test:
Microlisa HIV
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
A1 +
A1 positive
negative
Strategy/Algorithm II A (for surveillance)
2 test kits required
A1
A1 +
A1 Report negative
A2
A1 + A2 +
A1+ A2 Report positive Report negative
Strategy/Algorithm II B
(Diagnosis of an individual with AIDS indicator disease
symptoms)
3 test kits required
A1
A1 +
A1 report negative
A2
A1+ A2 +
report positive
A1+
A2 -
A3
A1+ A2 – A3+
indeterminate
A1+ A2 - A3report negative
Strategy/Algorithm III
To detect HIV infection in asymptomatic individuals (VCTCs, PPTCTs)
3 test kits required
A1
A1+
A1 -
Report negative
A2
A1+ A2 –
A1+ A2 +
A3
A1+ A2+ A3+
Report positive
A1+ A2+ A3indeterminate
A1+ A2- A3+
Indeterminate
A3
A1+ A2- A3report negative
 INDETERMINATE STATUS: Repeat test after 14-28 days.
Results continue to be indeterminate – WB/PCR
refer to NRL.
 EQUIVOCAL WB: Rpt. WB after 2 weeks
4 weeks
12 weeks
one year.
Correlate with high risk behaviour &
clinical
parameters.
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 2.
Failure to detect Ag when complexed with Antibody
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
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.
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.
Diagnosing HIV infection in an infant born to
HIV positive mother
Protocol 1
Asymptomatic
18 months-ELISA
Non-reactive
Conditionally consider
Non-infected
Re-test
NR
HIV Unifected
Reactive
confirm with a retest
PROTOCOL 2
SYMPTOMATIC
Early symptoms
Earlier than 12 mon.
Viral assay at 6 weeks
Whenever symptoms occur
Retest 1 month later
If both are reactive consider
reactive
AIDS
defined symptoms
same as for early
symptoms
ELISA at 12 months
Non reactive
Follow protocol 1
Retest at 18 months
Non reactive
HIV UNIFECTED
Reactive
conditionally consider HIV +ve
retest at 18 months
reactive
HIV INFECTED
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 14-28 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.