15. lab diagnosis of hiv

Presenter: Dr G Santhipriya
Moderator: Dr Rajesh T Patil
Integrated seminar
Dept. of pathology and microbiology
Lab diagnosis of HIV
7/25/20191 Integrated seminar-PESIMSR

Contents
 Background
 Course
 Collection, Storage, and Transport –Specimens
 Serological Diagnosis
 Molecular & Other Assays

Contents
 National Strategies and Algorithms
 Reference laboratories
 Legal and Ethical issues
 Summary
 References

Background- Structure of HIV

Background- Lifecycle of HIV

Course - HIV infection

Lab Diagnosis of HIV Infection
 Pre test counselling, informed consent- documented
 Specific exceptions for informed consent
 Post test counselling
 Confidentiality

Blood Collection, Storage & Transport- specimen
Serological test- serum/ plasma/ whole blood
CD4 enumeration test, DNA/ RNA PCR -only whole
blood in k2/k3 EDTA
Dried blood spots

Blood Collection, Storage & Transport-
specimen
Sera: 2o to 8o C , a week.
For longer storage of serum and plasma, frozen at –
20o C
Repeated freeze-thawing should be avoided
CD4 enumeration: should not refrigerate

Blood Collection, Storage & Transport- specimen

Modalities of tests
 Serological
Antibody detection
Antigen detection
 Molecular method
 PCR

Tests
 Specific
Screening
Confirmatory
 Nonspecific
Complete hemogram
Skin test for diminished CMI
Lymp node biopsy
 Monitoring
CD 4 counts
Viral load assay

Screening
 ELISA
 Rapid tests
Immunoconcentration/Dot Blot assay (vertical flow)
Agglutination assay
Immunochromatographic assay (lateral flow)
Dipstick and comb assay based on Enzyme Immune
Assay (EIA) 7/25/201914 Integrated seminar-PESIMSR

Confirmatory
 Western blot
 NAT
Qualitative detection of HIV-1 DNA or RNA,
Quantitative detection of HIV-1 RNA (viral load
determination)
 Virus isolation
 Antigen detection(P 24 antigen assay)

Screening tests

Enzyme Linked Immunosorbent Assay (ELISA)
Types
 Indirect
 Competitive
 Sandwich
 Capture 7/25/201917 Integrated seminar-PESIMSR

Indirect ELISA
 Most commonly used principle
 Colour change directly proportional to the
concentration of specific antibodies in the specimen

Competitive ELISA
 Advantage -Less time than indirect ELISA
 No pre-dilution of test specimen is required
 Low OD readings –HIV reactive
 High OD readings – HIV non-reactive

Sandwich ELISA
 Modification to improve sensitivity and specificity of
the indirect ELISA
 Advantage- all classes of HIV antibodies can be
detected
 Antibody sandwich ELISA’s that detect p24 antigens
are available

Capture ELISA
 Advantage –more specific than an indirect assay
 Developed to test specimens with low concentrations
of HIV antibodies- urine and saliva
 To detect a specific class of antibodies- IgG, IgM or
IgA

Factors which may affect the test results
Pre-analytical
 Haemolysed sample
 Grossly lipaemic samples
 Repeated freezing and thawing
 Contaminated samples and reagents
 Improperly stored, expired and deteriorated reagents

Factors which may affect the test results
Analytical Post-analytical
 Pipetting errors
 Improper incubation time
and temperature
 Improper washing
procedure
 Carry over from the
adjacent specimen
 Equipment malfunction
 Glove -powder aerosol
 Calculation errors
 Transcription errors

Results
False positive False negative
 Autoimmune diseases
 Alcoholic hepatitis
 Primary biliary cirrhosis
 Leprosy
 Multiple pregnancies
 Technical errors
 The test may be
negative during the
window period and
during the end stage of
the disease

Generation of anti-HIV antibody tests

Rapid Anti-HIV Tests
 Visual point of care tests, that do not require any
special equipment
 Smaller test packs
 Small number of samples
 Technically simple to perform
 Can be stored at an ambient temperature(20o c to 25o c)

Rapid Anti-HIV Tests- types
 Immunoconcentration / Dot blot immunoassay
(vertical flow)
 Immunochromatographic assay (lateral flow)
 Particle agglutination (e.g., gelatine or latex)
 Dipstick and Comb assay based on Enzyme Immune
Assay(EIA)

Immunoconcentration / Dot Blot immunoassay (vertical
flow)
 Solid phase immunoassay
 HIV antigens are immobilized on a porous membrane
 Conjugate binds to the Fc portion of the HIV
antibodies – coloured dot

Immunochromatography Tests
 Nitrocellulose strip
 Antigen and signal reagent- incorporate

Interpretation of Immunochromatography
tests

Particle Agglutination Tests
 Antibody detection
 Antigen is coated on a carrier particle and the antigen
antibody reaction is observed in clumps
 Carriers- red cells, latex particles, gelatine particles
and micro beads

Particle Agglutination Tests- interpretation
Lattice
network

Dipstick and Comb Assay
 Antigens “spotted” on a solid support
 Enzyme substrate reactions
 Production of colour at the site of antigen spotting
 Differentiate between HIV-1 and HIV-2 antibodies

Disadvantage of rapid antibody tests
 Antibodies are not detectable in the window period and
children<18 months of age
 Each test device cannot be quality controlled with an
external quality control sample
 External controls(known positives and negatives)
should be incorporated along with every batch of 15
samples or on a daily bases- in case the turn over of
samples is more than 15 per day

Disadvantage of rapid antibody tests
7/25/2019Integrated seminar-PESIMSR35
 Accuracy – different kits should be used in
combination based on testing strategies/ algorithm
 Every kit used should be monitored, from time to time,
for its performance using internal and external controls

Differences
ELISA RAPID TEST
Definition A plate based assay technique
designed to detect and quantify
substances such as peptides,
proteins, hormones, antigens and
antibodies
A rapid preliminary immuno
based screening test that utilizes
the same technique of ELISA but
with less sophisticated equipment
Specificity Very specific Comparatively less specific
Equipment
needed
Highly sophisticated equipment
are needed
Less sophisticated equipment
with non- regular maintennance
is needed
Sensitivity Highly sensitive Comparatively less sensitive
Technicians To carry out ELISA, skilled and
highly trained technicians are
required
No such requirement of highly
skilled technicians
Diagnostic
duration
Time consuming A fast approach with a time
period of 10-120 min7/25/201936 Integrated seminar-PESIMSR

Confirmatory tests

Western Blot Test
 Various HIV specific
recombinant or synthetic
antigens are adsorbed onto
nitrocellulose paper
 WB tests are a highly
specific conformational
test
 Antibodies against specific
HIV proteins

Criteria – western blot
Centre for Disease Control
and prevention
World Health
Organization
 Any two- p24, gp41, gp
120/160
 2 env with / without
gag/pol
Env ( gp160, gp 120, and gp 41)
gag ( p55, p 24, and p17)
Pol ( p66, p51, and p31)

Interpretation Criteria – western blot
Organization Criteria
ASTPHLD/ CDC
(Association of state and territorial
public health laboratory directors/
centres for disease control and
prevention
Any two: p24, gp41,
gp120/160
Du Pont p24+p31+gp41 or gp120/160
ARC( American red cross) Three or more bands, one
from each gene product
Group: gag, pol, env
CRSS(Consortium for retroviruses
serological standardization)
Two or more bands: p24 or
p31 and gp41 or gp120/160
WHO(World Health Organization) Two env bands with or
without gag or pol 7/25/201940 Integrated seminar-PESIMSR

 Will same tests apply for the adults and children <18
months?

Molecular & Other Assays
 Window period
 Infants born to HIV reactive mothers
 Nucleic Acid Testing (NAT)
 Qualitative detection of HIV-1 DNA or RNA,
 Quantitative detection of HIV-1 RNA (viral load
determination)
 Virus isolation
 Antigen detection

DBS cards
 Infant below 18 months of age
 Obtain consent from the parent/guardian after pre-test
counselling

Recommendations
 Children aged 6 weeks to 4
months: heel
 Children aged 4+ to 10
months: big toe
 Children aged 10+ to 18
months: finger prick-3rd or
4th finger
 Use 2mm lancets
 Take care not to hit the
bone of the child when
pricking

DBS

Qualitative PCR for HIV DNA
 Sensitivity -90 to 100 % by the age of 4- 6 weeks
 Qualitative detection of HIV DNA
 AMPLICOR HIV-1 DNA PCR Test, ver. 1.5 (Roche)-
DBS or Whole blood collected in EDTA. The Drug
Controller General of India (DCGI)

Steps of PCR
950c
50-550c
720c

Quantitative detection of HIV-1 RNA (viral
load determination)
 Gen- probe: APTIMA HIV 1 Qualitative RNA assay
licensed by US Food and Drug administration(FDA)
 Aid in detection of acute HIV 1 infection in plasma
specimen

Quantitative HIV-1 RNAAssays
 Quantitative HIV-1 RNA assays detect plasma (cell-
free) viral RNA by using various techniques.
 The methods of amplification of HIV-1 RNA include:
1) Target amplification by
a. Reverse transcriptase PCR (RT-PCR)
b. Real time PCR (qPCR)
c. Nucleic acid sequence-based amplification
(NASBA)
2) Signal amplification by branched-chain DNA
technique (bDNA)
 Cannot detect copy number below 20 per ml

Reverse transcriptase PCR (RT-PCR)
 Amplicor HIV-1 Monitor Test, version 1.5 (Standard/
Ultrasensitive/ COBAS) (Roche)
 FDA approved- quantitation of HIV-1 RNA in plasma
 HIV-1 RNA isolation →cDNA →Amplify cDNA
→Hybridization of amplified DNA → Colorimetric
detection assay
..

Reverse transcriptase PCR (RT-PCR)
 Known number of Quantitation Standard (QS) RNA
molecules+ lysis reagent
 Extrapolation of standard curve
 Specimen prepation: manual
 Amplication : automated
 Detection: manual ELISA/ automated ELISA reader

Roche Amplicor HIV-1 Monitor Test version 1.5 &
Gene Amp PCR System 9700

ELISA plate used for HIV-1 RNA quantification. 1 ,2 and 3
rows represent controls (negative control, low positive
control, high positive control). Rows 4 to 12 represent
samples.

Real time PCR (qPCR)
 Detects- during the process of amplification
 When amplicons generated →fluorescent dye labelled
probes uncoil as they hybridize to amplicons →
separates fluorescent dye from quencher →increase
fluorescence
 Fluorescence is proportional to original amount of
HIV-1 RNA in sample

Real Time HIV-1 Amplification (Abbott)

Nucleic acid sequence-based amplification
(NASBA)
 Directly amplifies HIV-1 RNA, without a PCR
 One step sandwich hybridization procedure
 Isothermal condition
 Electrochemiluminiscence
 Samples- plasma, CSF, lymphnode tissue, genital
secretions, cells
 Molecular testing (e.g., sequencing)

Branched-chain DNA technique (bDNA)
 Signal amplification
 HIV-1 is disrupted to release the viral RNA
 Series of hybridization → capture probes →b DNA
complex
 Enzyme substrate reaction
 Chemiluminescent substrate

Comparison of FDA approved assays
Method AMPLICOR
HIV-1
Monitor 1.5
COBAS
TaqMan HIV-1
Real Time HIV-
1
NucliSENS
HIV-1 QT
VERSANT
HIV-1
RNA 3.0
Manufac
turer
Roche Roche Abbott BioMerieux Siemens
Principle Target
amplification
( quantitative
PCR)
Target
amplification (
real time PCR
with TaqMan
probes)
Target
amplification (
real time PCR
with molecular
beacon probes)
Target
amplification
( NASBA)
Signal
amplificatio
n (branched
DNA)
Dynamic
range
(copies/
ml)
Standard 1.5
(400-750,000)
ultrasensitive
1.5(50-
100,000)
48-10,000,000 48-10,000,000 80-3,470,000 75-500,000
Specime
n type
Plasma in
ACD or
EDTA tube
Plasma , DBS Plasma , DBS Plasma in
ACD, EDTA ,
or heparin
tube
Plasma in
EDTA tube

Comparison of FDA approved assays
Method AMPLICOR
HIV-1
Monitor 1.5
COBAS
TaqMan
HIV-1
Real Time HIV-1 NucliSENS
HIV-1 QT
VERSAN
T HIV-1
RNA 3.0
Specimen
volume
Standard 1.5:
0.2 ml
Ultrasensitive
1.5: 0.5 ml
0.5 ml-1 ml 0.2 ml-1 ml 1.0 ml 1.0 ml-
2.0 ml
Detected
subtypes
Group M( A-
G)
Group M(A-
D, F-H; CRF
01_AE)
Group M(A-D,F,
G,H; CRF01_AE,
CRF02_AG),
Group N, Group o
Group
M(A-G)
Group M
(A-G)
Area of HIV
genome
selected for
amplification
Gag Gag Pol Gag pol

Quantitative HIV-1 RNAAssays
 To determine viral load
 Recommended cut off is > 10,000 copies/mL for
making HIV diagnosis
 Sensitivity is 25-50 % with in 1st few days of life,
100% by 6-12 wks of age
 Quantitative HIV 1 RNA assays- sensitive and more
expensive than qualitative PCR for HIV 1 DNA

Antigen detection
 Ultrasensitive p24 assay( Perkin Elmer)
 Anti p24 antibodies / free p24 antigens in blood
 Positive p24 tests confirms diagnosis, however a
negative test does not rule out HIV infection
 Based on ELISA
 Not yet been recommended for diagnosis

Viral isolation
 Co-cultivation of peripheral blood mononuclear cells
(PBMCs)
 Mitogen stimulated PBMCs
 Up to 6 weeks in a medium containing interleukin- 2
 37°C in a 5 percent CO2 atmosphere for up to 28 days
 p24 antigen by ELISA, or reverse transcriptase activity
in culture supernatant

Viral isolation
 More disadvantages
Labour intensive
Time consuming
Expensive
Requires contaminant facilities
Limited use as research tool

7/25/2019Integrated seminar-PESIMSR65
Algorithms and National Strategies
For HIV Testing

Algorithm for HIV testing- above 18 months of age

Algorithm for HIV testing - <18 months age
HIV exposed baby (born to HIV
positive mother) or baby
referred from Medical Officer
with unknown exposure status
presents at the ICTC (at least 6
weeks old)
6 weeks to less than 6 months
Collect Dried Blood Spot (DBS)
for HIV-1 PCR Test (at ICTC)
6 months old or more
Collect blood and test for HIV
antibodies using All 3
Serological tests. Also prepare a
Dried Blood Spot (DBS) for
HIV-1 PCR Test (at ICTC) at
the same time. 7/25/201967 Integrated seminar-PESIMSR

National Strategies For HIV Testing
 Based on situation
Blood /Organ donation safety
Surveillance
Diagnosis

Strategy 1
High sensitivity

Strategy 2A
High specificity

Strategy 2B
High specificity

Strategy 3

Roles and responsibilities of laboratories under
the NACO laboratory network

Legal and Ethical Issues In HIV Testing
 Ethics of Prevention and Care
 Discrimination/ Right to Work
 Confidentiality and HIV/AIDS

Summary

References
1. National Guidelines for HIV Testing 21Apr2016
2. CDC, 1993 revised classification system for HIV infection and expanded
surveillance case definition for AIDS among adolescents and adults. MMWR
Recomm Rep. 1992. 41: p. 1-19.
3. Gottlieb, M.S., et al., Pneumocystis carinii pneumonia and mucosal candidiasis in
previously healthy homosexual men: evidence of a new acquired cellular
immunodeficiency. N Engl J Med, 1981. 305(24): p. 1425-31.
4. UNAIDS, UNAIDS Report on the Global AIDS epidemic. 2010.
5. NACO, NACO Annual Report 2012-13. 2012-13.
6. Requejo, H.I., Worldwide molecular epidemiology of HIV. Rev Saude Publica,
2006. 40(2): p. 331-45.
7. Damond, F., et al., Identification of a highly divergent HIV type 2 and proposal for
a change in HIV type 2 classification. AIDS Res Hum Retroviruses, 2004. 20(6):
p. 666-72.
8. Pereyra, F., et al., Persistent low-level viremia in HIV-1 elite controllers and
relationship to immunologic parameters. J Infect Dis, 2009. 200(6): p. 984-90.
9. Candore, G., et al., Biological basis of the HLA-B8,DR3-associated progression
of acquired immune deficiency syndrome. Pathobiology, 1998. 66(1): p. 33-7.
10. Den Uyl, D., I.E. van der Horst-Bruinsma, and M. van Agtmael, Progression of
HIV to AIDS: a protective role for HLA-B27? AIDS Rev, 2004. 6(2): p. 89-96.

References
11. Shankarkumar, U., et al., Association of HLA B*3520, B*1801, and Cw*1507
with HIV-1 infection Maharashtra, India. J Acquir Immune Defic Syndr, 2003.
34(1): p. 113-4.
12. Verma, R., et al., Distribution of CCR5delta32, CCR2-64I and SDF1-3'A and
plasma levels of SDF-1 in HIV-1 seronegative North Indians. J Clin Virol,
2007. 38(3): p. 198-203.
13. MacDonald, K.S., et al., Vitamin A and risk of HIV-1 seroconversion among
Kenyan men with genital ulcers. Aids, 2001. 15(5): p. 635-9.
14. Djoba Siawaya, J.F., et al., Correlates for disease progression and prognosis
during concurrent HIV/TB infection. Int J Infect Dis, 2007. 11(4): p. 289-99.
15. Chun, H.M., et al., Hepatitis B virus coinfection negatively impacts HIV
outcomes in HIV seroconverters. J Infect Dis, 2012. 205(2): p. 185-93.
16. Laboratory Guidelines for HIV Diagnosis in Infants and Children < 18
months, NACO 2010.
17. Operational Guidelines for Integrated Counseling and Testing Centres, NACO
2007.

 Thank you!!

15. lab diagnosis of hiv

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