Laboratory diagnosis and monitoring of HIV

HIV
Laboratory diagnosis
and monitoring
Nguyen Thi Bich Huyen
1
1. Thet Su Win
2. Truong Van Hau
3. Le Van Chuong
4. Trinh Xuan Son
5. Nguyen Thi Bich Huyen
6. Myo Htet Thu
7. Saw Thu Wah
Group 2

2
Detection methods of
laboratory diagnosis
laboratory monitoring
Diagnosis Algorithm
Diagnosis Algorithm of
babies < 18 months
Contents

3
laboratory diagnosis

4
 Can be used in both clinical and nonclinical
 Four rapid HIV antibody tests approved by US
FDA
1. Oraquick rapid HIV-1/2 antibody tests
2. Reveal rapid HIV-1 antibody tests
3. Uni-Gold recombigen HIV test
4. Multispot HIV-1/HIV-2 rapid test
Rapid HIV tests

6
Serum, plasma
Screens for HIV-1
Perform test in 5 min
Reactive Negative
Reveal HIV-1 antibody test

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 Whole blood, serum, plasma
 Screens for HIV-1
 Results in 10 minutes
Uni-Gold Recombigen

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 Serum, plasma
 Distinguishes HIV-1 from HIV-2
 Perform test in 15 minutes
Negative Positive
Multispot HIV-1/HIV-2

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A rapid review of rapid HIV antibody tests, Current Infectious Disease Reports,
2006, Vol 8-2, pp 125-131
US FDA–approved rapid HIV antibody tests
for HIV-1 detection

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 Direct assay for HIV antibody
 Modification with the use of recombinant protein derived
from HIV-1 genome
 That are linked to polystyrene beads.
 High sensitivity
 High specificity
 Simple
 Rapid
Rapid Latex agglutination Assay

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 Oral Fluid
 Urine (IgG Ab to glycoprotein gp120 and
gp160)
 Vaginal mucosa (high risk seronegative
subjects had IgA in their genital mucosa)
Alternative Antibody Testing

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HIV Antibodies HIV-1 RNA HIV p24 Antigen
Most Common Test for
Established Infection
Rarely Used
Future use: 4th Generation
EIA
Used for Acute HIV and
Indeterminate WB
http://www.cdc.gov/
Types of HIV Diagnostic Tests

13
ELISA is the most commonly used type of test to
screen for HIV infection because of
 Its relatively simple methodology
 Inherent high sensitivity
 Suitability for testing large numbers of
samples
Enzyme-Linked Immunosorbent Assays
(ELISA)

14
 The sample with an unknown
amount of antigen is
immobilized on a solid support.
 The detection antibody is
added, forming a complex with
the antigen.
 The detection antibody can be
linked to an labeled molecule.
Enzyme-Linked Immunosorbent Assays
(ELISA)

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First Second Third *Fourth
Uses crude viral lysate Detects IgM and IgG in
“Sandwich” EIA
Uses recombinant HIV
antigens or peptides
Detects HIV antibodies
and p24 antigen
http://www.cdc.gov/
Generation of EIA Tests

Principle of ElectroChemiLuminescence assay
• ElectroChemiLumin
escence (ECL) is an
immune assay.
• ECL is based on the
use of a ruthenium-
complex and
tripropylamine
www.nature.com

17
 RT-PCR
 End-point assays
 Real-time assays
HIV nucleic acid detection

18
 Branched-chain DNA (b-DNA)
HIV nucleic acid detection

20
 Protein detection technique that combine the
separation power of SDS-PAGE together with
high recognition specificity of antibodies
 Identification is base on 2 properties:
+ Molecular weight
+ Antibody binding specificity
Western blotting

21
 The most common of the highly specific tests
used in confirmatory testing.
 Western blot HIV tests usually look for antibodies
against the following HIV proteins
Western blotting

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SDS-PAGE
1. Separation of proteins using
SDS-PAGE
2. Transfer of the proteins onto
membrane (nitrocellulose or PVDF)
3. Detection
Western blotting

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Africa Australia UK
USA
CDC1
USA
CDC2
USA
FDA
USA
Red
Cross
ENV
gene
gp160
gp120
gp41
Any two
One or
more
One or
more
gp160
gp120
& p41
gp160
gp120
& p41
One or
more
One or
more
POL
gene
p68
p53
p32
Optional Any three
p31* p31*
Any
one
GAG
gene
p55
p24
p17
p24 p24 p24
Any
one
 GAG: p17 [p18], p24, p55 (core)
 POL: p32 [p31] (Endonucleases)
p53, p65 [p68] (Reverse transcriptase)
 ENV: gp41 (Transmembrane protein)
gp120, gp160 (Envelope unit)
Varying Criteria for a Positive HIV
Western Blot

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laboratory monitoring

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Laboratory monitoring
 CD4+ T cell counts
 HIV RNA determinations
 HIV resistance testing
 Co-receptor tropism assay
 HLA-B* 5701 Screening
 Prognosis
 Monitoring response
to therapy
 Therapies
(Drugs) Option

26
CD4 T cell counts
Aims to:
 Monitor function of immune system
 Stage disease ( AIDS stage: CD4 T cells < 200 cells/mm3)
 Guidance on treatment therapies
CD4 T cell thresholds Clinical guidance
< 500 cells/mm3 Start to use ART (Antiretroviral)
< 200 cells/mm3 Start to use OI prophylaxis for PCP
(Pneumocystis jiroveci pneumonia)
< 50 cells/mm3 Start to use OI prophylaxis for MAC
(Mycobacterium avium complex )
OI: Opportunistic illness

27
CD4 T cell counts
Methods: Flow cytometry

28
CD4 T cell counts
When to be used
After
diagnosed
HIV infection
Before
applied ART
therapy
Every 3 to 6
months when
starting ART

29
HIV RNA determinations
Monitor
effectiveness
of therapy
Objectives
Real Time
PCR
Methods
• Before starting ART
• Every 3-6 months after
starting ART
When to use

30
HIV resistance testing
When to use
 HIV patients: not response to ART
Aims
 Detect drug-resistant in HIV infection
Method
Genotypic assay
(Sequencing method)
 Detect mutation
Phenotypic assay
 Measure drug
susceptibility

31
Phenotypic assay
Reverse
Transcription
Reverse
Transcription
Patient Virus Viral RNA Viral cDNA Amplified DNA
PCR
HIV-1 resistance
plasmid
HIV-1 resistance
Recombinant
Transfection
Quantification
& report
http://www.monogrambio.com/hiv-tests/phenotypic-assays
Susceptibility testing
(Series of drug dilution
+ Fixed viral titration )
CD4 MT4 cell
IC50
Calculation
( IC50,
Resistance)

32
Co-receptor tropism assay
 A blood test identifies strains of HIV by tropism
Virus only uses
CCR5=R5 Tropic virus
Virus only uses
CXCR4=X4 Tropic virus
Rapid
progression
Slow
progression
Virus that can use either receptor
= Dual Tropic virus
http://www.monogrambio.com/hiv-tests/tropism
HIV

33
Co-receptor tropism assay
HIV-1
from PBMS
MT-2 cells
(Human T-cell)
SI: Syncytium
induction
NSI: No Syncytium
induction
SI (With X4 HIV) NSI (Without X4 HIV)
MT-2 cell assay
Principle:
 Detect CPE (Cytopathic
effect) via microscope
 Used to detect X4 HIV
http://www.natap.org/2008/ResisWksp/ResisWksp_56.htm

34
HLA-B57 Screening
Aims:
 Detect B57 allele
 Avoid prescribing Abacavir
HLA-B35 Px
Rapid progression
(2-3 years)
 HLA-B37
 HLA-B57
Low progression
Allergic
hypersensitivity
to Abacavir
Methods:
 PCR
 Sequencing

36
 Proposed by CDC and APHL in March, 2010.
 Updated by Diagnostic Conference 2012.
 Guideline entitled by CLSI
HIV Laboratory Diagnostic
Testing Algorithm

37
 Initial testing with 4th generation HIV-1/2 antigen/antibody
combination immunoassay (IA).
 Reactive result followed by HIV-1/2 antibody differentiation
assay.
 Negative or indeterminate result undergo HIV-1 nucleic
acid test(NAT).
 Advantages
 Over conventional algorithm followed by Western Blot
Comfirmation- repeatedly reactive results
 Accurately classify HIV-1 and HIV-2 infection.
Algorithm Recommends

41
 Some general guidelines to follow
1. Lab should specify which assay are used.
2. If use the substituted assays for
recommended algorithm,should describe the
limitations.
3. If the entire algorithm is not
completed,should specify which tests are
pending or additional specimens required.
Guidance on Reporting to Health
Care Providers

43
Diagnosis Algorithm of
babies < 18 months

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HIV infection in infant
and children
http://www.tensteps.org/breastfeeding-hiv-pmtct.shtml
 During pregnancy, delivery and post partum through
breastfeeding, or through parenteral exposure.

45
Early diagnosis, treatment and
safe feeding can reduce mortality
and morbidity
http://degrees.fhi360.org/2012/12/preventing-mother-to-child-
transmission-of-hiv-in-zambia-replicating-success/
http://www.topnews.in/healthcare/content/21468mother-child-hiv-
transmission-decline-us-more-can-be-done
Preventing mother to child
transmission (PMTCT)

46
Serological test
 Maternal HIV antibody can persist for 18 months
 Positive test - confirm HIV exposure, but not definite
diagnosis, need virological confirmation test
 Negative test - suggest unexposed or uninfected
- risk of HIV in breastfeeding
 Determine Ag-Ab combo test
- have not been evaluated for early infant
- can detect acute infection in adult
HIV test for baby <18 months

47
Virological test
 HIV DNA from whole blood or dry blood spot
(DBS) - PCR
 HIV RNA from plasma - RTPCR
 p24 antigen from plasma and DBS-
Immunoassay

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Virological test
 HIV DNA from whole blood - PCR
 HIV RNA from plasma or dry blood spot
(DBS) - RTPCR
 P24 antigen from plasma and DBS-
Immunoassay

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Dry blood spot sample collection

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Status age Remark
Exposed infant 4-6 weeks Virological test (+)indicate infected in
utero
>9 month Recommend to perform serological
test, If (+) result – do virological test
Breastfeeding 6 week
after
cessation
of breast
feeding
Need age-appropriate retesting
Infant still be at risk of acquiring HIV
Non breastfed/
Never breastfed
At / above
9 month
Serological test negative rule out HIV
infection

51
Immediately start ART
Virological test (+)
Second sample for confirmation
Negative in 1/3 of child(+) confirmed
DBS not recommend
Third sample to solve
previous discordance results
HIVDNA test is needed for
reconfirmation and
before ART discontinued

52
Adult <18 month of
age
Serological test Diagnosis test Not reliable for
diagnosis
Virological test For monitoring
treatment
For confirmation
and monitoring
treatment
Ag/Ab combo
test
Use for detect
early infection
Have not been
validated

Laboratory diagnosis and monitoring of HIV

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