HIV-Current diagnostic approach

Dr Sarada Devi K.L

Professor
Dept of Microbiology

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Luc Montagnier & Francoise Barre Sinoussi

Isolated HIV in 1983

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 HIV and AIDS
The cellular and immunological picture - The course of the disease

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 Infection by HIV

Acute Sero conversion Illness

Asymptomatic Phase

Symptomatic Infection/ AIDS

Death

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HIV genome

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 Purpose of HIV Testing
• To identify asymptomatic individuals
• To diagnose HIV infection in those who
practice high risk behavior
• To prevent secondary transmission
• Donor screening for blood & tissue products
• For prophylaxis, Medical management,
Treatment
• For epidemiological surveillance
• To diagnose clinically suspected cases

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 National guidelines for HIV testing

National testing policy reiterates the following:

- No individual should be made to undergo a
mandatory testing for HIV
- No mandatory HIV testing should be imposed as a
precondition for employment or for providing health
care services and facilities
- Any HIV testing must be accompanied by a pretest
and post test counseling services

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 Testing strategies
• Unlinked & anonymous– Surveillance
• Voluntary & confidential
Asymptomatic
AIDS cases
Research
• Mandatory– Transfusion safety

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 Testing strategies

• Surveillance – by two tests based on

different antigen preparations/ principle
• Transfusion safety –single test.
• Voluntary – three different tests ELISA/Rapid/
Simple (E/R/S) based on different antigen
preparations /principle

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 Sequential HIV testing strategy
Why?
• To maximize both sensitivity & specificity for
detection of HIV
• An effective approach for diagnosis even in
low prevalence population
• High positive predictive value

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 Lab diagnosis

• Antibody detection
• Antigen detection
• Detection of viral nucleic acid
• Viral isolation
• Indirect predictors of HIV infection

HIV testing & counseling should always be

voluntary & confidential

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 Laboratory diagnosis of
HIV infection

• Direct demonstration of infective agent

- Virus isolation- virus culture
- Antigen detection- P24 detection
- viral nucleic acid detection- PCR

• Indirect demonstration of infective agent

-Anti -HIV antibody detection

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 Specimens to be collected for
Antibody detection
• Blood / Serum / Plasma
• Saliva / Urine

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 Window period
• Early ELISA & WB- 2.1 months(3wks- 3 mths)

• Sandwich ELISA (III gen ELISA)- 6wks

• IV generation ELISA- 16-18 days

• NAT- 12-14 days

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Early infant diagnosis by DBS

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•

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 Tests to detect HIV Antibodies
• Screening tests (ELISA, Rapid, Simple)
– ELISA (2-3 hours)
– Rapid tests (minutes)
• Dot blot assays
• Particle agglutination
• HIV Spot tests
– Simple (½ hour )
• Based on ELISA principle

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HIV - ELISA

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ELISA Washer

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ELISA READER

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 Rapid tests
Advantages:
1. Quick
2. Easy to perform
3. No sophisticated instruments are required
4. Can be done on single sample

Disadvantages:
1. Costly
2. Tedious if large no. samples have to be tested at one
time

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 The ICTC (integrated counseling & testing
centre) or Jyothis

• The ICTC (integrated counseling & testing

centre) or Jyothis
The main elements of ICTC :
• Pre-test counseling
• Testing
• Post-test counseling
• Partner notification
• Follow-up and referral

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 Rapid Tests
• Rapid tests can employ a variety of techniques
including:
– Particle agglutination
– Lateral flow membranes
– Through flow membranes
– Comb-dipstick based systems

• Most have sensitivities and specificities of 99%

and 98% respectively
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 Principle: Flow-through Devices
Micro particles immobilized in
membrane form test spots
Control
antibody:
HIV-2 peptide
HIV-1 peptide

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 Discriminating
TRIDOT Rapid tests

Tridot assay: HIV-1 www.similima.com

and 2 antibodies 27
Retroquic & HIV Comb

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Immunocomb

Con
HIV1
HIV2

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• Once established HIV seropositivity is typically
life long
Exceptions
1.Late in the course of infection
2.Rapid progressors
3.Early introduction of ART

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 Confirmatory Tests
Western Blot, Line immunoassay

• WB use antigens from whole virus lysates

electrophoretically transferred to a membrane

• LIA use recombinant or synthetic HIV antigens

mechanically applied on to support membrane

• Presence or absence of bands is scored

• Highly specific, Labor intensive, expensive -WHO criteria-

presence of at least 2 envelope bands (gp120, gp160, gp41)

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 Western blot
Gp 160
Gp 120

p66
p55
p51

Gp 41

p31
p24

p17
+ ve Indeterminate - ve
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Western blot

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Direct Methods of HIV diagnosis: p24 antigen
detection
• EIA for detection of p24 antigen in serum, plasma, CSF or cell
culture

• can detect infection in window period, in late stage of

disease ,and in newborns
• To monitor response to anti-retroviral therapy
• To monitor disease progression
• Sensitivity is limited (only 69% in patients with AIDS and low
in neonates < 1 month old)

• Negative test does not rule out HIV infection

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 Detection of HIV nucleic acid (RNA or
DNA)
• Polymerase chain reaction (PCR)
– To detect and quantitate the viral nucleic acid in infected
lymphocytes in blood, in serum and in culture supernate
• Application of PCR
– HIV detection in newborn
– Window period
– Resolution of indeterminate ELISA/WB
– Characterization of isolates
– Measurement of virus load.

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 Advantages of PCR over other
techniques
• Capable of detecting proviral DNA
• Highly sensitive can detect 10 ng of DNA
• Nucleic acid can be detected in fresh / archival
samples
• Results within few hours
• Less expensive than virus culture
• PCR requires less sample material

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 Viral Load
• Quantitative measurement of the numbers of
HIV- 1 RNA copies in a patient’s sample
• Usually the viral load test is carried out in
peripheral blood
• The test results are expressed “HIV RNA
copies per milliliter of plasma or body fluids”

Slide 37
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 virus isolation
• HIV can be cultured from blood (PBMC), semen, vaginal/cervical specimen,
tissue, CSF and plasma

• Direct stimulation of patient’s lymphocytes or co-cultivation of patient’s

lymphocytes with healthy lymphocytes

• 98% positivity

• Virus can be isolated in window period

• Procedure is expensive, labour intensive and time consuming

• Procedure is used only in research settings

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 Virus isolation

• CO-CULTURE METHOD – PBMCS from

heterologous HIV un-infected donors are
stimulated with PHA and after 48–72hrs the
stimulated cells are cultured along with the
PBMCS from the patient

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 Staging and progression of HIV / AIDS

• Clinical – markers (AIDS indicator diseases)

• Viral load estimation
• Surrogate markers of disease progression
– ↓CD4 cells  P24 antigen
– ↓P24antibody  serum neopterin
 2 microglobulin
–  neutrophils  platelets  Hb  ESR
–  IL 2 receptors

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 HIV Diagnosis during window period
• Need for laboratory diagnosis in window period
-Following untested blood transfusion
-Risky heterosexual/homosexual exposure
-Needle stick injury
• By demonstrating virus and virus components
-PCR
-p24 antigen assay (40%)
-Viral culture

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 Paediatric HIV Testing
• Only virological based tests (searching for
virus particles) such as:
– nucleic acid detection
• polymerase chain reaction (PCR)
• RT-PCR
• Nucleic acid sequence based assays (NASBA)
– viral culture and p24 antigen testing (dissociation
of Ag-Ab complexes)
will prove if they are infected or not as
maternal antibodies may take up to 18
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 Drug resistance & HIV
• with HIV, 2007 AdultsHIV…
• evolves rapidly within human body
• has a high replication rate
• has a high mutation rate

• Resistant strains can emerge within days if drug pressure is
not sufficient to suppress replication.
• Resistant strains persist indefinitely and can re-emerge if
same drugs are stopped and restarted (even if they are not
detected by standard resistance assays).
• and Children Estimated to be
Living with HIV, 2007
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 HIVDR is inevitable consequence of
ART

• It is impossible to eliminate or completely

prevent drug resistance, it is possible &
necessary to minimize drug resistance

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 In Which Conditions is DR More Likely?

• Treatment with <3 drugs

• Inappropriate selection of drugs
• Adding one drug to a failing regimen
• Interruption of treatment (even for a few
days)
• Prolonging a failing regimen

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 Advantages of drug testing

• Shows which drugs not to use

• Save costs associated with switching drugs too
early or using drugs that are no longer
effective
• Avoid toxicity of inactive drugs
• May avoid further development of resistance

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 Types of drug resistance assay

• Genotypic Testing: Prediction of drug

susceptibility based on sequence
• Phenotypic Testing: Measure of susceptibility to
specific drugs
– Recombinant Assays: RT/PCR portion of patient
virus and transfer into a vector
– PBMC Assay: Culture virus from patient
• Largely replaced by recombinant assays due to
difficulties in reproducibility.

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• Genotyping
advantages
• Can be performed rapidly (days)
• Relatively inexpensive
• Available in many labs
disadvantages
• Does not directly measure susceptibility
• Sometimes difficult to interpret results
• Not all patterns of resistance mutations are known (esp. for
new drugs and combinations)
• Generally qualitative

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 Phenotyping
advantages
• Direct measure of drug susceptibility
• Quantitative
• Can immediately test new RT and PR inhibitors
disadvantages
Longer time to obtain results (weeks)

Relatively complex technology

More expensive than genotypic assays
Available in fewer labs
Clinical
cutoff values for drug resistance not clearly
defined for all drugs
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