HIV tests in adults and children

Testing Related to HIV

in Adults & Children

and ART Monitoring


Session Objectives

At the end of the session, the participant should be able to:

• understand the general principles of HIV testing

• classify the testing procedures

• elaborate on the policy of three strategies of testing and its applications

• learn the tests for diagnosis of HIV-infection in children

• discuss the tests for monitoring disease progression in adults and children

2Testing Related to HIV

Objectives of Testing

• Diagnosis

– Adults

– Children

• Test blood/tissue/organ for transplantation safety

• Monitor trend of HIV infection and surveillance

• Research


General Principles of Testing

• Voluntary and part of overall comprehensive prevention and treatment programme

• Use of quality testing kits, standardised techniques, and QA/QC procedures

• Test kit and procedure must be appropriate to the field situation

• Cost-effective


Uses of Laboratory Testing of HIV/AIDS


• Establishing diagnosis

– Adults

– Children

• <18 months

• >18 months

• Monitoring progression of HIV disease and response to ART

– CD4 T-cell estimation (Indirect Marker)

– Viral load estimation (Direct Marker)

Types of HIV Diagnostic Tests

• HIV antibody test: Serology

• Viral antigen test: p24

• Viral isolation and culture

• Viral DNA PCR


10^6

10^2

HIV RNA Copies/ml

1 3 Wk about 6mths // 5yrs 10 yrs

Acute HIV

Opportunisticinfections

Asymptomatic

Minor HIV-relatedsymptoms

Virologic set-point Varies from patient to

patient

HIV antibodiesAcute HIV800

200

HIV antibodies

CD4 countcells/µl

Time Duration

Typical Course of Untreated HIV Infection

Death


Typical’ HIV-1 infection: Lab Markerssymptoms

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10

weeks years

HIV antibodies

Time following infection

HIV viral load

HIV proviral DNA

symptoms

‘window’period

1° infection

Testing Related to HIV 8

p66 & 31

HIV Structure

Image Courtesy GHTM , Tambaram, HIV Fellowship Programme


Tests for Diagnosing HIV


• Antibody Tests

• Screening Tests

– Rapid tests

– Enzyme linked immunosorbent assays (ELISA)

• Confirmatory/Supplemental Tests

– 2nd/3 rd R/E to confirm 1st R/E

– Western blot assay

• Same blood sample is utilised for performing all the tests for identifying HIV antibodies

Antibody Tests: HIV EIA/ELISA (Microwell Format)

Advantages:

• Used as a screening test since 1985

• Easy for mass screening

• Easy to automate

• Accurate

• Less costly than other tests

Disadvantages:

– Technical expertise

– Special equipment

– More stringent standardisation


ELISA Test: Microwell Format


Images Courtesy: GHTM, Tambaram, Chennai

After several incubation and wash steps, a colour reaction occurs if HIV antibody is present

An automated reader gives a measurement of optical density (presence of colour) for each well

ELISA Screening Tests

Generation Antigen / Antibody

First Generation Viral Lysates as Ag (Not in use anywhere)

Second Generation Synthetic peptides/Recombinant Ag

Third GenerationSynthetic peptides/Recombinant Ag, Double Ag binding,Enables IgM, IgA Ab detection

Fourth Generation Detects Ag and Ab to HIV


4 GENERATIONS OF HIV Ab TESTSRelative shortening of Window Period with different ELISA Kits

Image Courtesy Abbott Publication


Concept of Window Period• Window Period (WP) is present between time of

infection and the point at which evidence of infection is detected.

• Rapid tests to detect antibodies to HIV-1 and HIV-2 are in-vitro qualitative tests

• Can be performed on whole blood, plasma, serum and saliva. In India whole blood and serum are used

• Most rapid test kits come with all materials required to perform the test

HIV Rapid Tests


Overview of workflow at ICTC


Step Personnel Time

1.Pre test counselling and informed consent

Counsellor 15-20 min

2.Blood (2-5ml) collection in sterile vial

Lab Technician 5-10 min

3. Serum separation Lab Technician 30-60 min

4. HIV testing on serum sample Lab Technician 30-60 min

5. Report preparation Lab technician 30-60 min

6.Report dispensing with Post test counselling

Counsellor 15-20 min

All times excluding waiting times; Waiting time depends on client load; Reports will be available on the same day

16

HIV Rapid Tests

Immunological Principles Positive Result

(Visual)

Particle agglutination Clumping

Immunofiltration/dot Dot

Immunochromatography Line

ELISA based rapid tests Dot

Most rapid tests detect Antibodies to both HIV-1 & HIV-2


Rapid Test: Line Test (Immunochromatography)


Lateral Flow Devices

Sample pad

HIV Antigen

Control

Specimen Flow

18

Non- Reactive

Reactive

Sample Pad Test lineControl line

Rapid Test: Dot Test(Immunoconcentration)


HIV antibody links to bound HIV peptide antigens forming the colour spot

HIV-1 peptide

HIV-2 peptide

Internal Control

19

False Positive Test

Reasons for False Positive Test

Antibody based tests:

• Lab error

• Cross reacting antibodies (Autoimmunity)

• IV drug abuse

• Multiple pregnancies

• Recent immunisation

• Chronic alcoholics

• Cirrhosis of Liver


False Negative Test

Reasons for False Negative Test

Antibody based test:

• Window period

• Lab/Clerical error

• Other immunodeficiency states


HIV Testing Strategies• Strategy I:

– All samples tested with one ELISA/Rapid

• Strategy II: – All samples tested with one ELISA/Rapid (E/R)– Reactive samples tested again on different system

(different antigen or principle)

• Strategy III:

– All samples tested with one ELISA/Rapid (E/R)

– Reactive samples from the first test tested with different antigen or preparation

– Reactive samples from the second test again tested with third system of different antigen or principle


Testing

StrategyObjective of

Testing Type of Testing

Place of Testing

ITransfusion/ Donation safety

Mandatory Blood Bank

II SurveillanceUnlinked

AnonymousDesignated laboratories

IIIDiagnosis of patients at ICTC

Voluntary Counselling Informed

Consent Confidential

ICTC

HIV Testing Strategies


A1

A1+ A1-A2

A2+ A2-

A3

A3-A3+

A1+, A2+, A3+Report: Reactive

A1+, A2+, A3-(indeterminate)

Report: Non-Reactive

A3

A3-

A3+

Report: Non-Reactive

A1+, A2-,A3+ (indeterminate)

HIV Testing Strategy III


Tests of Choice for Infants & Children <18 Months of Age

Test Recommendation Reason

HIV antibody

HIV p 24 Antigen

HIV viral culture

HIV DNA PCR

Name the tests used for the diagnosis of HIV in infants and children aged <18 Months and give their relevance


Tests of Choice for Infants & Children <18 Months of Age

26

Test Recommendation Reason

HIV antibody NoFalse +ve due to persistent maternal antibodies

HIV p 24 Antigen Yes, butLower sensitivity than PCR (27% at 6 weeks)

HIV viral culture Yes, butCostly, result takes 2-4 wks, not readily available

HIV DNA PCR Yes98% sensitive from 6 weeks of age

Name the tests used for the diagnosis of HIV in infants and children aged <18 Months and give their relevance


• Positive HIV antibody (Ab) alone indicates there has been exposure to HIV: DOES NOT mean child is infected, can be circulating maternal antibodies

• Test to diagnose HIV in this population: HIV DNA PCR (Qualitative)

• Ideal: First positive virological test (HIV DNA PCR) should be confirmed by a repeat positive test on a separate specimen


Diagnosis of HIV Exposed Infants & Children

Diagnosis of HIV Exposed Infants & Children


6 weeks10 weeks

14 weeks6 months

9 months12 months

18 months

DNA PCR for all HIV exposed infants

All HIV infected and/ or symptomatic infants/children are to be referred to ART centre

DNA PCR HIV Antibody test followed by DNA PCR if HIV+

Final confirmatory Antibody Test for all HIV exposed infants irrespective of earlier testing results / treatment status

Birth

Schedule of visits at ICTC

28

• Follow two different diagnostic algorithms

A. Infants <6 months old and born to HIV positive mother

B. Child of age 6-18 months born to HIV positive mother

• First HIV DNA PCR test sample at ICTC (Dry blood spot)

• If DBS is positive, Second sample for DNA PCR test (whole blood) will be collected at linked ART Centre

– A positive DNA PCR test reveals the infant/child as HIV-1 infected

– A negative DNA PCR test reveals discordance between the first and second tests; needs a tie breaking third DNA PCR test from the whole blood sample to be taken at ART centre


Diagnosis of HIV Exposed Infants & ChildrenNational Guidelines: General Principles

• Sample for DNA PCR test (whole blood) taken at ART Centre for the tie-break

– A positive DNA PCR test reveals the infant/child as HIV-1 infected

– A negative DNA PCR test reveals the infant/child as HIV-1 uninfected

• Final confirmatory Antibody Test for all HIV exposed infants irrespective of earlier testing results / treatment status at 18 months


Diagnosis of HIV Exposed Infants & ChildrenNational Guidelines: General Principles

30

Children <18 months with unknown HIV status of Mother• In Children (<18months) with signs and symptoms

of HIV, whose exposure status is unknown, perform rapid test for HIV antibodies

• If negative, label child as uninfected

• If positive, follow algorithm A or B, depending on age of the child


• Tests are required to monitor

- Disease progression

- Staging of disease

- Response to ART

• Include - CD4 T-cell Assay

- Viral load assay

Tests for Monitoring


Standard Technologies for Monitoring ART

• CD4 by Flow Cytometry advised by NACO

– Variations across methods can occur

• Nucleic acid Amplification Technologies (NAT)

– Cannot be widely used in countries with limited resources because:

• Lack of adequate infrastructure

• Requires highly skilled lab personnel

• NAT testing is too expensive


FACS Machines for CD4 Estimation Available Through NACO

Images Courtesy: GHTM, Tambaram, Chennai

FACS Calibur FACS Count Partec


When to Perform CD4 Test

• All HIV patients accessing Hospitals - immediately after their HIV status known

• Pre-ART: once in 6 months till they are being initiated on ART

• During ART:

– Once in 6 months for monitoring

– As and when their clinical conditions demand

Testing Related to HIV 35

CD4 counts and CD4 %

• Used to assess immunological status of the HIV-infected child

• CD4 counts are higher in infants as compared to adults and fall to adult values by age 5

– Varies due to diurnal change, undercurrent illness, steroid treatment, splenectomy, after immunisations

– Test variability: repeated measurements are more informative than single value

• CD4% varies less than CD4 counts, hence considered more valuable in children <5 years of age


Key Points

• Encourage voluntary testing for HIV with pre-test and post-test counselling

• HIV testing should follow recommended strategies I/II/III depending on the situation

• HIV Ab test significance varies in adults and children <18 months; DNA PCR test is advised

• A CD4 test for monitoring must be done with a fresh sample and at the same lab

• Paediatric HIV monitoring and staging relies on CD4%


Health & Medicine

HIV tests in adults and children