LABORATORY DIAGNOSISOF

HIVTarun Prudvi B

MBBS 2nd Professional

HIV

AIDS has turned out to be Social Problem.Global Problem.

So precise diagnostic methods and strategies are designed now.

HIV

A pretest and post-test counseling is Growing

importance

Counseling gives the confidence!

PURPOSE OF TESTING FOR HIV

• A matter of great importance as in Blood donors to prevent infected blood being transfused.

• To diagnose the patients infected with HIV virus.

• For surveillance purpose.

• Persons with high risk behavior.

• In Pregnant women to prevent Mother to Child transmission.

• Patient's presenting with opportunistic infections.

• For persons who need to undergo a surgery.

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TYPES OF TESTS

Specific tests and Nonspecific tests.

In India, National AIDS Control Organisation (NACO) to ensure

quality and uniformity has provided guidelines for conducting

serological tests and their interpretation.

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Antigen detection

Specific HIV Tests

Antibody detection

Cultivation of Virus P24 Ag detection

Screening- ELISA; Sandwich ELISA, Indirect ELISA, Competitive ELISA.

RAPID TESTS; Cassette ELISA, Particle agglutination, Dot Blot.

Confirmatory/Supplementary- Immuno Blot, Western Blot, RIPA, IFA

HIV

The HIV virus has several antigens GP120, GP41, p24,

p31, p18, p15.

P24 is the earliest to appear in blood.

(Major core antigen);

In cases of small viral load, like needle prick injury it takes

much longer time and appearance of p24, viremia and IgM

coincides with seroconversion illness.

P24 antigen will be reactive only after 48 hrs.

SPECIFIC TESTS / Antigenic detection 1

HIV

Free p24 antigen disappears later and appears during the end stage while remaining

absent through out the long asymptomatic stage.

However, antibody bound p24 is demonstrable after dissociation.

The p24 capture essay that uses anti-p24 antibody can be used to demonstrate this.

It is positive in 30% cases. It is positive in first few weeks after infection and in terminal phase

it is uniformly positive.

It can be used for screening blood donors.

SPECIFIC TESTS / Antigenic detection2

2 4 8 12

A

B

C

p24

Ig

M

IgG

Temporal sequence in appearance of p24, IgM and IgG antibodies © Tarun Prudvi B

p24 can be detected in early phase of infection, it disappears later through out the asymptomatic

stage. IgM can be detected 4-6 weeks, followed by IgG.

HIV

In the HIV viral infection the antibodies are not readily detected in the serum immediately

after infection. It needs at least 2-8 weeks to several months to be detected.

During which the antibodies cannot be identified even though the individual is highly

infectious- the “WINDOW PERIOD”. ( In window period the infection can be detected by p24 assay.)

Once they appear they increase in titre. And IgM disappears following the

appearance of IgG.

So, antibody testing will have to be done after 2-6 months to ascertain whether the

infection has occurred or not.

SPECIFIC TESTS / Antibody detection1

HIV

SPECIFIC TESTS / Antibody detection2

Serological tests of HIV are of two types: screening test and Confirmatory/supplementary tests.

Screening Tests:

ELISA, Rapid tests, simple tests

They are not always positive may give false positive results. All positive results must be rechecked

before sample is confirmed positive.

HIV

Screening test

High degree of Sensitivity

Few false negatives

Confirmatory test

High degree of specificity

Few / No

false positive results.

ELISA- Indirect ELISA, Sandwich ELISA, Competitive ELISA

A common method used in Blood banks in mass screening of

Human blood.

Useful in large scale screening.

SPECIFIC TESTS / Antibody detection3

This is inexpensive, but false positive results may occur.

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1st generation

All antigens used to bind HIV antibodies, indirect immunoassay format employs labeled

antihuman IgG for detection of IgG antibodies.

2nd generation

Synthetic peptide or recombinant protein antigens alone are used to bind HIV

antibodies. Design of the specific antigenic epitopes improves sensitivity for HIV-1 group

O and HIV-2

3rd generation

Synthetic peptide or recombinant protein antigens are used to bind HIV antibodies in an

immunometric antigen sandwich format.

This allows detection of IgM and IgG antibodies.

Lower sample dilutions and the ability to detect IgM antibodies (which are expressed

before IgG antibodies) increase sensitivity during early seroconversion.

4th generation

Synthetic peptide or recombinant protein antigens are used in the same antigen

sandwich format as 3rd generation assays to detect IgM and IgG antibodies, and monoclonal antibodies are also included to detect p24 antigen. Inclusion of p24 antigen

capture allows detection of HIV-1 infection before seroconversion.

Evolution of HIV Immunoassay Technology

HIV

SEQUENCE OF APPEARANCE OF MARKERS OF HIV INFECTION

HIV

A growing importanceResults can be issued within < 20 minutes.Limited protocols, and less demanding technical skills.But needs confirmation with ELISA / Western Blot testing.Can differentiate HIV 1 and HIV 2.

RAPID TESTSCassette ELISA, Immune chromatography,

Coated particle agglutination tests,

Dot Blot test

SIMPLE TESTSThey are also based on ELISA principle. Takes 1-

2 hrs.

HIV

In patients in Labor whose Immune status is

not known,

Resource poor establishments.

But needs confirmation with ELISA / WB

DOT BLOT TEST

It is important to confirm all screening tests with Confirmatory

tests, or we brand some one without infection as infected,

Confirmatory tests differentiates false reactive tests and

identifies truly infected or not.

CONFIRMATORY/ SUPPLEMENTARY TESTS1

/ Western blot test

1. HIV proteins are

separated on

polyacrylamide gel by

electrophoresis

2. Blotted on to strips of

nitrocellulose paper.

3. These strips are reacted

with the test sera

4. The anti-human

globulins are added with

attached enzyme and

suitable substrate added,

produces colour

HIV

CONFIRMATORY/ SUPPLEMENTARY TESTS/ Western Blot2

The antibodies in the serum should react with at

least two of gp160/120, gp41, p24 antigens.

If does not meet requirements, marked as

indeterminate.

/ INTERPRETATION

SPECIFIC TESTS / Antibody detection3

False positive results:

A positive HIV test may be obtained in the absence of HIV antibodies in blood.• Influenza immunisation may temporarily cause false positive result• Autoimmune disorders RA /SLE• Presence of autoantibodies against lymphocytes• Sera stored for longer durations

All positive results must be conformed by other method.

Other tests in Antibody detection

RIFA

IFA

Immuno Blot

HIV

Recommended Laboratory HIV Testing Algorithm for Serum or Plasma Specimens

HIV

Reporting of all Positive results is a great

concern, Avoid casual reporting

PCR

Viral Load Tests

CD4 Count

Prognostic Tests

HIV

Nucleic acid test (NAT)

Nucleic-acid-based tests amplify and detect one or more of several target

sequences located in specific HIV genes

In the RT-PCR test, viral RNA is extracted from the patient’s plasma and is treated with

reverse transcriptase (RT) to convert the viral RNA into cDNA.

The polymerase chain reaction(PCR)process is then applied.

After PCR is complete, the resulting DNA products are hybridized to specific

oligonucleotides bound to the vessel wall, and are then made visible with a probe

bound to an enzyme.

HIV

Once infected the virus is present in circulation and in fluids. During asymptomatic phase

the viral titres are low and are antibody bound.

Their titres remain high in early infection and in the end stage.

Thus, infected person is infectious through out the life, infectivity being high in early and

end stages.

It can be isolated by co-cultivation of patients lymphocytes and uninfected

lymphocytes and the reverse transcriptase activity can be observed. However, it is

not used as a routine diagnostic test.

Viral isolation

HIV

Viral Load tests

HIV-1 RNA Polymerase chain reaction (PCR)

Multiplies amount of HIV RNA in a blood sample through use of an

enzyme. The resultant chemical reaction marks the virus and the

markers are measured to calculate the amount of HIV-1 RNA

Branched chain DNA (bDNA)

Uses a substance that produces light when it combines with

HIV particles.

The amount of light is measured and converted to a viral

count

Nucleic acid sequence-based amplification (NASBA)

Continues amplification of nucleic acid sequences

Advantage is that it is more sensitive and gives faster results

HIV

Steep rise in plasma HIV-1 RNA levels that reach a peak of between 105 and

106 copies/ml approx. 2 weeks after infection.

Once the host defences are mobilized against the virus, there is a slow

decline to a steady-state viral load, or set point, of between 104 and 105

copies/ml at approx. 4 months post infection.

Viral load in ADULTS

Viral load in infected infants may rise above one million

copies/ml within weeks of infection and remain at this level

during first year of life.

The reported levels of HIV-1 RNA viral loads in infants during primary

and early infection appear to be higher than those seen in adults.

A reduction in viral load produced by antiretroviral therapy is a

reliable marker of reduced risk of disease progression

HIV

When the counts drops < 20% we have to watch for

onset of opportunistic infections and malignancy.

Most widely used predictor of HIV progression.

Risk of progression to an AIDS defining illness, opportunistic

infections or malignancy is high, when the counts drop below

200/mcl.

CD4 Counts

Detected by Flowcytometry

HIV

< 200 : Die 80% within 4 yrs.

200 - 400 : Die 50% within 4 yrs.

Positive HIV NAT results at any age should be confirmed

by repeat testing as soon as possible on a new sample.

Two independent positive test results definitively

diagnose paediatric HIV infection in HIV-exposed infants

and subsequent testing is not necessary.

HIV nucleic acid testing (NAT) to detect HIV RNA or DNA should

be performed for early diagnosis of paediatric HIV infection at

the following ages

• Within 48 hours of birth

• At 2 weeks of life

• At 4 to 6 weeks of life

• At 4 to 6 months of life

EVALUATION IN PAEDIATRIC AGE GROUP

HIV

Two negative HIV NAT results, one obtained >4 weeks of

age and one obtained >4 months of age, definitively

exclude paediatric HIV infection in HIV-exposed infants.

State of infection p24 Anti HIV IgG Anti HIV IgM Western Blot pattern

Early infection - - - -

Acute (seroconversion

illness)

+ to - - To + + Partial: p24 and

gp120

Carrier

asymptomatic

- + - Full pattern

PGL + + - LOSS OF P24/

P55

AIDS + ABSENCE OF

P24; LOSS OF

OTHER

REACTIVITIES

Evolution of serological markers in HIV infection

Other tests /findings

Total Leukocyte count and lymphocyte count <2000 cells per cu.mm

T cell counts; CD4+T cell count <200 cells per cu.mm

Thrombocytopenia

Raised IgG & IgM Levels.

Mantoux –ve (diminished CMI in the fourth stage)

IgG

IgM

ThrombocytopeniaCD4+ T cell decreased

CMI

Hb To assess Anaemic conditions in AZT (azidothymidine; Zidovudine) treatment.

Bone marrow suppression.

Serum alanine or aspartate amino transferase

Assess possible hepatitis coinfection

And monitoring liver toxicity

Serum creatinine / BUN

Renal function assessment / renal toxicity

Serum glucose

Hyperglycaemia and DM in cases of PI based regimen (Atazanavir, Indinavir)

OTHER TESTS

Tarun Prudvi BHIV

Post test counseling a Must in all

Positive results

HIV infections cause production of antibodies and viral products which can be used to make diagnosis

Elisa and rapid tests are commonly used for diagnosis for HIV

CD4 counts can be used as a guide for initiating prophylactic medications and ART to provide information on the efficacy of ARV’S and to monitor HIV progression.

Viral Load tests can be used for early detection of new HIV infections, for determination of response to therapy and sometimes for when to initiate therapy.

SUMMARY

Tarun Prudvi BTarun Prudvi BHIV

AIDS is

Caused by fascinating Virus to - Scientists.

Friendly in approach to - Risk group.

Dangerous to Life to - Infected.