laboratory diagnosis of HIV infection. different tests performed. strategies in performing tests.

1. LABORATORY DIAGNOSIS
OF
HIVTarun Prudvi B
MBBS 2nd Professional

2. HIV
AIDS has turned out to be Social Problem.
Global Problem.
So precise diagnostic methods and strategies are designed now.

3. HIV
A pretest and post-test counseling is Growing
importance
Counseling gives the confidence!

4. 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.
Tarun Prudvi BHIV

5. 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.
Tarun Prudvi BHIV

6. 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

7. 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

8. 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 detection
2
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

9. 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 detection
1
HIV

10. SPECIFIC TESTS / Antibody detection
2
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.

11. 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 detection
3
This is inexpensive, but false positive results may occur.
Tarun Prudvi BHIV

13. 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

14. SEQUENCE OF APPEARANCE OF MARKERS OF HIV INFECTION
HIV

15. A growing importance
Results 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 TESTS
Cassette ELISA, Immune chromatography,
Coated particle agglutination tests,
Dot Blot test
SIMPLE TESTS
They are also based on ELISA principle. Takes 1-
2 hrs.
HIV

16. In patients in Labor whose Immune status is
not known,
Resource poor establishments.
But needs confirmation with ELISA / WB
DOT BLOT TEST

17. 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 TESTS
1
/ 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

18. CONFIRMATORY/ SUPPLEMENTARY TESTS/ Western Blot
2
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

19. SPECIFIC TESTS / Antibody detection
3
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

20. Recommended Laboratory HIV Testing Algorithm for Serum or Plasma Specimens
HIV

22. Reporting of all Positive results is a great
concern, Avoid casual reporting

24. PCR
Viral Load Tests
CD4 Count
Prognostic Tests
HIV

25. 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

26. 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

27. 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

28. 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

29. 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.

30. 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.

31. 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

32. 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

33. 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

34. Post test counseling a Must in all
Positive results

35. 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

37. AIDS is
Caused by fascinating Virus to - Scientists.
Friendly in approach to - Risk group.
Dangerous to Life to - Infected.