This document summarizes the structure, diagnosis, and testing of HIV and AIDS. It describes key events in the isolation and identification of HIV, including the discovery of HIV-1 and HIV-2. It outlines the structure and genome of HIV, its replication cycle, and the various tests used to diagnose infection and monitor disease progression, including ELISA, viral load testing, CD4 counts, drug resistance testing, and more.

1. STRUCTURE OF HIV & LAB
DIAGNOSIS

2. • In 1983, Luc Montagnier and his group in Pasteur
institute at Paris managed to isolate a retrovirus
from lymph node which they called
lymphadenopathy associated virus.
• In 1984, another group led by Dr. Robert Gallo at
National Cancer Institute in betheda confirmed
isolation and considerably expanded the
evidence linking this virus which they called the
human T lymphotrophic virus 3 to
immunodeficiency syndrom.
• It was subsequently renamed human
immunodeficiency virus(HIV 1 ) .
• In 1986 second immunodeficiency virus (HIV 2)
was isolated from west africa

3. Origin
• HIV 1 – SIV of chimpanzees found in forests of
central Africa
• HIV 2 – SIV of old world monkey inahabitating
in Western Africa

4. Structure
• Family- Retrovirdae
• Genus – Lentivirus
• It is 120 nm icosahedral , single stranded, positive
sense, envoloped virus
• Outer envolope- lipid bilayer with uniformly
arrranged 72 spikes of glycoprotein 120 and 41
• Inside envolope- protein core containing 2 copies
of RNA and viral enzymes (reverse transcriptase,
integrase, protease)

5. Hiv genome
• Structural proteins-
1. Gag (group specific antigen)- protiens that
form core of virion
2. Pol (RNA dependent DNA polymarase)-
reverse transcriptase, integrase, protease,
ribonuclease
3. Env (enevelop) – gp 160

6. Regulatory proteins -
1.Tat (transcriptional transactivator)- promote
the elongation phase of hiv transcription
2. Rev (regulator of viral expression)- induce
transition from early to late phase of HIV gene
expresion

7. Accessory proteins -
1. Nef – decreases expression of CD4
2. Vif – replication in peripheral blood
lymphocytes , macroaphages
3. Vpr- transport of provirus in to nucleus
4. Vpu- release of virions . Not found in HIV 2

9. Types of HIV
• HIV 1 – major cause
• - 11 subtypes
• - type C is prevalent in india
• HIV 2 – restricted to certain geographic
areas like west Africa
• - less transmissibility
• - long incubation period
• - better prognosis

10. Peculiarities of HIV
• Genetically diverse in variety of biologic,
serologic, molecular features.
• Cellular tropism
• Multiplies in presence of antibodies
• High level viral replication even during latency
• Mutagenic
• Persists in reservoir sites

11. Replication cycle of HIV
• HIV entry –
1. Binding – binding of gp 120 to CD4 receptors
2. Fusion – CCR5 AND CXCR exposes gp 41
which undergoes confirmational change to
form hairpin structure
• Postfusion events-
1. Reverse trancriptase
2. Integrase
3. Transcription and translation
4. Viral assembly and release

12. Lab diagnosis
• Mandatory testing-
1. blood and blood products safety
2. donors of sperms, organs and tissue
• Voluntary testing-
1.clinically suspected individuals
2.prevention of parent to child
transmission
3. individuals with high risk behavior
• Unlinked anonymous testing-
1. epidemiological purpose
2. research and surveys

13. Direct predictors of HIV infection
• Specific antibody detection
1. Screening tests-
A . ELISA tests – blood banks and tertiary care centres
 Carried out by using 3rd and 4th generartion kits
 Takes 2 to 3 hours
 Positive after 3-4 weeks of infection
 Advantages –
 Easy to perform
 Adaptable to large no of samples
 Sensitive and specific
 Cost effective

14. • False positive results of ELISA-
• Hematologic malignancy
• DNA viral infections
• Autoimmune disorders
• Multiple pregnancies
• Multiple blood transfusions
• Hepatitis
• Chronic alcoholics
• primary biliary cirrhosis
• Multiple myeloma
• Technical errors
• Hepatitis and influenza immunization
• Hypergammaglobulonemia
• Steven- johnson syndrom

15. • False negative results-
• window period
• Late stage disease
• Immunosuppressive therapy
• Malignant disorders
• B cell dysfunction
• Bone marrow transplantation
• Technical errors

16. B . Rapid tests-
• Detects antibodies against HIV 1 and 2 in
serum, plasma, whole blood, saliva , urine
• Indications-
• When ELISA is not available
• In emergency cases
• Remote blood banks

17. Confirmatory tests-
• When a serum specimen is reactive by any
one of the screening tests it has to be tested
again by different system by using different
antigens or tests with different principle. If the
specimen is reactive in two different systems
then it is confirmed by confirmatory tests .

18. Westorn blot test
• It identifies with specific antibodies, the proteins
that have been separated from one another
according to their size by gel electroforesis.
• The blot is a membrane, almost always of
nitrocellulose or polyvinylidene fluride.
• The gel is placed next to the membrane and
application of an electric current induces the
proteins in gel to move to the membrane where
they adhere.
• The membrane then a replica of the gels protein
pattern , and is subsequently stained with
antibodies.

19. Recent advances-
• Oral mucosal transudate tests-
• Specimens – oral fluids, whole blood obtained
from finger puncture or a venipuncture and
plasma, swab from gum line
• Specificity - 99.8%
• Sensitivity – 99.3 %
• Any positive tests needs confirmtion by doctor

20. Direct detection of HIV infection-
• Indications –
1. during window period
2. in health care workers following accidental
exposure to contaminated blood.
3. babies born to HIV positive mothers
4. indeterminate ELISA/WB
5. discordant results on antibody testing

21. Specific antigen detection tests-
• Specimen – blood / serum / plasma / CSF /
tissue culture fluid
• Antigens – p24 and reverse transcriptase
• Positive in 1 week to 3-4 weeks
• Indicates active infection
• Once antibodies are produced tests becomes
negative

22. Detection of viral nucleic acid -
• Polymerase chain reaction-
• Indications – window period
- equivocal WB test
- neonate
- CNS infection
- late stage
- monitor response to ART
- vaccine efficacy studies
- subtyping of HIV

23. • Advantages –
1. Capable of detecting proviral DNA
irrespective of viral expression
2. Highly sensitive
3. Fresh or archival samples
4. Results within 24-48 hours
5. Less expensive than viral culture
6. Less sample material

24. Recent advance
• Nucleic acid amplification tests –
-PCR based tests
-Used in acute infection when antibodies
are still undetectable

25. Culture of virus-
• Specimen – blood , Tissue,plasma, CSF
• Predominantly research tool
• Methods- 1. direct
2. co-culture

26. Strategies for HIV testing in India-
• Strategy I- serum is subjected once to ELISA/
rapid / simple tests . If negative serum is
considered free of HIV
• Strategy II – If first ELISA is reactive , it is
subjected to second ELISA with different
principle. It is reported reactive only when
second ELISA is also reactive.
• Strategy III – third reactive ELISA is required
for a sample to be reported HIV positive

27. Diagnosis during window period
• P24 antigen
• PCR
• Viral culture
• Need – 1. untested blood transfusion
2. risky sexual exposure
3. needle stick injury

28. Diagnosis in children less than 18 months
• HIV positive mother- 1st DNA PCR if positive
then do 2nd DNA PCR if report is positive, then
it is confirmed to be HIV positive , if it is
negative then repeat and regular follow up
• If 1st DNA PCR is negative , if breast fed repeat
2nd PCR after 6 to 8 weeks after stopping
breast feeding, if not breast fed then repeat
2nd PCR at 6 months

29. Lab tests for monitoring , staging and
progression of HIV infection
• Plasma HIV RNA load-
• Copies of HIV in 1 ml of blood
• Best viral load tests result is undetectable, it does
not mean that there is no virus in blood . It just
means that it is not detectable by tests
• Methods- 1. PCR
2. branched DNA
3. nucleic acid sequence based
amplification assay

30. Indications –
• To monitor effect of ART
• For prognosis
• For initial evaluation of newly diagnosed HIV
infection
• For surveillance of patients who are not
receiving ART
• In pregnant women it determines risk of
transmission

31. Immunological markers-
• CD4 cell count
• Indications –
1. To estimate the level of immune competence of
an individual
2. To stage HIV disease
3. Initiation of ART
4. Monitoring response to ART
5. To initiate chemoprophylaxis against
opportunistic infections

32. Serological testing algorithm for recent
HIV seroconversion-
• Only on already confirmed HIV positive
specimens
• To discriminate recent from chronic infections
• It is done by deliberately combining highly
sensitive and less sensitive tests
• Recent infection – positive on highly sensitive
tests and negative on less sensitive

33. Capture enzyme immunoassay-
• Principle – relative proportion of anti HIV IgG
antibodies on the total IgG antibodies
• Early infection lower proportion
• After 2 years of infection proportion increases

34. Drug resistance tests-
• It should be performed at under following
circumstances-
1. At baseline
2. Before initiation of ART
3. In patients experiencing treatment failure

35. Genotyping -
• The genetic code of HIV has mutations that
are linked to drug resistance
• Adv – rapid
• Disadvantage – actual viral phenotype may be
different from result

36. Phenotyping -
• Provides direct measure of drug resistance
• Method - DNA recombinant method is used
to measure ability of virus to grow in presence
of drug
• Requires 3-5 weeks
• Minimum viral load requirement
• More accurate

37. Coreceptor trophism analysis-
• Determines the type of cellular co-receptor
that an HIV infected individual’s dominant
viral population uses to gain access to host
cell.
• Recently infected individuals – CCR5