HIV Structure and Lab Diagnosis

HIV Structure and Lab Diagnosis

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

8. 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

9. 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

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

11. 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

12. Direct predictors of HIV infection Specific antibody detection Screening tests- A . ELISA tests – blood banks and tertiary care centres Carried out by using 3 rd and 4 th 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

13. 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

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

15. 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

16. 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 .

17. 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.

18. 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

19. 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

20. 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

21. 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

22. Advantages – Capable of detecting proviral DNA irrespective of viral expression Highly sensitive Fresh or archival samples Results within 24-48 hours Less expensive than viral culture Less sample material

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

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

25. 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

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

27. Diagnosis in children less than 18 months HIV positive mother- 1 st DNA PCR if positive then do 2 nd 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 1 st DNA PCR is negative , if breast fed repeat 2 nd PCR after 6 to 8 weeks after stopping breast feeding, if not breast fed then repeat 2 nd PCR at 6 months

28. 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

29. 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

30. Immunological markers- CD4 cell count Indications – To estimate the level of immune competence of an individual To stage HIV disease Initiation of ART Monitoring response to ART To initiate chemoprophylaxis against opportunistic infections

31. 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

32. 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

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

34. 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

35. 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

36. 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

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