1. DIAGNOSIS OF HIV INFECTION THE LABORATORY BY DR. K.BUJJIBABU.MD.

2. METHODOLOGIES AVAILABLE SEROLOGICAL ASSAYS SUPPLEMENTAL ASSAYS ANTIGEN DETECTION VIRAL CULTURE MOLECULAR METHODS SURROGATE MARKERS

3. SEROLOGICAL ASSAYS Depends on rise of antibody levels to detectable range. 2- 8 weeks of acquiring the infection( not useful for early infections) IgM- Gag proteins. IgG- p 24 antigen and the gp120…. gp 41 Persistently undetectable antibodies more than 3 months – rare.

4. SEROLOGICAL ASSAYS ELISA Format: First generation: Whole viral lysate. Second generation: Recombinant antigens Third generation:- synthetic peptides. Sens: >99% Specificity: >98.5% Principles: Sandwich assay Competitive assay Capture assay principle

5. False negative serology results Very early during the course of infection. Immunocompromised patients. Errors in collection, labeling and handling of the specimens. Unusual HIV strains ( O group HIV strain) Recent Exchange transfusion. Postulated to be due to a lack of B cell “ help”

6. False positive Serology Results 1. Human Error. 2. Lipaemic or hemolysed samples. 3. VDRL Positive. 4. Autoimmune disorders. 5. Hypergammaglobulinemia. 6. Multiple myeloma.

7. SEROLOGICAL ALTERNATIVES Latex agglutination Immunochromatography. Other spot tests.

8. SUPPLEMENTAL ASSAYS WESTERN BLOT ASSAY INDIRECT IMMUNOFLUORESCENCE RIPA ( Radio immunoprecipitation assay) Specific Blots for the differentiation of the subtypes of HIV-1

9. WESTERN BLOT Relevant antigens are separated from electrophoresis and transferred to a membrane. Antibody reactivity is visible as bands. Subjectivity is a problem and causes diagnostic confusion.

10. Interpretation of Western Blot assay CDC Criteria. WHO Criteria. American Red Cross Criteria. ASPTHLD Criteria. Reactive Borderline reactive. Non Reactive.

11. Problems in Interpretation 1. False positive results- Hyperbilirubinemia, Connective tissue disorders, Polyclonal gammopathies. 2. Assays with no Group O proteins tend to miss the Non Type B infections of HIV-1 3. 20% of the HIV- 2 assays may give a false negative result. 4. No clear patterns to predict seroconversion.

12. Problems in Interpretation A single band in the blot. Strong positive ELISA with a borderline reactive blot. Weak positive ELISA with a borderline reactive blot. Technical errors causing diagnostic confusion.

13. ANTIGEN DETECTION Almost obscure today- replaced by the RNA based molecular diagnostic tests. Both false negative results and false positive results seen. Average sensitivity of the assays- 10- 30pg/ml. Heat or glycine mediated dissociation of the immune complexes- more sensitive.

14. VIRUS CULTURE Cumbersome but highly specific for the diagnosis of HIV-1 Infection. Co-cultivation of the PBM’C of a donor with those of the patient. RT activity or the p 24 antigen are measured. Drug resistance studies can be done with the viral culture and strain typing with differentiation can be performed. Time consuming to be of clinical use.

15. MOLECULAR DIAGNOSIS Detection of HIV- 1 RNA in plasma using RNA RT PCR( Lower limit of detection 20- 50 copies of the genome). Detection of the HIV- 1 Subtypes is still far from reality. Window period diagnosis can be settled with these assays, although not validated by the US FDA for clinical use.

16. MOLECULAR DIAGNOSIS Detection of the proviral DNA in PBMC’S 9 Helpful in the neonatal HIV infection) More sensitive and rapid than culture Technically more cumbersome and tedious than RNA RT PCR.

17. Quantification of the HIV- Viral oad Measurement of Virological set point and prognosis. Monitoring response to therapy. Predicting resistance to anti- retroviral drugs. Predict progression of disease. Predict maternal vertical transmission.

18. Specialised Testing Procedures 1. Screening for mutations in CCR5 gene( homozygotes versus heterozygotes) 2. Detection of HIV strains with genomic defects ( Deletions in the nef gene) 3. NSI/ SI phenotypes- inability to infect lymphoblastoid cell lines- low multiplication rate.

19. SURROGATE MARKERS 1. CD4/ CD8 ratios ( useful but superseded by the viral load tests) 2. Beta –2 microglobulin. 3. Serum neopterin levels. 4. Serum IgA levels. 5. High CD8 counts reflective of slower disease progression.