Lab diagnosis hiv

LAB DIAGNOSIS OF HIV

1. LABORATORY TESTS FOR HIV UPAMA SISHAN

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3. Structure of HIV Virus core Major capsid protein – p24. Nucleocapsid protein – p18 2 copies of genomic RNA. 3 viral enzymes : Protease Reverse transcriptase I ntegrase Genes : gag, env , pol others: tat, rev , vif , nef,vpr , vpu Virus envelope 2 glycoproteins: gp 120 gp 41 Envelop glycoproteins exhibit genetic variability – development of vaccine is difficult. Protein p17, beneath the envelope . Matrix

4. Viral RNA gp 120 CD4 Receptor Co-receptor (CCR5 or CXCR4) New HIV Particle Viral Proteins Viral DNA Nucleus Cell DNA CD4 + T-Cell B – lymphocytes M acrophages Monocytes Glial cells - CNS LIFE CYCLE OF HIV

5. Purpose of Testing for HIV Infection To avoid contaminated blood transfusion . For surveillance purpose. Persons with high risk behavior. In Pregnant women to prevent Mother to Child transmission. Patient's presenting with opportunistic infections.

6. Current routine lab diagnosis of HIV is mainly based on detection of these specific anti-HIV antibodies The period following the entry of HIV into the body and the appearance of detectable levels of antibodies with the available tests is called the “ window period ”. During the window period, the patient is highly infectious but the antibody test is negative . S eroconversion : the point when the infective status of the person starts from HIV negative to HIV positive and the body starts producing antibodies against the virus.(4-8 WEEKS).

7. WHO Recommended Strategies Strategy I Test all samples with one EIA Strategy II Strategy I with all reactives retested in a more specific test with different principle and/or antigen. Strategy III Strategy II with reactives tested in a third test differing from the first two tests.

8. The Importance of Maintaining a Strategy Consistency of laboratory records &results Clarity of results to doctors Maintaining data base to assess performances Avoiding common false reactivity Avoiding technical errors Reducing costs

9. Although many tests can be used to detect virus in general, specific tests has been described for detecting HIV-1 & 2

10. 1. HIV -1 ANTIBODY SCREENING ASSAYS: a) Enzyme Linked Immunoasorbant Assay [ELISA ]: used to detect antibody-antigen reactions whole viruses extract from infected cells / recombinant proteins of HIV-1 that are affixed to plastic surface at the bottom of micro-titre well. enzyme reaction as a marker for the proof of reaction .

11. INDIRECT ELISA 13

12. 14 SANDWICH ELISA

13. COMPETITIVE ELISA 15

14. 4 Generations of ELISA testing : 1 st generation - use Ag derived from viruses grown in human lymphocytes 2 nd generation - use artificially derived Ag from bacteria or fungi 3 rd generation – use synthetic peptides 4 th generation - simultaneous detection of HIV p24 Ag and anti HIV Ab (Combo test) 17

15. 6-8 weeks 5 week 3 week 2 week

16. Purchase the kit Prick your finger - lanclet Allow the blood to drop into a small circle on the absorbent paper Send the properly packaged blood sample in the enclosed envelopes Your results are given and access to a counselor is available . B) HOME ACCESS HIV -1 TEST SYSTEM/DRIED BLOOD SPOT :

17. C) RAPID TESTS: p rovides results earlier than other tests A. “ Agglutination tests” use different types of particles to produce clumping or settling patterns of the particles when a specimen is positive. 1. An “Autologous red cell agglutination” - detects HIV antibodies with a hybrid antigen antibody reagent which agglutinates the particles in red blood cells. 2. “Latex particle agglutination” detects HIV antibodies by the agglutin - ation of minute latex particles when mixed with the patient’s blood . 3. A “Newer method” uses fluid capillary action to enhance and quicken particle agglutination.

18. B . “ Flow through cassettes / “ membrane immuno concentration devices capture & detect HIV antibody in a specimen flowing through a porous membrane . A visible dot or line forms on the membrane when HIV antibodies are present. C. Solid phase tests include dipstick “comb” assay. This assay uses a solid plastic matrix to which an HIV antigen is fixed. When HIV antibodies are present, a spot or dot will be visible when processed with a signal reagent .

19. D. “ Immunochromatographic strip (ICS)” a one-step method the patient’s blood specimen is combined with a signal reagent & migrates through a special membrane . A positive reaction is seen as development of a line on the membrane

20. Four rapid HIV antibody tests approved by US Food and drug administration (FDA). They are: 1 . Oraquick rapid HIV-1/2 antibody tests (Fig 2) 2 . Reveal rapid HIV-1 antibody tests 3 . Uni -Gold recombigen HIV test 4 . Multispot HIV-1/HIV-2 rapid tests. Oraquick is mainly used in : Labour & delivery emergency departments, hospital inpatient services occupational exposures & in military battlefield operations

21. Oraquick advance rapid HIV test: 25

22. E) DOT BLOT ASSAY Rapid screening test as an alternative to ELISA and western blot A lysate of viral antigens is prepared from HIV-1, harvested from cell culture and is dotted onto a grid of absorbent nitrocellulose paper. Appropriate dilutions of test sera are spotted onto the areas containing viral antigens & allowed to react The dots are developed by the addition of an appropriate substrate for the bound enzyme, resulting in a colorimetric reaction . HIV TRI DOT -Commonly used commercially available

23. D) RAPID LATEX AGGLUTINATION ASSAY: Modification of standard latex agglutination – 5 MINS LATEX BEADS COATED WITH VIRAL ANTIGENS ( gp 120 gp 41 etc ) COLLOIDAL SUSPENSION OF LATEX BEADS

24. 2) HIV-1 ANTIBODY CONFIRMATORY ANTIBODY ASSAYS: Algorithm for the use of ELISA & western blot in the diagnosis of HIV-1 or HIV-2 infection.

25. Confirmatory tests differentiates false reactive tests and identifies truly infected or not. a) Western blot : PRINCIPLE Hiv infected cells are ruptured & its proteins(antigens) are separated according to their size in gel electrophoresis. Blotted on to strips of nitrocellulose paper These strips are reacted with the patient serum antibody to get attached with specific antigen. Secondary antibody conjugated with peroxidase is added to bind primary antibody. Coloured band visualized on membrane strip on addition of substrate.

26. Relation of HIV structure and Western Blot configuration

27. Interpretation of Western Blot. The antibodies in the serum should react with at least two of following antigens: gp 160/120 If does not meet gp 41 requirements, marked p 24 as indeterminate.

28. Interpretive criteria for a positive HIV-1 western blot test

29. Limitation of Western Blot Test If not designed for HIV 2 inclusion, we miss HIV2 infections Can give Indeterminate results in Pregnancy Malaria After administration of Tetanus Toxoid. Autoimmune conditions.

30. B) INDIRECT IMMUNOFLUORESCENCE ASSAY: T he detection of virus antigen in clinical specimens, as well as the detection of virus specific IgG or IgA or IgM antibody . Antigens placed on the slide Primary antibody (from patients serum Anti – IgG / IgM Florecin labelled Anti – human secondary antibody

31. C) RADIO IMMUNOPRECIPITATION ASSAY [R IPA]: An alternative test that is sometimes used as a confirmatory assay over the conventional western blot. Here the infected lymphocyte cells such as H9-T cells are grown in presence of amino acids radiolabelled with S–methionine & S- cystine to permit incorporation of radiolabel into HIV-1 proteins . D) LINE IMMUNOASSAY: Here recombinant / synthetic peptide antigens are applied on a nitrocellulose strip rather than electrophorosed as in the western blot.

32. Alternative Antibody Testing Technologies Non invasive methods have high patient acceptance . increased safety and reduced costs. Saliva GCF oral mucosal transudates urine

33. Collection devices: A. Salivette B.Orapette C.Omni -SAL D.OraSure . Several devices are commercially available for the collection of saliva & oral mucosal transudate specimens for the detection of HIV antibodies

34. Screening for HIV Antibody in Oral Fluids: A number of different screening assays have been employed for the detection of HIV antibodies in oral fluids . These include both - C onventional enzyme immunoassays (EIA ). Rapid tests designed for use with serum or plasma samples , IgG antibody capture radioimmunoassay (GACRIA) Enzyme-linked immunosorbent assay (GACELISA )

35. b . Urine analysis: A high prevalence of antibodies to the glycoproteins gp120 and gp160 in urine samples among seropositive specimens led to the proposition of detection of antibodies specific for these glyco -proteins . Several studies have utilized IgG antibody capture particle adherence tests and have concluded that performance of urine antibody screens is similar to that of serum tests . c . Antibodies against HIV in vaginal mucosa: A study has shown that “high- risk seronegative ” subjects had IgA in their genital mucosa, that it likely was produced locally , and that it probably served as a barrier to systemic HIV-1 infection.

36. VIRAL IDENTIFICATION ASSAYS: 1 ) Polymerase Chain Reaction [PCR]: PCR has emerged as one of the most powerful tools for the amplification of genes and their RNA transcripts. Advantages: Sensitivity and specificity - 97 % to 98%. Disadvantages : Highly subjective to false positivity (contamination ) 2 ) Virus culture: The qualitative culture enables one to isolate HIV and may be used to confirm infection in an individual with equivocal serologic studies, or in infants born to seropositive mothers. The quantitative assay enables one to determine the number of infected cells or titer of infectious virus in a given sample by evaluating serial dilutions of infected peripheral blood mononuclear cells (PBMC) or plasma.

37. Useful in several clinical settings such as : Characterization of isolates of virus mainly with regard to antiviral sensitivities Evaluation of the efficacy of experimental antiretroviral agents 3 ) p24 antigen capture assay: This antigen can be detected relatively early after HIV-1 exposure in many patients & detection often precedes the process of seroconversion by several weeks. This rise in measurable p24 antigen correlates with the burst in viral replication .

38. MONITORING TESTS: Lymphocyte analysis: The CD4 T cell count is an important determinate of disease stage and prognosis in HIV infected individuals Lymphocyte subsets are usually quantitated in two related ways . The first - is a percentage of the total T lymphocyte (CD3 bear- ing ) population. This value is generated directly from the fluorescence activated cell sorter (FACS). The second measurement (more commonly used) of the CD4 subset is the total CD4 cell count , as an absolute number derived by multiplying the FACS‟s percentage of CD4 cells by the total lymphocyte count.

39. Nonspecific markers such as CD4 cell count, is expressed either - an absolute value (normal adult range 600-1700 /mm3) - as a CD4:CD8 ratio (normal adult ratio 1.2: 3.5). CD4 T cell count < 350/µl is indication of initiating antiretroviral Rx Decline in CD4 T cell count of > 25 % - change in therapy. CD4 T cell count is < 200 /µl , and patients should be placed on a regimen for pneumocystis carnii pneumonia prophylaxis. count < 50 /µl , primary prophylaxis for MAC (mycobacterium avium complex) infection is indicated.

40. Based on CD4 cell count . CD4+ T cell categories Stage A Asymptomatic acute (1°) HIV or PGL Stage B Symptomatic Not A or C Stage C AIDS indicator conditions >/= 500/mm 3 A1 B1 C1 200-499/mm 3 A2 B2 C2 <200/mm 3 (AIDS indicator T cell count) A3 B3 C3

41. Other surrogate markers : The following are few surrogate markers which help in assessing the progress of the disease. They are as follows β2 – Microglobulin levels Neopterin levels Interleukin-2 receptor levels Titres of antibody to p24 antigen CD8 cell counts Serum IgA levels Total serum immunoglobulins Erythrocyte sedimentation rate Acid labile endogenous interferon levels Soluble CD16 levels Tumour necrosis factor levels gp120 antigen levels Anti gp120 antibody levels

42. VIRAL LOAD ASSAY: They target different regions of the HIV genome such as the gag and po l genes & use of a variety of technologies, such as Reverse transcriptase polymerase chain reaction (RT-PCR) analysis with colorimetric detection (Roche ) DNA hybridization & branched DNA signal amplification. Nucliec acid sequence based assay. Colorimetric detection ( Organon / bioMerieux ).

43. Reverse transcriptase polymerase chain reaction (RT-PCR) In RT-PCR, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA ) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional or real-time PCR. 48

44. HIV-2 tests: The envelope antigens of HIV-2 are different to those of HIV-1, but the molecular weights may vary slightly. As with HIV-1 screening tests, a variety of test formats are available to detect antibodies to HIV-2 ELISA beads, ELISA microtitre & rapid/simple assays . Commercially available HIV-1/2 "combination tests," which incorporate antigens from both viruses, can be used to screen sera in an attempt to identify either infection.

45. HIV-2 confirmatory tests include the Western blot and the RIPA . For HIV-2 confirmation the WHO requires reactivity to at least 2 HIV-2 env elope antigens , whereas other organizations require reactivity to p26 (gag ) and to gp34 or gp105 ( Env ). To conclude, if a specimen is tested by both HIV-1 and HIV-2 Western blot, the blot exhibiting the strongest reactivity to envelope antigens usually indicates which infection is present.

46. LABORATORY DIAGNOSIS OF HIV IN INFANTS I n infants - complicated - all infants born to infected women are seropositive due to passively acquired maternal antibodies, irrespective of their infection status. Sensitive diagnostic tests designed to detect small amounts of HIV antibodies, give positive results in uninfected infants for 12 to 15 months, till they seroconvert.

47. Therefore, a definitive diagnosis using traditional ELISA and Western Blot can be made only after 18 months of age Several approaches have been used for early diagnosis of HIV infection in newborns. They are as follows: Viral culture HIV-1 p24 antigen detection HIV-1 DNA detection HIV-1 RNA detection Enzyme linked immunospot assay (ELISPOT) and In vitro antibody production (IVAP) HIV specific IgA IgG capture EIA

48. SEROLOGICAL ASSAYS : ADVANTAGES DISADVANTAGES ELISA HIGHLY AUTOMATED PROCEDURE HIGH- FALSE POSITIVE SCREENING - LARGE NO EASY TO PERFORM SIMPLE TECHNIQUE SENSITVITY – 99.5% HOME HIV SIMPLE TECHNIQUE EXPENSIVE TEST HIGH SENSITIVITY RAPID TEST RESULTS - EARLIER HIGH- FALSE POSITIVE RAPID LATEX MIN: EQUIPMENTS NON SPECIFIC RESULTS AGGLUTINATION TIME - 5 MIN TEST DOT BLOT SIMPLE TECH HIGH- FALSE POSITIVE ASSAY HIGH SENSITIVITY & SPECIFICITY(ELISA)

49. MOLECULAR ASSAY: ADVANTAGES DISADVANTAGES PCR RAPID PROCEDURE HIGH – FALSE POSITIVE (4 HRS) (CROSS CONTAMINATION) SENSITIVITY & SPECIFICITY – 97% -98% P24 ANTIGEN HIGHLY SPECIFIC FALSE POSITIVE ASSAY (COMPARING TO SEROLOGICAL TEST) CONFIRMATORY ASSAYS WESTERN HIGHLY SPECIFIC & EXPENSIVE BLOT: DEFINES ANTIBODIES TO LABOUR INTENSIVE SPECIFIC HIV PROTEIN NEEDS EXPERTISE TO EXPERTISE TO INTERPRET IFA TURNS POSITIVE IN EXPENSIVE MICROSCOPE THE EARLY COURSE OF NO SPECIFIC PATTERN INFECTION OF Ag- Ab reactions provided THAN WESTERN B BETTER SENSITIVITY

50. ADVANCES IN HIV DIAGNOSIS: BIOSENSORS: HIGHLY SPECIFIC COMMERCIALLY NOT AVAILABLE ECONOMICAL AT PRESENT. RAPID & SENSITIVE

51. CONCLUSION Among these supplemental tests, the western blot is the most informative and it is the current “gold standard” for confirmation of HIV serological assays . CD4 cell enumeration and HIV-1 antigen capture assay are useful in predicting the course of HIV-1 infection and in monitoring anti retroviral therapies . Non-invasive tests using saliva, GCF, mucosal transudates requires further research for the improvement & standardization of these alternative techniques. When choosing an HIV test, the sensitivity, specificity, and cost of the test, the prevalence of HIV infection in the community, the return rates for test results, HIV risk of the patient being tested and the patient’s personal preferences should all be considered. To conclude, we advocate continued development of new technologies that will increase the objectivity, specificity and sensitivity of tests used for the diagnosis of HIV infections.

52. Non-invasive tests using saliva, GCF, mucosal transudates requires further research for the improvement and standardization of these alternative techniques . When choosing an HIV test, the sensitivity, specificity, and cost of the test, the prevalence of HIV infection in the community, the return rates for test results, HIV risk of the patient being tested and the patient’s personal preferences should all be considered . To conclude, we advocate continued development of new technologies that will increase the objectivity, specificity and sensitivity of tests used for the diagnosis of HIV infections.

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