serological test of HIV/ AIDS and their application.pptx

I am graduated BSc in medical laboratory science with very great distinction and awarded gold medal for academic achievement.currently I am graduate assistant at Salale university and pursuing my master degree in medical microbiology at Jimma university . I want to conduct researches and give lectures on different areas of infectious disease, medical microbiology, diagnostic techniques

1. Common serological test for viral infection by Desalegn Ashenafi 1

2. Outline Serological tests for HIV/AIDS Characteristics , composition, structural genes , replication and immunology of HIV Common HIV antibody tests Current HIV test algorithm Common HIV antigen tests Western blot Molecular technique(Viral load ) 2

3. Learning Objectives At the end of this chapter the students should be able to: Describe serological tests HIV/AIDS Describe antibody test , antigen test and molecular techniques for HIV Explain current HIV test algorithm. Explain Characteristics , composition , structural genes , replication and immunology of HIV . perform serologic tests of viral infections(HIV/AIDS) Identify factors affecting each serologic test and minimize this effect Explain how to read, interpret and report the results of each serologic test 3

4. Serologic tests for HIV/AIDS Characteristics of HIV HIV is a RNA virus and its a member of the family retroviridae HIV is retrovirus , i.e it is the RNA virus but when infect the cell it converts it's genetic materials from RNA to DNA. HIV-1 and HIV-2 are medically important viruses The two viruses are 40% similar in their overall structures, and both can cause AIDS CD4 is receptors of HIV virus The surface glycoprotein(gp120) of HIV virus attaches to the CD4 of human lymphocte cell 4

5. cont.... The virus transmits through sexual intercourse, blood transfusion or by contaminated needle. Sexual transmission, either heterosexual or male-to-male is a well-documented route of transmission. Children born to women with HIV have a 20% to 30% risk of HIV infection. Infected mothers can also spread HIV to their newborn infants by breast-feeding. 5

6. Cont... The quantity of CD4 lymphocytes continues to diminish as the disease progresses and when the number of cells reaches a critically low level the risk of opportunistic infection increases. Clinical symptoms of the later phase of the disease include extreme weight loss, fever and multiple secondary infections. The end stage of AIDS is characterized by the occurrence of opportunistic infection like M. tuberculosis, Salmonella, P. carinii, etc. 6

7. Composition and structural genes of HIV HIV virus have two basic components ,these are: Genetic material ,RNA which is called genome ,and protein which surrounds the genomes ,called a capsid. The genomes consists of two identical copies of ss RNA. HIV genomes consists of three structural genes Gag gene ;Gag genes encode the precursor protein p55 ,which is cleaved by viral protease to form matric protein ( p17) capsid protein(p24 ) nucleocapsid protein (p7&p9) gag gene helps to form core of the virus 7

8. 2. pol gene ;encode the precursor protein p100 which is cleaved to form protease, reverse transcriptase ( p66 & p55) a nd endonuclease( p31) . 3. Env gene ;encodes the precursor protein gp 160 which is cleaved to form surface spike glycoprotein ( gp 120) and transmembrane protein (gp41). 8

9. Composition of HIV 9

10. HIV replication CD4 is the receptor of HIV virus on T-lymphocyte gp120 is the glycoprotein on the surface of HIV that attach to the CD4 of the host cell . The virus have six stage of replication 1.Attachment : the virus attaches to the host cell .different Viruses have different attachment site. HIV attaches to the CD4 of the cell by using it's gp120. 10

11. cont... 2.Penetration :the virus cross the plasma membrane of the host cell either by fusion or endocytosis 3.Uncoating : the virus release it's genetic materials by uncoating the capsid or protein
Enzyme reverse transcriptase convert the RNA into DNA 4 . Biosynthesis: During this phases nucleic acid, capsid, regulatory protein, spike and enzymes are synthesized. 5.Assembly : During this stage the structural molecules synthesized in the step 4 start assembling to form anothers many viruses. 6: Release: the cell will rupture and release the viruses and they infect another cell . 11

12. HIV Life Cycle: Reverse Transcriptase Converts RNA into DNA 12

13. Serological Diagnosis of HIV Several laboratory methods are available to screen blood,diagnose infection, and monitor disease progression in individuals infected by HIV. These tests can be classified into those that: 1) detect antibody, 2) identify antigen, 3) detect or monitor viral nucleic acids, and 4) provide an estimate of Tlymphocyte numbers (cell phenotyping ). 13

14. Cont... The isolation of HIV, its nucleic acid and methods used to detect HIV antigen are mainly used to detect early HIV infection before antibodies develop 14

15. HIV Antibody test 1 . ELISA test 2 . Western blot 3 . rapid immunochromatographic tests 15

16. Common HIV Antibody Test 1. Enzyme Linked Immunosorbent Assays (ELISA) Enzyme Linked Immunosorbent Assays relies on a primary antigen-antibody interaction. ELISA have four generation assays: First generation assays were based on purified HIV whole viral lysates, have poor sensitivity and specificity Second generation assays used HIV recombinant proteins/or synthetic peptides which enabled the production of assays capable of detecting HIV-1 and HIV-2. It has had improved specificity, but has similar overall sensitivity to that of first generation assay 16

17. cont... Third-generation assays used the solid phase coated with recombinant antigens and/or peptides and , similar recombinant antigens and peptides conjugated to a detection on enzyme or hapten that could detect HIV-specific antibodies bound to a solid phase. These assays could detect IgM early antibodies to HIV, in addition to IgG , thus resulting in a reduction of the sero -conversion window (2-4 week time period) Fourth generation assays are very similar to third generations test, but have the ability to detect simultaneously HIV antibodies and antigens. Hence, reduce window period. 17

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19. Characteristics of ELISA performed at a regional or national laboratory since they require: well-trained and skilled laboratory technicians, technologically advanced equipment like incubators, Washers and spectrophotometers most efficient for laboratories that process a large number of specimens (100 or more) daily or for batch testing which is common in HIV sentinel surveillance. have limited application in rural settings where the laboratory infrastructure and equipment may be insufficient 19

20. Performing an ELISA It can be performed with serum, plasma, urine, oral fluids, or dried blood spots (once eluted). can take from 2 to 4 hours to perform including , specimen preparation and dilution and, an additional 3 to 4 hours if a screening result has to be confirmed. Manufacturer’s instructions provided with the specific ELISA used should be followed. 20

21. General steps for performing an ELISA 1. Dilute the specimen in the specimen buffer and put it in a micro well plate containing HIV antigen already bound to the plate. 2. Incubate the plate as per protocol and then wash as indicated. 3. Add antihuman immunoglobulin-enzyme conjugate, which will react with the HIV specific antibody if present 4. Incubate 5. Wash the plate, add the substrate and incubate as prescribed 6. Add a stopping solution to terminate the enzyme reaction and read the absorbance of the solution using spectrophotometer. 21

22. Reading Result of ELISA A positive reaction has occurred if the specimen in the specimen well changes color or becomes colored Positive reaction indicates the presence of HIV-specific antibody in the specimen. The reaction is best read quantitatively with an ELISA plate using spectrophotometer (ELISA reader). 22

23. Critical to the success of conducting an ELISA: Use of test kits that are not expired Calibrated and well-maintained equipment Adherence to dilution and incubation times described in the manufacturer’s instructions. Use of deionized water Use of a spectrophotometer to read results accurately and objectively Training with the technology being used Consistent source of power without outages that would affect the storage of reagents or the functioning of equipment. 23

24. Quality control Run controls with the patient sample as per the manufacturers’ instructions. It is advisable to include known reactivity sera as well 24

25. Principle of ELISA As its name suggests, the ELISA uses an enzyme system to show the specific combination of an antigen with its antibody. The enzyme system consists of: An enzyme, which is labeled, or linked, to specific antibody or antigen A substrate, which is added after the antigen antibody reaction. This substrate is acted on (usually hydrolyzed) by the enzyme attached to the antigen antibody complex, to give a color change. The intensity of the color gives an indication of the amount of bound antigen or antibody. 25

26. Types of ELISA There are four types of ELISA ,these are: Direct ELISA Indirect ELISA Sandwich ELISA competitive ELISA Direct ELISA In general, all ELISAs are quantitative tests to determine the concentration of unknown specific antibodies or antigens. Direct ELISA requires the immobilization of patient antigens on a microplate well, and complementary antibodies to the patient antigens 26

27. Cont... Complementary antibodies are conjugated to an enzyme that reacts with a substrate. The substrate is converted into detectable product after interacting with the enzyme and the colour changes. Advantage of direct ELISA simple and requires less time than other ELISA Disadvantages of direct ELISA less sensetive than others types of ELISA 27

28. Indirect ELISA Like direct ELISAs, indirect ELISAs require the immobilization of patient antigens on the surface of microplate well . However, an indirect ELISA uses two types of antibodies instead of one. The first is primary antibodies, which are complementary and specific to the patient antigen, and form complexes with the patient antigen on the surface of the microplate well. The second antibodies are specific to the Fc region of the primary antibodies, and conjugated to an enzyme that reacts with a substrate to produce detectable products More sensitive than direct ELISA since it have two antibodies 28

29. Sandwich ELISA Specific antibodies (called capture antibodies or complementary antibodies) to the antigen of interest are adsorbed to the microplate well. Therefore, the sandwich ELISA overcomes the issue of reduced sensitivity that is associated with both direct and indirect ELISA Patient antigens bind to capture antibodies, secondary antibodies, conjugate to an enzyme (called conjugate) and specific to the patient antigen, binds to the patient antigens that are on the capture antibodies. Finally, the enzyme conjugated to secondary antibodies reacts with the added substrate to produce products. It is more sensitive than direct and indirect ELISA ,but it is costy . 29

30. Competitive ELISA This method depends on the competition between two antigens for binding on limited available antibodies site. The first antigen is the patient antigen the second one is usually similar to that antigen in patient serum but labeled with biotin or directly labeled with an enzyme. Here, the antigens compete for the same binding sites on the antibodies, and the number of antigens in the sample is inversely proportional to the level of signals generated. 30

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32. 2. Western Blot Is a technique in which proteins are; separated electrophoretically , transferred to membranes, and identified through the use of labeled antibodies specific for protein of interest 32

33. Principle of Western blot In the western blot (WB) assay, HIV virus is disrupted and HIV proteins are separated by molecular weight into discrete bands by electrophoresis on polyacrylamide gels. The viral proteins are then transferred onto nitrocellulose sheets and cut into strips. Individual strips are incubated overnight with patient serum, washed, and incubated with anti-human immunoglobulin conjugated with enzymes or biotin. After the addition of the appropriate substrate, color develops to show discrete bands where antigen - antibody reactions have occurred 33

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35. Interpretation of the results of the tests The Centers for Disease Control recommends that tests be considered positive when the gp41 band appears alone or an envelope antibody (gp41, gp120, or gp160) appears in combination with another HIV characteristic band (p15, p18, p24, gp41, p51, p55/ p66, gp120, or gp160). The test is considered negative when no bands appear. the test result is considered doubtful (or indeterminate). If there is isolated reactivity to a single HIV protein or a pattern of reactivity to multiple proteins from the same viral gene product (i.e. polymerase gene products: p31, p51/66 only), In this case, a follow-up specimen from the patient should be collected in 6 months and test repeated in case the patient is in the early stages of HIV infection 35

36. Negative no bands at locations of viral Ags positives standard is that there must be Ab to two of the major Ags, p24,p31,gp41, gp120/160 if a specimen has positive bands but doesn’t follow above rule th e n the result is indeterminate early infections may do this healthy false positive individuals may do this Autoimmune diseases 36

37. Abs to p24, p55 appear first then decrease Abs to gp31, gp41, gp120, gp160 remain throughout disease reliable disease indicators pure HIV Ag important in Western blot assay p17, p24, p31, gp41, p51, p55, p66, gp120 and gp160 must be present for a valid assay. 37

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40. Cont... False- positive WB reactions may occur in infections in bilirubinemia , in connective tissue diseases, and in-patients with human leukocyte antigen (HLA) antibodies. Disadvantages of Western blot it is cumbersome to perform, it requires overnight incubation and interpretation is often difficult 40

41. 3.Rapid Tests or immunochromatography Most HIV rapid tests contain antigens to HIV-1 and HIV-2 and detect antibodies to both. A positive test result is indicated by a spot dot or line depending on the test format. The sensitivity and specificity of the latest generation of rapid tests are similar to those of ELISA. Many rapid tests are under evaluation or are currently in use in developing countries for screening, diagnostic and surveillance purposes. 41

42. Characteristics of Rapid Tests Rapid tests are useful; for small laboratories that routinely perform fewer than 100 HIV tests per day for laboratories without electricity or equipment, and for geographic areas with limited laboratory infrastructure. In some instances, even if a laboratory performs more than 100 tests per day, but only during a limited time in a year, rapid tests may be more appropriate than ELISA. A result can usually be obtained in less than 45 minutes, and it is easy to interpret. However, some training is required to correctly perform the test and interpret the results. 42

43. cont.... The test kits generally contain all reagents needed to run the assay, no additional reagents or equipment is required. Many rapid tests do not require electricity, special equipment, refrigeration, or highly skilled staff although a few require refrigeration for heat- sensitive reagents 43

44. Interpretation of HIV Antibody Rapid Test Results All serum/plasma is first tested with one rapid assay (T1) which is highly sensitive. Serum that is non-reactive on the first test is considered HIV antibody negative. Any serum found reactive on the first assay (T1) is retested with a second highly specific rapid assay (T2) based on a different antigen and/ or different test principle. Serum that is reactive on both tests is considered HIV antibody positive Any serum that is reactive on the first test but non reactive on the second test should be retested with the first test kit (T1). If the result is non- reactive, then it should be considered HIV antibody negative. The reactive result at the first (T1) was probably a technical error 44

45. 6. When serum that is reactive on the first test, (T1) and negative on the second test (T2) is again reactive with first test (T1), then one should repeat the second test (T2) in order to rule out a technical error. If the result is positive with (T2), then it is considered HIV antibody positive. It means that there was a technical error associated to (T2). If however, the test result is negative with T2, then a tie- breaker (T3) is needed. If the result of the tie_x0002_breaker test is positive the sample is reported as HIV antibody positive or if the result is negative the sample is considered negative remote populations for whom HIV test results 45

46. Positive Negative 46

47. Current HIV test Algorithm 47

48. Possible outcome in serial algorithm 48

49. Meaning of HIV Antibody Test Result A negative test result means that the person is either not infected or is so recently infected, that the test could not detect the infection. In the later case, the person could be in the ‘ window period’ . During this period, which may last 3 to 6 months after the initial infection, the person could be infected with HIV and can infect others, but will have a negative test and possibly with no physical complaints. A positive test result means that the person is infected with HIV and can transmit it to others 49

50. HIV antigen tests p24 antigen is the HIV antigen test It reduces the window period of HIV so it detect the virus early p24 antigen test detects the presence of the p24 protein of HIV (also known as CA), the capsid protein of the virus. Monoclonal antibodies specific to the p24 protein are mixed with the person's blood. Any p24 protein in the person's blood will stick to the monoclonal antibody and an enzyme-linked antibody to the monoclonal antibodies to p24 causes a color change if p24 was present in the sample. 50

51. Cont... The p24 antigen test is not useful for general diagnostics, as it has very low sensitivity and only works during a certain time period after infection before the body produces antibodies to the p24 protein. the HIV antibody test give false negative in window period.but the p24 can decrease the window period and also reduce the false negativity since the objective was to reduce the risk of false negatives in the window period. Nucleic acid testing (NAT) is more effective for this purpose, and p24 antigen testing is no longer indicated if a NAT test is performed. 51

52. Molecular Techniques(PCR) Or Viral Load(Nucleic Acid Test (NAT)) Nucleic-acid-based tests amplify and detect one or more of several target sequences located in specific HIV genes, such as HIV-I GAG, HIV-II GAG, HIV- env , or the HIV-pol Viral load, also known as viral burden, viral titre or viral titer, is a numerical expression of the quantity of virus in a given volume It is often expressed as viral particles, or infectious particles per mL depending on the type of assay. higher viral burden, titre , or viral load often correlates with the severity of an active viral infection. 52

53. Cont …. There are many different molecular based test methods for quantifying the viral load using NATs EDTA plasma is the best source of cell-free viral RNA for RNA-based viral load testing. EDTA plasma can be stored at room temperature for 30 hours, 14 days at 4 °C and extended periods of time at -70 °C without significant decreases in viral load signal Viral load is typically reported as copies of HIV in a milliliter (mL) of blood 53

54. Viral load monitoring for HIV is the regular measurement of the viral load of individual HIV-positive people as part of their personal plan for treatment of HIV/AIDS. A count of the viral load is routine before the start of HIV treatment Viral load is used to predict how long an individual will remain healthy, or how quickly the disease will progress viral load greater than 100,000 copies/mL of blood within six months of seroconversion indicates a greater likelihood of developing AIDS within five years. A viral load less than 10,000 copies/mL of blood in the early stages indicates a decreased risk of developing AIDS. 54

55. Cont., A nyone with a viral load greater than 100,000 copies/mL of blood should begin treatment. A CD4 test quantifies Helper T cells and is often combined with viral load testing to monitor the progression of HIV CD4 testing shows the strength of the immune system, but does not report viral activity. person with HIV and a CD4 count below 200 or a CD4 percentage below 14% is considered to have AIDS A decreased CD4 count, in combination with higher numbers on a viral load test, indicates an increased risk of getting sick from opportunistic diseases 55

56. CD4 T-cell count CD4 T-cell count is not an HIV test, but rather a procedure where the number of CD4 T-cells in the blood is determined. CD4 count does not check for the presence of HIV. It is used to monitor immune system function in HIV-positive people Declining CD4 T-cell counts are considered to be a marker of progression of HIV infection . A normal CD4 count can range from 500 cells/mm3 to 1000 cells/mm3 56

57. Cont … HIV-positive people, AIDS is officially diagnosed when the count drops below 200 cells/ μL or when certain opportunistic infections occur. 57

58. Saliva Antibody test ( Ora quick) While antibody to HIV can be found in saliva (and urine), its concentration is usually lower than that found in serum or plasma. While sufficiently sensitive for surveillance testing, saliva tests are not recommended for diagnostic testing. For diagnosis, a serum, plasma or whole blood HIV antibody test should be used. 58

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