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Premium member Presentation Transcript HIV INCIDENCE IN OUR HOSPITAL & DIAGNOSTIC POINT OF VIEW : HIV INCIDENCE IN OUR HOSPITAL & DIAGNOSTIC POINT OF VIEW Dr.N.RAJKUMAR MD.MICROBIOLOGY DEPT OF MICROBIOLOGY DSMCH,PERAMBALURDEPT OF CLINICAL MICROBIOLOGY: DEPT OF CLINICAL MICROBIOLOGY Started doing ELISA for HIV and HBSAG on 14 th March 2011. ELISA - HIV detection for 2167 pts till date. Detected 43 pts to be REACTIVE. PERCENTAGE OF REACTIVE CASES :- 01.98% 2 08-09-2011PowerPoint Presentation: MONTH PATIENTS TESTED FOR HIV BY ELISA REACTIVE CASES MARCH 222 02 APRIL 345 09 MAY 335 05 JUNE 385 11 JULY 500 12 AUGUST 323 01 SEPTEMBER 57 03 TOTAL 2167 43 PERCENTAGE OF REACTIVE CASES :- 01.98 % 3 08-09-2011Elisa…..: Elisa….. 4 th generation ELISA Extremely sensitive and specific An in-vitro qualitative enzyme immunoassay – detection of antibodies to HIV-1 and HIV-2 & HIV-1 p24 antigen in human serum or plasma. Based on “Sandwich ELISA” 4 08-09-2011Elisa contd….: Elisa contd…. Detects the anti-HIV ENV (envelope) antibodies to HIV-1 and / or HIV-2 with equal reactivity and HIV-1 antigen . Detects 1)HIV-1 p24 antigen & 2)antibodies to gp41, C terminus of gp120 HIV-1, gp36 for HIV-2 5 08-09-2011Elisa contd…..: Elisa contd….. Use recombinant proteins; - Gp41 – envelope protein - C terminus of gp120 & gp36 representing the immunodominant regions of HIV-1 and HIV-2 envelope gene structure respectively & - HIV-1 p24 antibodies. coated onto microtiter wells 6 08-09-2011Elisa contd…. Serological Events Following HIV Infection : Elisa contd…. Serological Events Following HIV Infection Antigen appears before antibody – due to SEROCONVERSION, antigen is lost, Antibody develops within 1-2 months after infection & thereby the level of the antibody increases ….. Antigen can generally be detected in acute phase & during symptomatic phase of AIDS Antibodies can be detected throughout the infection 7 08-09-2011PowerPoint Presentation: 8 08-09-2011Elisa contd….: Elisa contd…. Core protein of HIV-1 and HIV-2 show cross reactivity Envelope proteins are more type specific Antibodies against the envelope gene products can be found in almost all the infected people 9 08-09-2011PowerPoint Presentation: Elisa washer Elisa reader 10 08-09-2011PowerPoint Presentation: 11 08-09-2011TRIDOT: TRIDOT In cases of emergency situations, we do CARD TEST Screening test Rapid visual test for the qualitative differential detection of antibodies (IgG) to HIV-1 &HIV-2 in Serum/Plasma The device is an immunofiltration membrane 12 08-09-2011Tridot contd….: Tridot contd…. Separate dots for HIV-1 &HIV-2 and CONTROL These dots contain HIV-1 & HIV-2 antigens immobilized on an immunofiltration membrane. It has been developed and designed using gp41, C terminus of gp 120 and gp36 representing the immunodominant regions of HIV-1 and HIV-2 envelope gene structure respectively. 13 08-09-2011Tridot ….Principle : Tridot ….Principle HIV antigens are immobilized on a porous immunofiltration membrane. Sample and reagents pass through the membrane and are absorbed into the underlying absorbent. 14 08-09-2011PowerPoint Presentation: CONTROL HIV-1 HIV-2 CONTROL CONTROL CONTROL HIV-1 HIV-2 HIV1 HIV2 HIV1 HIV2 NONREACTIVE REACTIVE FOR HIV1 REACTIVE FOR HIV2 REACTIVE FOR HIV1&HIV2 TRI-DOT RESULTS 15 08-09-2011INTRODUCTION: INTRODUCTION Etiologic agent of ACQUIRED IMMUNODEFICIENCY SYNDROME. Still an WORLDWIDE EPIDEMIC, expanding in SCOPE AND MAGNITUDE. Origin – likely from pan troglodytes troglodytes species of CHIMPANZEES. Hence an zoonotic disease 16 08-09-2011HISTORY – Important landmarks: HISTORY – Important landmarks 1981 – AIDS - first recognized case reports. 1982 – CDC named the condition as AIDS. 1983 – ISOLATION of the etiological agent of AIDS was first reported in MAY 1983. (Named it as LAV - became to be known as HIV later in 1986). 1985 - detection of anti-HIV antibodies by Elisa. 1986 - HIV-2 was 1 st identified in west African pt. 1 st case of AIDS detected in INDIA. 1987 – national AIDS control programme started. Old names of HIV – LAV, HTLV-III, ARV. 17 08-09-2011PowerPoint Presentation: 18 08-09-2011PowerPoint Presentation: Professor LUC MONTAGNIER &Virologist FRANCOISE BARRE-SINOUSSI 2008 NOBLE PRIZE WINNERS for MEDICINE 19 08-09-2011DISCOVERERS OF HIV : DISCOVERERS OF HIV Professor LUC MONTAGNIER & Virologist FRANCOISE BARRE-SINOUSSI. French scientists from PASTEUR INSTITUTE, PARIS. Isolated this virus from a west african pt with persistent generalised lymphadenopathy. Awarded the 2008 NOBLE PRIZE for MEDICINE on 6 th OCT 2008. 20 08-09-2011TAXONOMY: TAXONOMY FAMILY - RETROVIRIDAE (RE-TR-O-VIRIDAE) RE- reverse TR- transcriptase containing viruses (Greek retro - backwards) SUBFAMILY - LENTIVIRINAE GENUS - LENTIVIRUS 21 08-09-2011TYPES OF HIV & differences between them: TYPES OF HIV & differences between them Two antigenic types of HIV has been identified. HIV-1 represents the original LAV/HTLV3, mcc of AIDS. HIV-2 was isolated from West Africa, causes similar spectrum of disease. -Displays only about 40% nucleotide sequence similarity with HIV-1. -Much less virulent than HIV-1. -Confined to west africa, isolated cases seen in western & southern INDIA. vpu gene absent in HIV-2, but has vpx gene which is absent in HIV-1 major difference. 22 08-09-2011Morphology & Properties of HIV: Morphology & Properties of HIV Characteristic feature – cylindrical bulletshaped nucleocapsid in mature virion Hall mark of HIV :- an RNA virus, Reverse transcription of its genomic RNA to DNA - RT Virion :- spherical, enveloped, 90-120nm diameter, NC is cylindrical-bulletshaped, 3 layers with 72 external spikes 23 08-09-2011Morphology Contd…: Morphology Contd… Genome :- 2 identical copies of ssRNA, with Reverse transcriptase. contains 3 major genes & 6 additional replicational genes RNA with RT & integrase is packed in coneshaped, nucleocapsid protein ( p24 -inner core), surrounded by p6 protein (regulates the release of virus from cell). 24 08-09-2011Morphology Contd…: Morphology Contd… RNA in NC core,along with PROTEASE is surrounded by a p17 matrix protein (outer core). Entire virion is enclosed in host cell membrane derived phospholipid bilayer envelope (PLBPE)- contains numerous external spikes. Matrix protein 25 08-09-2011Morphology Contd…: Morphology Contd… major viral envelope proteins gp120 (ext surface) & gp41 (transmembrane) form the external spikes. They are embedded in the PLBLE. Since PLBLE is derived from host cell memb, various host proteins (MHC class I & II DR molecules) are incorporated into PLBLE. REPLICATION :- RT makes DNA copy from genomic RNA; Provirus DNA - template for viral RNA. genetic variablity is common. 26 08-09-2011MAJOR GENE PRODUCTS of HIV that are useful in diag of infection: MAJOR GENE PRODUCTS of HIV that are useful in diag of infection gp160 – precursor of envelope glycoproteins gp120–outer envelope glycoprotein of virion , SU. gp41–Trans-membrane envelope glycoprotein . p66 – Reverse transcriptase & RNase activity. p51 – Reverse transcriptase, RT. p32 – integrase, IN. p24 – nucleocapsid (outer core) protein of virion, CA. p17 – matrix (inner core) protein of virion, MA. Antibodies to gp41 & p24 - the most commonly detected. 27 08-09-2011OUTSTANDING CHARACTERISTICS OF HIV : OUTSTANDING CHARACTERISTICS OF HIV Non-oncogenic Maybe cytocidal. Infect cells of the immune system. Provirus's remain permanently asso with cells. Viral expression restricted in some cells in vivo. Cause slowly progressive, chronic diseases. 28 08-09-2011Major genes: Major genes Virus contains 3 major genes required for replication gag, pol, env. Each gene codes for 2 or more polypeptides. 1)gag gene :- g roup-specific A g g ene, encodes precursor protein p55, which is cleaved to Nucleocapsid protein p24 (inner core) & matrix protein p17 (outer core). 29 08-09-2011genes contd…: genes contd… 2)env gene :- Env elope gene - Encodes large 160kda precursor viral glycoprotein- gp160 , cleaved into gp120 (external) & gp41 (transmembrane) envelope glycoproteins. gp120 forms the surface spike & gp41 forms the trans-membrane anchoring protein. 3)pol gene :- Pol ymerase gene , encodes enzymes incl RT, protease, & endonuclease, expressed as precursor protein, which is cleaved into p31, p51,& p66 . 30 08-09-2011AUXILLARY GENES: AUXILLARY GENES 6 additional genes - regulate viral expression. tat, rev, nef, vpr, vpu, vif. encode proteins involved in regulation of various steps of viral replication tat gene:- +ve regulator of transcription, responsible for transactivation, encodes p24. rev gene:- regulator of structural gene expression, encodes p19. 31 08-09-2011AUXILLARY GENES contd…: AUXILLARY GENES contd… nef gene:- Increases viral replication, downregulates host cell CD4, encodes p27. vpr gene:- +ve regulator of early viral transcription, encodes p15(protein R). vpu gene:- Efficient viral assembly and budding, encodes protein U (p16). vif gene:- promotes infectivity of viral particle, encodes p23 (infectivity protein). 32 08-09-2011Lab diagnosis & ETHICS: Lab diagnosis & ETHICS WHO recommends HIV testing for 1) Screening of blood, bld products, organs & tissues for transplantation, 2) Epidemiological Surveillance (ES), 3) Diag of symptomatic inf among those clinically suspected of having AIDS, 4) Early diag of HIV inf among asymptomatic persons who would like to know their HIV status 33 08-09-2011Contd… : Contd… Informed consent is required for last two situations with maintenance of confidentiality, & counselling - pretest & posttest No place in national AIDS prevention & control programmes for testing without informed consent Forcing someone to undergo medical testing of any kind is an invasion of privacy & a violation of human rights - WHO 34 08-09-2011Contd… : Contd… If a person tests +ve for HIV, it means that he\she has HIV inf & will ultimately develop AIDS, a fatal dis for which there is at present no cure. This can take 10-15yrs & some would prefer to live those healthy yrs without knowing their diag. In addn, pt with HIV inf or AIDS can suffer from exclusion, discrimination & unemployment. HIV testing just gives a diag, & a diag alone cannot stop an epidemic. 35 08-09-2011Contd… : Contd… Lab testing only helps if there are ways of breaking the chain of transmission. Testing of bld for transfusion & of tissues or organs for transplantation is the only area where testing needs to be compulsory. No mandatory HIV testing should be imposed as a precondition for employment or for providing health care services & facilities. B’se of this potential consequences, informed consent is mandatory, 36 08-09-2011LAB DIAGNOSIS: LAB DIAGNOSIS SCREENING TESTS (E\R\S) (a) E LISA (b) R apid tests - Dot blot assays - particle agglutination (gelatin, RBC, latex, micro beads) - HIV spot and comb tests - Fluorometric micro particle technologies (c) S imple tests - (Also based on ELISA principle but takes 1-2 hrs.) 37 08-09-2011Contd… : Contd… 2) CONFIRMATORY TESTS (a) western blot assay (b) immunofluorescence test (c) virus isolation (d) detection of p24 antigen (e) detection of viral nucleic acid -in situ hybridization -PCR 38 08-09-2011SCREENING TESTS : SCREENING TESTS These are serological tests which are used to screen antibodies against HIV ANTIBODIES TO HIV appear in the circulation 4 to 8 wks after primary HIV infection. Mean time to seroconversion after HIV infection is 3-4 wks. (a) ELISA (b) Rapid tests (c) Simple tests 39 08-09-2011ELISA Enzyme linked immuno-sorbant assay : ELISA Enzyme linked immuno-sorbant assay Extremely good screening test & a standard procedure for diagnosing HIV infection. Test kits are commercially available for measuring antibodies. Most commercial ELISA kits contain Antigens from both HIV-1 & HIV-2. kits use both natural and recombinant antigens. If properly performed, ELISA has a sensitivity of 99% and specificity exceeding 98%. 40 08-09-2011ELISA Contd… : ELISA Contd… When Elisa based antibody tests are used for screening populations with a low prevalence of HIV infections (eg, blood donors) a positive test or indeterminate serum sample must be confirmed by a repeat ELISA test. If the repeat ELISA test is reactive, a confirmative test (western blot or PCR) is performed to rule-out false-positive ELISA results. 41 08-09-2011PowerPoint Presentation: 42 08-09-2011ELISA Contd…: ELISA Contd… Though ELISA is sensitive, false-negative results do occur. False negative – occurs with individuals infected by group O HIViruses. Hence if necessary kits incorporated with antigens from group O viruses are used. uncommon, usually occur during the WINDOW PERIOD (WP) 43 08-09-2011ELISA Contd…WINDOW PERIOD: ELISA Contd… WINDOW PERIOD period between infection and seroconversion , i.e. the interval between time of exposure to HIV & development of detectable levels of antibodies 1-3 months, rarely exceeds 6 months. Hence screening of bld in this period is not 100% safe, invariably gives false-negative results, b’se sufficient antibodies have not yet been produced for detection. 44 08-09-2011ELISA - WP Contd…: ELISA - WP Contd… The 3 rd generation HIV antigen sandwich – Elisa can detect antibodies at about 3 – 4 wks after infection Pre Sero Conversion Period (PSCP) – 3-4 wks of WP. It can be shortened by about 2 wks if p24 antigen detection assay is used. WP can still be shortened by using nucleic acid detection methods, such as PCR. 45 08-09-2011False-positive ELISA for HIV : False-positive ELISA for HIV The factors associated with false-positive ELISA for HIV are 1) antibodies to class II molecules, 2) autoantibodies, 3) hepatic diseases, & 4) recent INFLUENZA infections. 46 08-09-2011PowerPoint Presentation: 47 08-09-2011RAPID TESTS: RAPID TESTS Total reaction time of less than 30 minutes. Used to detect HIV antibodies, used in ill-equipped labs, can be performed on bld or oral fluids. 1) Dot blot assays 2) particle agglutination (gelatin, RBC, latex, micro beads) 3) HIV spot and comb tests 4) Fluorometric micro particle technologies 48 08-09-2011TRI-DOT: TRI-DOT Rapid visual test for the differential detection of HIV-1 & HIV-2 in human serum or plasma. Based on flow-through technology. Pts antibodies against HIV-1\2 Ag binds to the immobilized HIV-1\2 Ag Protein A conjugate signal reagent pink color dots 49 08-09-2011PowerPoint Presentation: CONTROL HIV-1 HIV-2 CONTROL CONTROL CONTROL HIV-1 HIV-2 HIV1 HIV2 HIV1 HIV2 NONREACTIVE REACTIVE FOR HIV1 REACTIVE FOR HIV2 REACTIVE FOR HIV1&HIV2 TRI-DOT RESULTS 50 08-09-2011CONFIRMATORY TESTS: CONFIRMATORY TESTS These are also serological tests which are used to detect antibodies against HIV Recommended for validation of the +ve results of screening tests. They are (a) western blot assay (b) immunofluorescence test, (c) detection of p24 antigen, (d) detection of viral nucleic acid 1) In situ hybridization, 2) PCR. e) virus isolation, 51 08-09-2011CONFIRMATORY TESTS contd…: CONFIRMATORY TESTS contd… Tests confirm HIV inf in an individual who is either sero+ve or has equivocal results from various serological tests. High degree of specificity. Can give few false -ve results. Purpose – to ensure that uninfected individuals, who test reactive in the screening assays aren’t incorrectly labelled as being +ve 52 08-09-2011a) WESTERN BLOT ASSAY: a) WESTERN BLOT ASSAY Highly specific & sensitive assay. The most widely used supplemental test. An excellent confirmatory test for HIV inf in pts with a +ve or indeterminate ELISA, but a poor screening test for HIV. Here antibodies to HIV proteins of specific molecular weights can be detected. Most commonly detected ones are antibodies to 1) viral core protein – p24, 2) envelope glycoproteins gp41,gp120,gp160. 53 08-09-2011WB procedure : WB procedure HIV proteins (Ag) from detergent disrupted purified virions are separated according to their electrophoretic mobility &molecular wt by PAGE Separated proteins blotted on to nitrocellulose membrane by standard blotting procedure. Membrane is then cut into strips having a full complement of viral proteins A serum sample found +ve by screening test is reacted with the strip followed by washing. 54 08-09-2011WB procedure Contd…: WB procedure Contd… Antibodies which attach to separated viral Ag on strip are detected by anti-human gamma globulin ab to which enzyme has been tagged. Enzyme substrate is added - indicates +ve test. Substrate changes color in the presence of enzyme & permanently stains the strip. Position of the band on the strip indicates the antigen with which the antibody has reacted 55 08-09-2011PowerPoint Presentation: 56 08-09-2011PowerPoint Presentation: 57 08-09-2011WB procedure Contd…: WB procedure Contd… NEGATIVE RESULT :- No band seen in nitrocellulose paper. In a pt with positive or indeterminate ELISA result for HIV, a negative Western blot result concludes that ELISA reactivity was false. POSITIVE RESULT :- antibodies to all 3 major Ag (gag,pol,env) conclusive evidence of HIV inf. WB can be considered +ve for HIV-1, if it contains bands to atleast two of the gene products, p24, gp41, & gp120\160. 58 08-09-2011PowerPoint Presentation: 59 08-09-2011PowerPoint Presentation: 60 08-09-2011WB – INDETERMINATE RESULT: WB – INDETERMINATE RESULT Patterns of reactivity doesn’t fall into +ve or -ve One or more bands may be present, but that bands don’t fulfill the criteria for positivity. Explanation – pt has antibodies that cross-react with one of the HIV proteins. (MC p24 & p55) Indeterminate individuals have identical indeterminate profile for longer period - don’t predict early infection. WHO recommends - retest the individual after 2 wks & after 1-6 months. Impt to test 1 st indeterminate serum also along with 2 nd serum sample to obtain a clear indication of changes in reactivity. 61 08-09-2011WB indeterminate result Contd…: WB indeterminate result Contd… In addition, p24 Ag capture ELISA or one of the tests for HIV RNA can be done to confirm a diag of HIV infection. If an intermediate individual is retested over a period of 6 months & becomes negative, or the band profile don’t progress, HIV inf in the individual can be ruled out. Some individuals who are initially indeterminate, later seroconvert & become +ve during retesting These individuals were probably just infected at the time of 1 st testing. (i.e. early HIV infection). 62 08-09-2011WB indeterminate result Contd…: WB indeterminate result Contd… Intermediate WB – due to 1) non-specific reactions, 2) hypergammaglobulinemia, 3) presence of autoantibodies, 4) presence of other cross reactive antibodies, 5) presence of infection by an unknown, but related RETROVIRUS. 63 08-09-2011WB Contd…: WB Contd… The response pattern against specific viral Ag changes over time, as pts progress to AIDS. Antibodies to envelope glycoproteins (gp41, gp120,gp160) are maintained,but those directed against the Gag proteins (p17,p24,p55) decline. The decline of anti-p24 heralds the beginning of clinical signs and other immunologic markers of progression. 64 08-09-2011FACTS (in serodiag of HIV): FACTS (in serodiag of HIV) If two ELISAs are used then accuracy is 99.93% If one ELISA and western blot are used then accuracy is 99.99%. Mean time to seroconversion after HIV infection is 3-4 wks. All individuals will be positive within 6 months. HIV infection for longer than 6 months without a detectable antibody response is very uncommon. 65 08-09-2011CONFIRMATORY TESTS contd…: CONFIRMATORY TESTS contd… Tests which confirm HIV inf in an individual who is either sero+ve or has equivocal results from various serological tests. High degree of specificity. Can give few false -ve results. Purpose – to ensure that uninfected individuals, who test reactive in the screening assays aren’t incorrectly labelled as being +ve a) virus isolation, (b) detection of p24 antigen, (c) detection of viral nucleic acid -in situ hybridization, PCR 66 08-09-2011a) Virus isolation: a) Virus isolation Cultured from lymphocytes in pts peripheral bld (occasionaly plasma, CSF, genital secretions, specimens from other site - brain, bone marrow) The most sensitive virus isolation tech is to co-cultivate the test sample with UNINFECTED MITOGEN-STIMULATED PERIPHERAL BLOOD MONONUCLEAR CELLS. Needs T-cell growth factor (interlukin-2), Presence of virus is detected by assays for RT & p24 Ag in the culture fluid. Time consuming (takes 3-6 wks) & laborious, limited to research. 67 08-09-2011b) Detection of p24 antigen: b) Detection of p24 antigen p24 Ag can be detected in the serum by ELISA in 30% pts during WP, 10% of asymptomatic pts, & 50-60% of AIDS pts. As the antibody response builds up, Ag tests become –ve, but in late stages of infection p24 Ag may reappear. 68 08-09-2011c) DETECTION OF VIRAL NUCLEIC ACID : c) DETECTION OF VIRAL NUCLEIC ACID Molecular based Amplification assays – detect viral RNA in clinical specimens. PCR – most sensitive Commonly used ones :- RT-PCR, DNA-PCR. RT-PCR - uses enzymatic method to ampilify RNA. Very sensitive, basis for plasma viral load determination. HIV RNA levels are important predictive markers of disease progression & valuable tool to monitor the effectiveness of antiviral therapies 69 08-09-2011PCR : PCR HIV study- two forms of PCR used. 1) DNA PCR - detects single copy of proviral DNA from infected cells. 2) RNA PCR – detects single copy of RNA. Valuable diag test in pts with +ve or indeterminate ELISA & indeterminate WB. Also useful in pts in whom serologic testing is unreliable – pts with hypogammaglobulinemia. Quantitative PCR measures the viral load in the peripheral bld as number of viral copies\ml of bld 400 or less viral copies\ml cannot be detected by the presently available methods PROBLEM – artifacts due to contamination. 70 08-09-2011DNA-PCR : DNA-PCR Peripheral bld MNC lysed proviral DNA of HIV is amplified using proper primer pairs from relatively constant regions of HIV genome. Primer pairs from both the gag & LTR region are usually employed. It is possible to determine whether or not HIV proviral DNA is present at a frequency of one copy per 10,000 – 1,00,000 cells 71 08-09-2011RNA - PCR : RNA - PCR To make an early diag of HIV To monitor changes in the level of virus after infection 72 08-09-2011PowerPoint Presentation: 73 08-09-2011PowerPoint Presentation: 74 08-09-2011FACTS: FACTS Early diag of HIV inf in infants born to infected mothers can be done using plasma HIV-1 RNA tests. The presence of maternal antibodies makes serologic tests uninformative Low levels of circulating HIV-1 p24 Ag can be detected in plasma by ELISA soon after inf. The Ag often becomes undetectable after Antibodies develop (b’se the p24 protein is complexed with p24 antibodies) but may reappear late in course of inf, indicating a poor prognosis. 75 08-09-2011Strategies of HIV testing : Strategies of HIV testing Strategy I:- used for transfusion safety. The serum is tested with one E\R\S test & if reactive, sample is considered +ve & if non-reactive it is considered –ve. Use a highly sensitive & very reliable test kit . Strategy II:- used for HIV surveillance. The sr reactive with one E\R\S test is retested with a second E\R\S test with higher specificity, based on diff Ag preparation & diff test principle. If found +ve on 2 nd test also, it is reported as +ve, otherwise –ve 76 08-09-2011Strategies of HIV testing – contd… : Strategies of HIV testing – contd… Strategy III:- The sr reactive with 2 E\R\S tests is retested with a third E\R\S test. This 3 rd test should again be based on diff Ag preparation or test principle. a) A sr testing reactive with all three tests is reported +ve. b) A sr sample non-reactive in 3 rd test is considered equivocal\borderline. Such individuals should be retested after 3 wks. If this sample also provides an equivocal result, the person is considered to be HIV –ve. 77 08-09-2011Strategies of HIV testing – contd…: Strategies of HIV testing – contd… c) For symptomatic HIV inf, it is necessary to confirm the diag with 3 tests. d) Symptomatic inf with opportunistic infections, the pt may be subjected to two tests. The 1 st test selected for any of the strategies should be of highest sensitivity & 2 nd & 3 rd test selected should be of highest specificity. 78 08-09-2011PowerPoint Presentation: 79 08-09-2011In the field of observation chance favours only prepared minds – LOUIS PASTEUR: 08-09-2011 80 In the field of observation chance favours only prepared minds – LOUIS PASTEUR THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
HIV.ppt4- (3) drnrkmd Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 121 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: November 25, 2011 This Presentation is Public Favorites: 0 Presentation Description hiv, aids, discoverers, p24, genes, morphology, diagnosis, Comments Posting comment... Premium member Presentation Transcript HIV INCIDENCE IN OUR HOSPITAL & DIAGNOSTIC POINT OF VIEW : HIV INCIDENCE IN OUR HOSPITAL & DIAGNOSTIC POINT OF VIEW Dr.N.RAJKUMAR MD.MICROBIOLOGY DEPT OF MICROBIOLOGY DSMCH,PERAMBALURDEPT OF CLINICAL MICROBIOLOGY: DEPT OF CLINICAL MICROBIOLOGY Started doing ELISA for HIV and HBSAG on 14 th March 2011. ELISA - HIV detection for 2167 pts till date. Detected 43 pts to be REACTIVE. PERCENTAGE OF REACTIVE CASES :- 01.98% 2 08-09-2011PowerPoint Presentation: MONTH PATIENTS TESTED FOR HIV BY ELISA REACTIVE CASES MARCH 222 02 APRIL 345 09 MAY 335 05 JUNE 385 11 JULY 500 12 AUGUST 323 01 SEPTEMBER 57 03 TOTAL 2167 43 PERCENTAGE OF REACTIVE CASES :- 01.98 % 3 08-09-2011Elisa…..: Elisa….. 4 th generation ELISA Extremely sensitive and specific An in-vitro qualitative enzyme immunoassay – detection of antibodies to HIV-1 and HIV-2 & HIV-1 p24 antigen in human serum or plasma. Based on “Sandwich ELISA” 4 08-09-2011Elisa contd….: Elisa contd…. Detects the anti-HIV ENV (envelope) antibodies to HIV-1 and / or HIV-2 with equal reactivity and HIV-1 antigen . Detects 1)HIV-1 p24 antigen & 2)antibodies to gp41, C terminus of gp120 HIV-1, gp36 for HIV-2 5 08-09-2011Elisa contd…..: Elisa contd….. Use recombinant proteins; - Gp41 – envelope protein - C terminus of gp120 & gp36 representing the immunodominant regions of HIV-1 and HIV-2 envelope gene structure respectively & - HIV-1 p24 antibodies. coated onto microtiter wells 6 08-09-2011Elisa contd…. Serological Events Following HIV Infection : Elisa contd…. Serological Events Following HIV Infection Antigen appears before antibody – due to SEROCONVERSION, antigen is lost, Antibody develops within 1-2 months after infection & thereby the level of the antibody increases ….. Antigen can generally be detected in acute phase & during symptomatic phase of AIDS Antibodies can be detected throughout the infection 7 08-09-2011PowerPoint Presentation: 8 08-09-2011Elisa contd….: Elisa contd…. Core protein of HIV-1 and HIV-2 show cross reactivity Envelope proteins are more type specific Antibodies against the envelope gene products can be found in almost all the infected people 9 08-09-2011PowerPoint Presentation: Elisa washer Elisa reader 10 08-09-2011PowerPoint Presentation: 11 08-09-2011TRIDOT: TRIDOT In cases of emergency situations, we do CARD TEST Screening test Rapid visual test for the qualitative differential detection of antibodies (IgG) to HIV-1 &HIV-2 in Serum/Plasma The device is an immunofiltration membrane 12 08-09-2011Tridot contd….: Tridot contd…. Separate dots for HIV-1 &HIV-2 and CONTROL These dots contain HIV-1 & HIV-2 antigens immobilized on an immunofiltration membrane. It has been developed and designed using gp41, C terminus of gp 120 and gp36 representing the immunodominant regions of HIV-1 and HIV-2 envelope gene structure respectively. 13 08-09-2011Tridot ….Principle : Tridot ….Principle HIV antigens are immobilized on a porous immunofiltration membrane. Sample and reagents pass through the membrane and are absorbed into the underlying absorbent. 14 08-09-2011PowerPoint Presentation: CONTROL HIV-1 HIV-2 CONTROL CONTROL CONTROL HIV-1 HIV-2 HIV1 HIV2 HIV1 HIV2 NONREACTIVE REACTIVE FOR HIV1 REACTIVE FOR HIV2 REACTIVE FOR HIV1&HIV2 TRI-DOT RESULTS 15 08-09-2011INTRODUCTION: INTRODUCTION Etiologic agent of ACQUIRED IMMUNODEFICIENCY SYNDROME. Still an WORLDWIDE EPIDEMIC, expanding in SCOPE AND MAGNITUDE. Origin – likely from pan troglodytes troglodytes species of CHIMPANZEES. Hence an zoonotic disease 16 08-09-2011HISTORY – Important landmarks: HISTORY – Important landmarks 1981 – AIDS - first recognized case reports. 1982 – CDC named the condition as AIDS. 1983 – ISOLATION of the etiological agent of AIDS was first reported in MAY 1983. (Named it as LAV - became to be known as HIV later in 1986). 1985 - detection of anti-HIV antibodies by Elisa. 1986 - HIV-2 was 1 st identified in west African pt. 1 st case of AIDS detected in INDIA. 1987 – national AIDS control programme started. Old names of HIV – LAV, HTLV-III, ARV. 17 08-09-2011PowerPoint Presentation: 18 08-09-2011PowerPoint Presentation: Professor LUC MONTAGNIER &Virologist FRANCOISE BARRE-SINOUSSI 2008 NOBLE PRIZE WINNERS for MEDICINE 19 08-09-2011DISCOVERERS OF HIV : DISCOVERERS OF HIV Professor LUC MONTAGNIER & Virologist FRANCOISE BARRE-SINOUSSI. French scientists from PASTEUR INSTITUTE, PARIS. Isolated this virus from a west african pt with persistent generalised lymphadenopathy. Awarded the 2008 NOBLE PRIZE for MEDICINE on 6 th OCT 2008. 20 08-09-2011TAXONOMY: TAXONOMY FAMILY - RETROVIRIDAE (RE-TR-O-VIRIDAE) RE- reverse TR- transcriptase containing viruses (Greek retro - backwards) SUBFAMILY - LENTIVIRINAE GENUS - LENTIVIRUS 21 08-09-2011TYPES OF HIV & differences between them: TYPES OF HIV & differences between them Two antigenic types of HIV has been identified. HIV-1 represents the original LAV/HTLV3, mcc of AIDS. HIV-2 was isolated from West Africa, causes similar spectrum of disease. -Displays only about 40% nucleotide sequence similarity with HIV-1. -Much less virulent than HIV-1. -Confined to west africa, isolated cases seen in western & southern INDIA. vpu gene absent in HIV-2, but has vpx gene which is absent in HIV-1 major difference. 22 08-09-2011Morphology & Properties of HIV: Morphology & Properties of HIV Characteristic feature – cylindrical bulletshaped nucleocapsid in mature virion Hall mark of HIV :- an RNA virus, Reverse transcription of its genomic RNA to DNA - RT Virion :- spherical, enveloped, 90-120nm diameter, NC is cylindrical-bulletshaped, 3 layers with 72 external spikes 23 08-09-2011Morphology Contd…: Morphology Contd… Genome :- 2 identical copies of ssRNA, with Reverse transcriptase. contains 3 major genes & 6 additional replicational genes RNA with RT & integrase is packed in coneshaped, nucleocapsid protein ( p24 -inner core), surrounded by p6 protein (regulates the release of virus from cell). 24 08-09-2011Morphology Contd…: Morphology Contd… RNA in NC core,along with PROTEASE is surrounded by a p17 matrix protein (outer core). Entire virion is enclosed in host cell membrane derived phospholipid bilayer envelope (PLBPE)- contains numerous external spikes. Matrix protein 25 08-09-2011Morphology Contd…: Morphology Contd… major viral envelope proteins gp120 (ext surface) & gp41 (transmembrane) form the external spikes. They are embedded in the PLBLE. Since PLBLE is derived from host cell memb, various host proteins (MHC class I & II DR molecules) are incorporated into PLBLE. REPLICATION :- RT makes DNA copy from genomic RNA; Provirus DNA - template for viral RNA. genetic variablity is common. 26 08-09-2011MAJOR GENE PRODUCTS of HIV that are useful in diag of infection: MAJOR GENE PRODUCTS of HIV that are useful in diag of infection gp160 – precursor of envelope glycoproteins gp120–outer envelope glycoprotein of virion , SU. gp41–Trans-membrane envelope glycoprotein . p66 – Reverse transcriptase & RNase activity. p51 – Reverse transcriptase, RT. p32 – integrase, IN. p24 – nucleocapsid (outer core) protein of virion, CA. p17 – matrix (inner core) protein of virion, MA. Antibodies to gp41 & p24 - the most commonly detected. 27 08-09-2011OUTSTANDING CHARACTERISTICS OF HIV : OUTSTANDING CHARACTERISTICS OF HIV Non-oncogenic Maybe cytocidal. Infect cells of the immune system. Provirus's remain permanently asso with cells. Viral expression restricted in some cells in vivo. Cause slowly progressive, chronic diseases. 28 08-09-2011Major genes: Major genes Virus contains 3 major genes required for replication gag, pol, env. Each gene codes for 2 or more polypeptides. 1)gag gene :- g roup-specific A g g ene, encodes precursor protein p55, which is cleaved to Nucleocapsid protein p24 (inner core) & matrix protein p17 (outer core). 29 08-09-2011genes contd…: genes contd… 2)env gene :- Env elope gene - Encodes large 160kda precursor viral glycoprotein- gp160 , cleaved into gp120 (external) & gp41 (transmembrane) envelope glycoproteins. gp120 forms the surface spike & gp41 forms the trans-membrane anchoring protein. 3)pol gene :- Pol ymerase gene , encodes enzymes incl RT, protease, & endonuclease, expressed as precursor protein, which is cleaved into p31, p51,& p66 . 30 08-09-2011AUXILLARY GENES: AUXILLARY GENES 6 additional genes - regulate viral expression. tat, rev, nef, vpr, vpu, vif. encode proteins involved in regulation of various steps of viral replication tat gene:- +ve regulator of transcription, responsible for transactivation, encodes p24. rev gene:- regulator of structural gene expression, encodes p19. 31 08-09-2011AUXILLARY GENES contd…: AUXILLARY GENES contd… nef gene:- Increases viral replication, downregulates host cell CD4, encodes p27. vpr gene:- +ve regulator of early viral transcription, encodes p15(protein R). vpu gene:- Efficient viral assembly and budding, encodes protein U (p16). vif gene:- promotes infectivity of viral particle, encodes p23 (infectivity protein). 32 08-09-2011Lab diagnosis & ETHICS: Lab diagnosis & ETHICS WHO recommends HIV testing for 1) Screening of blood, bld products, organs & tissues for transplantation, 2) Epidemiological Surveillance (ES), 3) Diag of symptomatic inf among those clinically suspected of having AIDS, 4) Early diag of HIV inf among asymptomatic persons who would like to know their HIV status 33 08-09-2011Contd… : Contd… Informed consent is required for last two situations with maintenance of confidentiality, & counselling - pretest & posttest No place in national AIDS prevention & control programmes for testing without informed consent Forcing someone to undergo medical testing of any kind is an invasion of privacy & a violation of human rights - WHO 34 08-09-2011Contd… : Contd… If a person tests +ve for HIV, it means that he\she has HIV inf & will ultimately develop AIDS, a fatal dis for which there is at present no cure. This can take 10-15yrs & some would prefer to live those healthy yrs without knowing their diag. In addn, pt with HIV inf or AIDS can suffer from exclusion, discrimination & unemployment. HIV testing just gives a diag, & a diag alone cannot stop an epidemic. 35 08-09-2011Contd… : Contd… Lab testing only helps if there are ways of breaking the chain of transmission. Testing of bld for transfusion & of tissues or organs for transplantation is the only area where testing needs to be compulsory. No mandatory HIV testing should be imposed as a precondition for employment or for providing health care services & facilities. B’se of this potential consequences, informed consent is mandatory, 36 08-09-2011LAB DIAGNOSIS: LAB DIAGNOSIS SCREENING TESTS (E\R\S) (a) E LISA (b) R apid tests - Dot blot assays - particle agglutination (gelatin, RBC, latex, micro beads) - HIV spot and comb tests - Fluorometric micro particle technologies (c) S imple tests - (Also based on ELISA principle but takes 1-2 hrs.) 37 08-09-2011Contd… : Contd… 2) CONFIRMATORY TESTS (a) western blot assay (b) immunofluorescence test (c) virus isolation (d) detection of p24 antigen (e) detection of viral nucleic acid -in situ hybridization -PCR 38 08-09-2011SCREENING TESTS : SCREENING TESTS These are serological tests which are used to screen antibodies against HIV ANTIBODIES TO HIV appear in the circulation 4 to 8 wks after primary HIV infection. Mean time to seroconversion after HIV infection is 3-4 wks. (a) ELISA (b) Rapid tests (c) Simple tests 39 08-09-2011ELISA Enzyme linked immuno-sorbant assay : ELISA Enzyme linked immuno-sorbant assay Extremely good screening test & a standard procedure for diagnosing HIV infection. Test kits are commercially available for measuring antibodies. Most commercial ELISA kits contain Antigens from both HIV-1 & HIV-2. kits use both natural and recombinant antigens. If properly performed, ELISA has a sensitivity of 99% and specificity exceeding 98%. 40 08-09-2011ELISA Contd… : ELISA Contd… When Elisa based antibody tests are used for screening populations with a low prevalence of HIV infections (eg, blood donors) a positive test or indeterminate serum sample must be confirmed by a repeat ELISA test. If the repeat ELISA test is reactive, a confirmative test (western blot or PCR) is performed to rule-out false-positive ELISA results. 41 08-09-2011PowerPoint Presentation: 42 08-09-2011ELISA Contd…: ELISA Contd… Though ELISA is sensitive, false-negative results do occur. False negative – occurs with individuals infected by group O HIViruses. Hence if necessary kits incorporated with antigens from group O viruses are used. uncommon, usually occur during the WINDOW PERIOD (WP) 43 08-09-2011ELISA Contd…WINDOW PERIOD: ELISA Contd… WINDOW PERIOD period between infection and seroconversion , i.e. the interval between time of exposure to HIV & development of detectable levels of antibodies 1-3 months, rarely exceeds 6 months. Hence screening of bld in this period is not 100% safe, invariably gives false-negative results, b’se sufficient antibodies have not yet been produced for detection. 44 08-09-2011ELISA - WP Contd…: ELISA - WP Contd… The 3 rd generation HIV antigen sandwich – Elisa can detect antibodies at about 3 – 4 wks after infection Pre Sero Conversion Period (PSCP) – 3-4 wks of WP. It can be shortened by about 2 wks if p24 antigen detection assay is used. WP can still be shortened by using nucleic acid detection methods, such as PCR. 45 08-09-2011False-positive ELISA for HIV : False-positive ELISA for HIV The factors associated with false-positive ELISA for HIV are 1) antibodies to class II molecules, 2) autoantibodies, 3) hepatic diseases, & 4) recent INFLUENZA infections. 46 08-09-2011PowerPoint Presentation: 47 08-09-2011RAPID TESTS: RAPID TESTS Total reaction time of less than 30 minutes. Used to detect HIV antibodies, used in ill-equipped labs, can be performed on bld or oral fluids. 1) Dot blot assays 2) particle agglutination (gelatin, RBC, latex, micro beads) 3) HIV spot and comb tests 4) Fluorometric micro particle technologies 48 08-09-2011TRI-DOT: TRI-DOT Rapid visual test for the differential detection of HIV-1 & HIV-2 in human serum or plasma. Based on flow-through technology. Pts antibodies against HIV-1\2 Ag binds to the immobilized HIV-1\2 Ag Protein A conjugate signal reagent pink color dots 49 08-09-2011PowerPoint Presentation: CONTROL HIV-1 HIV-2 CONTROL CONTROL CONTROL HIV-1 HIV-2 HIV1 HIV2 HIV1 HIV2 NONREACTIVE REACTIVE FOR HIV1 REACTIVE FOR HIV2 REACTIVE FOR HIV1&HIV2 TRI-DOT RESULTS 50 08-09-2011CONFIRMATORY TESTS: CONFIRMATORY TESTS These are also serological tests which are used to detect antibodies against HIV Recommended for validation of the +ve results of screening tests. They are (a) western blot assay (b) immunofluorescence test, (c) detection of p24 antigen, (d) detection of viral nucleic acid 1) In situ hybridization, 2) PCR. e) virus isolation, 51 08-09-2011CONFIRMATORY TESTS contd…: CONFIRMATORY TESTS contd… Tests confirm HIV inf in an individual who is either sero+ve or has equivocal results from various serological tests. High degree of specificity. Can give few false -ve results. Purpose – to ensure that uninfected individuals, who test reactive in the screening assays aren’t incorrectly labelled as being +ve 52 08-09-2011a) WESTERN BLOT ASSAY: a) WESTERN BLOT ASSAY Highly specific & sensitive assay. The most widely used supplemental test. An excellent confirmatory test for HIV inf in pts with a +ve or indeterminate ELISA, but a poor screening test for HIV. Here antibodies to HIV proteins of specific molecular weights can be detected. Most commonly detected ones are antibodies to 1) viral core protein – p24, 2) envelope glycoproteins gp41,gp120,gp160. 53 08-09-2011WB procedure : WB procedure HIV proteins (Ag) from detergent disrupted purified virions are separated according to their electrophoretic mobility &molecular wt by PAGE Separated proteins blotted on to nitrocellulose membrane by standard blotting procedure. Membrane is then cut into strips having a full complement of viral proteins A serum sample found +ve by screening test is reacted with the strip followed by washing. 54 08-09-2011WB procedure Contd…: WB procedure Contd… Antibodies which attach to separated viral Ag on strip are detected by anti-human gamma globulin ab to which enzyme has been tagged. Enzyme substrate is added - indicates +ve test. Substrate changes color in the presence of enzyme & permanently stains the strip. Position of the band on the strip indicates the antigen with which the antibody has reacted 55 08-09-2011PowerPoint Presentation: 56 08-09-2011PowerPoint Presentation: 57 08-09-2011WB procedure Contd…: WB procedure Contd… NEGATIVE RESULT :- No band seen in nitrocellulose paper. In a pt with positive or indeterminate ELISA result for HIV, a negative Western blot result concludes that ELISA reactivity was false. POSITIVE RESULT :- antibodies to all 3 major Ag (gag,pol,env) conclusive evidence of HIV inf. WB can be considered +ve for HIV-1, if it contains bands to atleast two of the gene products, p24, gp41, & gp120\160. 58 08-09-2011PowerPoint Presentation: 59 08-09-2011PowerPoint Presentation: 60 08-09-2011WB – INDETERMINATE RESULT: WB – INDETERMINATE RESULT Patterns of reactivity doesn’t fall into +ve or -ve One or more bands may be present, but that bands don’t fulfill the criteria for positivity. Explanation – pt has antibodies that cross-react with one of the HIV proteins. (MC p24 & p55) Indeterminate individuals have identical indeterminate profile for longer period - don’t predict early infection. WHO recommends - retest the individual after 2 wks & after 1-6 months. Impt to test 1 st indeterminate serum also along with 2 nd serum sample to obtain a clear indication of changes in reactivity. 61 08-09-2011WB indeterminate result Contd…: WB indeterminate result Contd… In addition, p24 Ag capture ELISA or one of the tests for HIV RNA can be done to confirm a diag of HIV infection. If an intermediate individual is retested over a period of 6 months & becomes negative, or the band profile don’t progress, HIV inf in the individual can be ruled out. Some individuals who are initially indeterminate, later seroconvert & become +ve during retesting These individuals were probably just infected at the time of 1 st testing. (i.e. early HIV infection). 62 08-09-2011WB indeterminate result Contd…: WB indeterminate result Contd… Intermediate WB – due to 1) non-specific reactions, 2) hypergammaglobulinemia, 3) presence of autoantibodies, 4) presence of other cross reactive antibodies, 5) presence of infection by an unknown, but related RETROVIRUS. 63 08-09-2011WB Contd…: WB Contd… The response pattern against specific viral Ag changes over time, as pts progress to AIDS. Antibodies to envelope glycoproteins (gp41, gp120,gp160) are maintained,but those directed against the Gag proteins (p17,p24,p55) decline. The decline of anti-p24 heralds the beginning of clinical signs and other immunologic markers of progression. 64 08-09-2011FACTS (in serodiag of HIV): FACTS (in serodiag of HIV) If two ELISAs are used then accuracy is 99.93% If one ELISA and western blot are used then accuracy is 99.99%. Mean time to seroconversion after HIV infection is 3-4 wks. All individuals will be positive within 6 months. HIV infection for longer than 6 months without a detectable antibody response is very uncommon. 65 08-09-2011CONFIRMATORY TESTS contd…: CONFIRMATORY TESTS contd… Tests which confirm HIV inf in an individual who is either sero+ve or has equivocal results from various serological tests. High degree of specificity. Can give few false -ve results. Purpose – to ensure that uninfected individuals, who test reactive in the screening assays aren’t incorrectly labelled as being +ve a) virus isolation, (b) detection of p24 antigen, (c) detection of viral nucleic acid -in situ hybridization, PCR 66 08-09-2011a) Virus isolation: a) Virus isolation Cultured from lymphocytes in pts peripheral bld (occasionaly plasma, CSF, genital secretions, specimens from other site - brain, bone marrow) The most sensitive virus isolation tech is to co-cultivate the test sample with UNINFECTED MITOGEN-STIMULATED PERIPHERAL BLOOD MONONUCLEAR CELLS. Needs T-cell growth factor (interlukin-2), Presence of virus is detected by assays for RT & p24 Ag in the culture fluid. Time consuming (takes 3-6 wks) & laborious, limited to research. 67 08-09-2011b) Detection of p24 antigen: b) Detection of p24 antigen p24 Ag can be detected in the serum by ELISA in 30% pts during WP, 10% of asymptomatic pts, & 50-60% of AIDS pts. As the antibody response builds up, Ag tests become –ve, but in late stages of infection p24 Ag may reappear. 68 08-09-2011c) DETECTION OF VIRAL NUCLEIC ACID : c) DETECTION OF VIRAL NUCLEIC ACID Molecular based Amplification assays – detect viral RNA in clinical specimens. PCR – most sensitive Commonly used ones :- RT-PCR, DNA-PCR. RT-PCR - uses enzymatic method to ampilify RNA. Very sensitive, basis for plasma viral load determination. HIV RNA levels are important predictive markers of disease progression & valuable tool to monitor the effectiveness of antiviral therapies 69 08-09-2011PCR : PCR HIV study- two forms of PCR used. 1) DNA PCR - detects single copy of proviral DNA from infected cells. 2) RNA PCR – detects single copy of RNA. Valuable diag test in pts with +ve or indeterminate ELISA & indeterminate WB. Also useful in pts in whom serologic testing is unreliable – pts with hypogammaglobulinemia. Quantitative PCR measures the viral load in the peripheral bld as number of viral copies\ml of bld 400 or less viral copies\ml cannot be detected by the presently available methods PROBLEM – artifacts due to contamination. 70 08-09-2011DNA-PCR : DNA-PCR Peripheral bld MNC lysed proviral DNA of HIV is amplified using proper primer pairs from relatively constant regions of HIV genome. Primer pairs from both the gag & LTR region are usually employed. It is possible to determine whether or not HIV proviral DNA is present at a frequency of one copy per 10,000 – 1,00,000 cells 71 08-09-2011RNA - PCR : RNA - PCR To make an early diag of HIV To monitor changes in the level of virus after infection 72 08-09-2011PowerPoint Presentation: 73 08-09-2011PowerPoint Presentation: 74 08-09-2011FACTS: FACTS Early diag of HIV inf in infants born to infected mothers can be done using plasma HIV-1 RNA tests. The presence of maternal antibodies makes serologic tests uninformative Low levels of circulating HIV-1 p24 Ag can be detected in plasma by ELISA soon after inf. The Ag often becomes undetectable after Antibodies develop (b’se the p24 protein is complexed with p24 antibodies) but may reappear late in course of inf, indicating a poor prognosis. 75 08-09-2011Strategies of HIV testing : Strategies of HIV testing Strategy I:- used for transfusion safety. The serum is tested with one E\R\S test & if reactive, sample is considered +ve & if non-reactive it is considered –ve. Use a highly sensitive & very reliable test kit . Strategy II:- used for HIV surveillance. The sr reactive with one E\R\S test is retested with a second E\R\S test with higher specificity, based on diff Ag preparation & diff test principle. If found +ve on 2 nd test also, it is reported as +ve, otherwise –ve 76 08-09-2011Strategies of HIV testing – contd… : Strategies of HIV testing – contd… Strategy III:- The sr reactive with 2 E\R\S tests is retested with a third E\R\S test. This 3 rd test should again be based on diff Ag preparation or test principle. a) A sr testing reactive with all three tests is reported +ve. b) A sr sample non-reactive in 3 rd test is considered equivocal\borderline. Such individuals should be retested after 3 wks. If this sample also provides an equivocal result, the person is considered to be HIV –ve. 77 08-09-2011Strategies of HIV testing – contd…: Strategies of HIV testing – contd… c) For symptomatic HIV inf, it is necessary to confirm the diag with 3 tests. d) Symptomatic inf with opportunistic infections, the pt may be subjected to two tests. The 1 st test selected for any of the strategies should be of highest sensitivity & 2 nd & 3 rd test selected should be of highest specificity. 78 08-09-2011PowerPoint Presentation: 79 08-09-2011In the field of observation chance favours only prepared minds – LOUIS PASTEUR: 08-09-2011 80 In the field of observation chance favours only prepared minds – LOUIS PASTEUR THANK YOU