Objectives. Seven days of separation. Clinical situation a transplant. The Donor COOMBS MD PHD 1. Laboratory Diagnosis & Monitoring of HIV Infection

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1 Laboratory Diagnosis & Monitoring of HIV Infection An Introduction to Diagnostic HIV Testing Robert W Coombs MD, PhD, FRCPC Professor, Departments of Laboratory Medicine & Medicine University of Washington, Seattle, Washington bcoombs@u.washington.edu Objectives Clinical case Background Acute infection Diagnosis: the seronegative window how to close it with HIV-1 nucleic acid testing (NAT) From laboratory-to-bedside testing: Simple, rapid HIV tests for point-of-care (POC) testing how close are we? 1 2 Seven days of separation The Japanese Tsunami was 11MAR11 A laboratory diagnostic Tsunami occurred on 18MAR11: To err is human, to forgive, not so divine. After, Alexander Pope ( ) Question: Why is seven days so ironic? Clinical situation a transplant The kidney recipient Hemodialysis-dependent adult male received a kidney transplant from a living donor in 2009 (New York) Tested negative for HIV-1 antibodies 12-days before transplant; no known risk factors for HIV-1 infection One-year later, hospitalized with advanced immunological suppression, which was attributed at that time to his anti-rejection therapy A screening test for HIV-1 antibody was reactive and confirmed with a positive Western blot 3 4 MMWR/60/No.10/ /March 18, 2011 The Donor Adult male; history of sex with men; syphilis HBV, HCV and HIV-1 negative 79 days before transplant One year following transplant, asks for STD testing and is found to be HIV-1 infected! 5 6 COOMBS MD PHD 1

2 Implications of this case CDC/MMWR March 18, 2011 What we already know: Routine laboratory screening for organ donors for HIV infection introduced in 1985 BUT no national policy exists for the type or timing of HIV screening tests used for living donors Why? What is added by this case: First documented case in the United States of HIV transmission through transplantation of an organ from a living donor, despite screening using serological testing!! Could there be limitations to serological testing and how do we overcome these limitations? Implications (cont d) What are the Public Health Implications? All prospective living organ donors should have initial serological tests supplemented with repeat testing with a combination of an HIV serological test and a nucleic acid test no longer than 7 days before organ donation What are the implications for the laboratory? Advise living donors of their obligation to avoid behaviors that would put them at risk for acquiring HIV before organ donation Nothing replaces a good medical history and frank conversation with the donor about HIV-risk behavior! 7 8 HIV-1/-2: A review of the basics VIRUS HIV is a human retrovirus diploid, plus-stranded, enveloped RNA-virus that uses a virus-encoded reverse transcriptase to transcribe the ssrna genome to double-stranded DNA, which integrates into the cell genome to form the PROVIRUS; transcription of DNA to mrna and genomic RNA followed by translation to viral proteins; zoonotic origin from African primate species (e.g., HIV-1 linked to Chimpanzees, Pan troglodytes troglodytes infected with SIVcpz) CLINICAL HIV-1: World-wide pandemic with more than 33 million persons infected and over 2 million deaths IMPORTANCE per year; major epidemic in Black American males and females; transmission via blood & body fluids, sex & injection drug use, mother-to-child, rare via blood transfusions with appropriate blood product screening in developed countries; produces immune deficiency (AIDS); HIV-2: limited epidemic to areas of Western Africa & India with importation of cases into Europe and Unites States; slower disease progression than HIV-1. DIAGNOSIS & Serological (EIA and Western blot) with virus-specific peptides to distinguish HIV-1 from -2; MONITORING polymerase chain reaction (PCR) amplification for detecting HIV RNA before antibody is detected; viral capsid (antigen) detection; virus may be grown by using mixed-lymphocyte co-culture; HIVinfection is monitored with plasma HIV RNA level & blood CD4 cell count. TREATMENT No cure or vaccines exist for HIV: for HIV-1, antiretroviral (ARV) drugs directed against virusspecific envelope fusion & chemokine-receptor binding; block reverse transcriptase, protease & integrase enzymes; prophylaxis for opportunistic infections when CD4 cell count is <200 cells/mcl; ARV therapy for HIV-2 somewhat more limited; post-exposure prophylaxis recommended (PrEP?) 9 Background to the virus Basic pathophysiology Life-long, chronic viral infection Viral replication drives the disease Progressive immune dysfunction defines the disease Successful antiretroviral therapy decreases transmission prolongs life We monitor the disease by following viral RNA levels and CD4 cell counts 10 HIV-1 Pathophysiology Infects CD4+ T-cells and macrophages Virus integrates into genome of cell Lifelong, chronic infection High rates of replication, recombination and variation Immune activation favors viral replication Vigorous immune response does not clear virus; sadly, there are NO cases of spontaneous viral clearance or cure Untreated, chronic infection results in progressive immunodeficiency with eventual death from wasting, opportunistic infections and malignancies HIV establishes a life-long, chronic/latent infection An infected cell reservoir is established within days of infection The gastrointestinal tract is depleted of CD4 cells early in infection An infected cell may return to a resting state as a memory T-cell, which maintains the reservoir of latently infected cells for life COOMBS MD PHD 2

3 Immune response to HIV is vigorous but ineffective Sequence of events in HIV The CD4 T-cell is the immune system conductor and orchestrates all other components of the immune response HIV targets the CD4 T-cell; ergo, a disabled conductor results in immune dysfunction & immunodeficiency Persistent viremia can be used to monitor infection and define prognosis -- the viral set point Ultimately, the decline in CD4 cell count defines the risk for opportunistic infections & malignancies Therapeutic interventions are directed at: Total CD4 cell count 13 Lymphoid tissue & CD4 cells in terminal ileum Suppressing viral replication to as low as possible Elevating the CD4 cell count to >200 cells/mcl 14 CD4 count and survival Brenchley et al., J Exp Med 2004; 200: Disease progression most marked with lowest CD4 cell counts Implications for when to start therapy 15 Viral RNA set-point and progression to AIDS COOMBS MD PHD Slow progressors (20%) Rapid progressors 16 Time to AIDS & death: a train speeding towards a precipice HIV RNA level and survival <350 cell/µl The speeding train analogy: CD4 CELL COUNT HIV RNA LEVEL 50% of patients in highest viral RNA quartile died in 5 years, which contrasts with <10% of patients in lowest quartile who died within 5 years of study entry These data are from a natural history study; subjects were not on antiretroviral therapy 17 Length of track remaining Speed at which the train is moving towards the AIDS/ Death precipice Multisystem effects related to above 18 3

4 The decline in CD4 cell count defines disease progression Acute HIV infection Issue to discuss (RNA level begins to increase) Laboratory diagnosis of acute HIV infection; what are the available tools? AIDS Laboratory Diagnosis & Monitoring Serology Enzyme immunoassay (EIA) and confirmatory immunoblot (Western blot) or immunofluorescence antigen detection (IFA) Viral nucleic acid DNA (cell-associated proviral) RNA (cell-free plasma, virion-associated, genomic RNA) CD4 cell count Viral life cycle and targets for diagnosis Infectious virus Viral p24 antigen RNA genome Viral DNA Reverse transcriptase Protease YY Anti-HIV antibodies directed against viral proteins Key components of the virus used to establish a laboratory diagnosis of HIV infection Timing of blood markers of HIV infection following exposure/infection Generally takes 3-6 months before viral RNA set-point is reached Eclipse Serological diagnosis of HIV-1 infection is based on demonstrating viral specific antibodies against at least two of the three viral proteins: p24, gp41 and gp Seronegative window 24 COOMBS MD PHD 4

5 Simple/rapid testing Revised guidelines for HIV antibody testing/ screening for the health-care setting released in 2006 MMWR 2006:55(No. RR-14):1-17 The effort here is to detect the 250,000 or so HIVinfected persons in the United States who do not know that they are HIV infected! Requirement for more point-of-care (POC) HIV antibody screening Guidelines don t include laboratory screening for acute infection (i.e., role of HIV NAT pooling as a public health intervention) HIV Testing Several anti-hiv antibody testing technologies approved by FDA enable testing of different fluids (i.e., whole blood, serum, plasma, oral fluid and urine) in the point-of-care setting: Enable specimen collection procedures that are less invasive and more acceptable than venipuncture Enable provision of rapid HIV test results during a single visit Increase the convenience of HIV testing Acute HIV infection Issues to discuss Laboratory Diagnosis: the seronegative window what is it & how to close it Viral eclipse -innate immunity -exposed seronegatives Dissemination & Seroconversion Submucosal propagation of HIV: the eclipse phase Zhang et al., PNAS 2004; 101: Blood markers of HIV acute/early infection Fiebig Stage (lower 95% CI) Generally takes 3-6 months before viral RNA setpoint is reached Eclipse Marker (median appearance) 1. RNA (5 days) 2. P24 Ag (10 days) 3. IgM Ab (14 days) 4. IND WB (19 days) 5. p31 Ab (89 days) 6. Full WB (indefinite) Donor gets infected Seronegative window COOMBS MD PHD 5

6 Fiebig et al., AIDS 2003; 17: HIV Enzyme immunoassays (EIA) Current third-generation assays are recombinant-antigen/ peptide sandwich EIAs that can detect all classes of antibodies to HIV-1 (i.e., IgM & IgG); Fourth-generation assays combine detection of anti-hiv antibody with p24 antigen (J Clin Microbiol 1998;36: ; Eur J Clin Microbiol Infect Dis 2001;20:104-10); Current third-generation & fourth-generation assays have greater sensitivity than either first or second-generation EIAs in detecting HIV-1 antibodies in the early stages of infection (CID 2009;49:444-53); Opportunity to combine 3 rd Generation EIA with a simple/ rapid test to provide clinicians with a presumptive positive test result while awaiting confirmation by WB (in progress) Virus Recombinant -antigen; IgG Peptides+IgM/IgG 3 rd + monoclonal p24 antibody HIV-1 RNA Window period estimates & reduction using different HIV detection assays [Am J Med 1997;102:115-6] See also CID 2009; 49: Plasma HIV-1 RNA for diagnosing primary infection Now approved for clinical diagnosis of HIV infection; clearly, has an important role in diagnosing acute infection (MMWR, December 5, 2008 / 57(RR10);1-8) Occasional false-positives result in a specificity of only 97.4% (AIM 2001;134:25-29; AIM 1999;130:37-9) but this has improved because of closed amplification/detection systems Because of potential for social & legal ramifications of a false-positive test result, consider confirmation with p24 Ag, HIV DNA PCR and importantly, follow-up testing; False-positives in 2.6%-5% of NAT with <2,500 RNA c/ml & contrasts with most primary infections, which are associated with >100,000 RNA c/ml or more (AIDS 2002; 16: ); HIV RNA amplification used to screen donated blood: only 4 of 12.6 million donations that were p24 Ag and HIV antibody-negative were found to be HIV RNA positive (Transfusion 2000;40: ) Donor gets infected ROOT CAUSE ANALYSIS CDC revised case definition of HIV infection (MMWR, December 5, 2008 / 57(RR10);1-8) Positive result from an HIV antibody screening test (e.g., reactive enzyme immunoassay [EIA]) confirmed by a positive result from a supplemental HIV antibody test (e.g., Western blot or indirect immunofluorescence assay test) or Positive result or report of a detectable quantity (i.e., within the established limits of the laboratory test) from any of the following HIV virological (i.e., non-antibody) tests: HIV nucleic acid (DNA or RNA) detection test (e.g., polymerase chain reaction [PCR]) HIV p24 antigen test, including neutralization assay HIV isolation (viral culture) COOMBS MD PHD 6

7 Testing for HIV infection Issues to discuss Antibody testing is sensitive and cost-effective Simple, rapid antibody tests for point-of-care testing what are the limitations? Simple, rapid nucleic acid tests for point-of-care testing how close are we? and by the FDA for use in Serological testing - summary Most HIV infection is diagnosed by demonstrating antibody specific for HIV-1 or HIV-2; Assays can detect antibody in serum, plasma, whole blood, saliva, urine and dried blood collected on filter paper; The mean time to seroconversion in patients with primary infection (3 rd generation EIA) is 20 days and most having detectable antibodies within 6-12 weeks of infection and almost all by 6 months (Am J Med 1997;102:117-24); 4 th generation assays shorten detection of seroconversion by 5 days or so compared to 3 rd generation assays (see table 1: CID 2007;44:460; SLIDE 33) Excessive delay in development of antibody response following infection at a mucosal surface is likely due to eclipse phase of clinical infection; Reactive EIA results are repeated and then confirmed by immunoblot or indirect immune fluorescence antibody (IFA) detection; Given the social & legal importance of a correct diagnosis, a repeat blood specimen should be obtained to exclude specimen mislabeling. HIV EIA for screening False positive test results Low positive predictive (PPP) value (e.g., 70%) in a population with a low HIV seroprevalence (0.5%) (MMWR 1989;36:833-45); Primarily due to cross-reactivity to contaminating bacterial or yeast proteins (due to use of recombinant peptides) but now less likely to be due to HLA-specific antibodies associated with rheumatoid arthritis, systemic lupus erythematosus, polyclonal gammopathy, and antibodies to HLA-DR as was seen with earlier generation EIA testing (however, many health departments still screen with 1 st generation assays!); With the addition of confirmatory testing (e.g., Western blot), the PPP is increased to almost 100% (JAMA 1986;256: and NEJM 1988;319:961-64) Simple/rapid testing Revised guidelines for HIV antibody testing/ screening for the health-care setting released in 2006 MMWR 2006:55(No. RR-14):1-17 The effort here is to detect the 250,000 or so HIV-infected persons in the United States who do not know that they are HIV infected! Role for Rapid HIV Tests Increase receipt of test results Increase identification of HIV-infected pregnant women so they can receive effective prophylaxis Increase feasibility of testing in acute-care settings with same-day results Increase number of venues where testing can be offered to high-risk persons The down side: with low seroprevalence, there will be many false-positive results given to people! Inefficient for large numbers of specimens Quality assurance/control are required; operator errors are an issue COOMBS MD PHD 7

8 Simple/Rapid Test Devices (S/ RTDs) for HIV antibody testing Simple/rapid test formats Immunoconcentration Immunochromatography In the United States, the OraQuick Advance Rapid HIV-1/2 Antibody Test device (OraSure Technologies) and the Uni-Gold Recombigen HIV Test are two of the six FDAapproved S/RTDs although only the OraQuick, Uni-Gold and Clearview tests are CLIAwaived and suitable for point-of-care (POC) testing; the others are of moderate complexity and more suitable for the laboratory setting. There is a compelling need to improve POC testing for HIV in the United States and in resource-poor settings. Reveal G3 Rapid HIV-1/2 Antibody Test device; Multispot HIV-1/2 Rapid Test; Clearview HIV-1/2 Stat-Pak; and Clearview HIV-1/2 Complete 43 Flow-through devices: Mulitspot & Genie II Lateral-flow devices: OraQuick, Uni-Gold, Determine & Hema-strip Particle agglutination Latex agglutination: Capillus & Serodia Branson, CDC Branson, CDC Trinity Biotech Uni-Gold Recombigen Uni-Gold Recombigen Clearview Complete HIV 1/2 Clearview Complete HIV 1/2 Multispot HIV-1/HIV-2 MedMira Reveal G3 Multispot HIV-1/HIV-2 Clearview HIV ½ Stat Pak Reveal G3 OraQuick Advance 45 The most compelling rationale for simple/rapid test devices (S/ RTDs) with a point-of-care application is to: Clearview HIV ½ Stat Pak OraQuick Advance Rapid serological testing for HIV Rapid nucleic acid testing for HIV Test parturient women & identify HIV-1 infected women in a timely fashion so that antiretroviral therapy can be started to prevent mother-to-child transmission (MCTC) of HIV at the time of labor & delivery (JAIDS 2004;35:151-54); Identify unrecognized, asymptomatic HIV-1 infection in a more timely way and in nearpatient situations: pharmacies, malls, bathhouses, etc. Quantitative vs qualitative Diagnostic (acute/early adult & neonate/infant) and clinical management capabilities HIV-1 and HIV-2 No clade restrictions Simple (self-contained reagents), rapid (< 1 hour) and minimal technologist training Adaptable to resource-limited setting Stable reagents; power requirements should be portable; electronic data output; internal QA controls Cohen et al., AIDS 2003;17: COOMBS MD PHD

9 S/R POC: LIAT (IQuum, Inc) JID 2010;201(Suppl 1): Clinical specimens (N=39) LIAT value = *Abbott value; r 2 = S/RTDs: Summary Occupational post-exposure prophylaxis Identify HIV serostatus of source and reduce the need for postexposure prophylaxis if the source is negative. STD clinics Only one-third of patients who test positive for HIV-1 by traditional methods return for test results (MMWR 2003;52:329-32); S/RTD testing improves the effectiveness of counseling and testing (AIDS 1997;11: ). Other point-of-care settings Clinics, pharmacies, emergency rooms, labor & delivery, assisted reproductive technology, vaccine-inducedseropositives (VISP), etc. S/RTDs: Summary continued Resource-limited settings Cost effectiveness including: absence of requirements for expensive and specialized equipment, maintenance, and the overall cost savings compared with conventional screening and confirmatory testing and ease of use by minimally trained non-laboratory personnel; Develop strategies to combine S/RTDs with laboratorybased EIAs to confirm HIV infection (AIDS 1997;11:369-75); now incorporated into UWMC HIVtesting algorithm Major need for quantitative s/r HIV RNA testing Conclusions Future diagnostic algorithms should incorporate HIV nucleic acid testing; hence, a need for simple/ rapid HIV RNA tests! [Remember the organ donor!] Finally, to reduce potential harm for vaccine trial participants, HIV testing outside of a vaccine research study protocol should be approached with caution until health care providers are educated about HIV vaccines and the need for diagnostic algorithms that incorporate HIV nucleic acid testing [Not really discussed but an obvious caution] Lecture Epilogue Serology still remains the mainstay of HIV diagnosis; Simple/Rapid Test Devices (S/RTDs) for HIV antibody detection are very useful for point-of-care HIV testing in both resource-rich & resourcelimited settings but the limitations of the S/RTDs must be appreciated; Along with CD4 cell count, HIV RNA has an established role for monitoring disease progression & response to therapy Successful antiretroviral therapy is associated with suppressed HIV-1 replication; Viral nucleic acid detection for diagnosis is evolving Remember the organ donor case; Development of simple/rapid HIV RNA quantitative (or semi-quantitative) assays would be helpful for HIV diagnosis as HIV NAT is necessary to identify early HIV infection Remember the seronegative window COOMBS MD PHD 9

10 What we covered today Reviewed a landmark clinical case Provided a background for understanding the basis of HIV diagnostic testing algorithms Reviewed acute infection Identified the importance of the seronegative window and how to close it with HIV-1 nucleic acid testing (NAT) Reviewed aspects of the laboratory-to-bedside paradigm and showed that simple, rapid antibody and NAT assays for HIV point-of-care (POC) testing has arrived and offers an important role in both the resource-rich and resource-poor settings. An Introduction to Diagnostic HIV Testing Now, I m NOT so DUMB after all!! COOMBS MD PHD 10