New HIV Tests and Algorithm: A change we can believe in

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1 New HIV Tests and Algorithm: A change we can believe in Esther Babady, PhD, D (ABMM) Memorial Sloan-Kettering Cancer Center New York, New York

2 Learning Objectives After this presentation you should be able to: List new methods used for diagnosis of HIV infections Explain the advantages of new methods in diagnosis of HIV infections Describe the most recent CDC/APHL laboratory testing recommendations for HIV diagnosis

3 Case 68 yo woman Developed a large parotid mass on neck Radiologic studies: Abnormal Fine needle aspirate of parotid Enlarged, atypical cells and abundant necrosis Suggestive of B cell lymphoma

4 Case Non-Hodgkin s Lymphoma confirmed Pre-chemotherapy Infectious Diseases Evaluation Hepatitis serologies HIV serologies

5 Case Tests Results Reference range Hepatitis B surface antigen Negative Negative Hepatitis B surface antigen Positive Positive* antibody Hepatitis B core antibody (Total) Positive Negative Hepatitis B core antibody IgM Negative Negative Hepatitis B quantitative PCR Negative [20-170,000,000 IU/mL] Hepatitis C antibody Negative Negative HIV-1 and HIV-2 antibodies Negative Negative

6 Case Patient admitted 1 week later Started on chemotherapy protocol Tests Results Reference range HIV-1 and HIV-2 Negative Negative Tests antibodies Results Reference range HIV-1 quantitative PCR 120,000 [20-10,000,000 copies/ml]

7 Memorial Sloan-Kettering Cancer Center

8 Background: The virus Enveloped positive-sense ss RNA virus Retroviridae Lentivirus ~100 nm diameter 9.7 kb genome 5 and 3 LTR, 9-10 genes

9 Background: HIV Phylogeny Groups Major (M) Subtypes A, B, C, D, E,F, G, H, J, K HIV-1 Non M/O (N) Outlier (O) Pending (P) Circulating recombinant forms A HIV-2 B C,D,E,F,G,H

10 Background: HIV Distribution Subtypes Subtype A Subtype B Subtype C Subtype D Subtype F Subtype G Subtype H Subtype J Subtype K HIV-2 Geography West Africa North America and Europe South Africa and S/E Asia Eastern and central Africa Central Africa, South America and Eastern Europe Africa and Central Europe Central Africa Africa and Caribbean DRC and Cameroon West Africa McCutchan, F.E. JMV 2006

11 Background: Stages of Infection Acute Flu-like symptoms Highly infectious Weeks Established Mild or No symptoms Infectious Years AIDS Low CD4 count Opportunistic infections Years Naif, H.M. Infect Dis Rep. 2013

12 Background: Epidemiology Memorial Sloan-Kettering Cancer Center

13 Background: Epidemiology Memorial Sloan-Kettering Cancer Center

14 Trends in Annual Age-Adjusted* Rate of Death Due to HIV Infection, United States, Memorial Sloan-Kettering Cancer Center Note: For comparison with data for 1999 and later years, data for were modified to account for ICD-10 rules instead of ICD-9 rules. *Standard: age distribution of 2000 US population

15 Stage 3 (AIDS) Classifications and Deaths of Persons with HIV Infection Ever Classified as Stage 3 (AIDS), among Adults and Adolescents, United States and 6 Dependent Areas Note. All displayed data have been statistically adjusted to account for reporting delays, but not for incomplete reporting. Deaths of persons with HIV infection, stage 3 (AIDS) may be due to any cause.

16 Need for new recommendations?

17 Need for new recommendations? Better tests Simpler tests More commercially available tests More treatment options

18 Laboratory testing sequence CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, Accessed [04/2015]

19 CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, Accessed [04/2015]

20 Laboratory testing: Immunoassays Utility: Screening Diagnostics Additional testing: Acute vs Established 5 LTR gag pol vif/vpr/tat rev/vpu env tat nef 3 LTR

21 Laboratory Testing: Immunoassays 1 st generation HIV-1 IgG ab Viral culture cell lysates 2 nd generation HIV-1/2 IgG ab Viral culture cell lysates Synthetic/Recombinant ag 3 rd generation HIV-1/2 IgM/IgG ab Synthetic/Recombinant ag 4 th generation HIV-1/2 IgM/IgG ab p24 ag Synthetic/Recombinant ag CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, Accessed [04/2015]

22 Laboratory Testing: Western blot Cambridge Biotech HIV-1 Western Blot Kit PI

23 Laboratory Testing: Rapid HIV tests Manufacturer Rapid test: Test CLIA waived Methods Target Date Alere Scarborough Bio-Rad Laboratories, Inc Alere Determine HIV- 1/2 Ag/Ab Combo Geenius HIV 1/2 Supplemental Assay Lateral flow Immunoassay Immunochromatograph HIV-1 HIV-2 p24 HIV-1 HIV Accessed 4/2015

24 Laboratory Testing: Rapid HIV tests Manufacturer Test Methods Target Date Bio-Rad Laboratories, Inc. OraSure Technologies Multispot HIV- 1/HIV-2 Rapid Test OraQuick ADVANCE Rapid HIV-1/2 Antibody Immunoconcentration Microparticle Lateral flow Immunoassay HIV-1 HIV-2 HIV-1 HIV Accessed 4/2015

25 Laboratory Testing: Immunoassays Manufacturer Test Methods Generation Date cleared Abbott Diagnostics Bio-Rad Laboratories, Inc. Ortho Clinical Diagnostics Siemens Healthcare diagnostics Architect HIV Ag/Ab combo GS HIV Combo Ag/Ab Vitros Anti-HIV 1+2 Advia Centaur HIV 1/0/2 Chemiluminescence Microparticle Immunoassay Enzyme Immunoassay Chemiluminescence Immunoassay Chemiluminescence Immunoassay 4 th th rd rd 2006 Accessed 4/2015

26 Laboratory Testing: Molecular tests Utility Diagnosis of HIV infection (Qualitative) Baseline viral load before initiation of treatment (Quantitative) Monitoring of response to antiviral treatment (Quantitative) 5 LTR gag pol vif/vpr/tat rev/vpu env tat nef 3 LTR

27 Laboratory Testing: Molecular tests Examples of Qualitative Molecular Methods Manufacturer Test Methods LOD Hologic Aptima HIV-1 RNA TMA 100 qualitative assay HPA copies/ml Accessed 4/2015

28 Laboratory Testing: Molecular tests Manufacturer Test Methods Linear range Abbott Molecular Realtime HIV-1 RT-PCR 40-10,000,000 copies/ml Roche Molecular Siemens Cobas Ampliprep/Cobas Taqman quantitative HIV-1 v.2.0 COBAS TaqMan HIV Test v2.0 For Use With The High Pure System Cobas Amplicor HIV Monitor v1.5 VERSANT HIV 3.0 Assay (bdna) RT-PCR RT-PCR RT-PCR bdna signal amplification 20-10,000,000 copies/ml 34-10,000,000 copies/ml 50-75,000 copies/ml ,000 copies/ml ,000 copies/ml Accessed 4/2015

29 Laboratory Testing: Molecular Tests Highly specific and excellent sensitivity Expected viral loads log IU/mL Measured viral loads log IU/mL ULOQ LLOQ LOD

30 Laboratory Testing: Studies Comparison of WB to a rapid antigen test to on specimens reactive by a 4 th generation IA Philadelphia, PA Accuracy Rapid antigen test vs WB: 98.2% vs. 57.9% Specificity Rapid antigen test vs WB: HIV-2 misidentified as HIV-1 by WB. Cardenas, A.M. et al., Journal of Clinical Virology 58S1 (2013), e97 e103

31 Laboratory Testing: Studies Comparison of a rapid antigen test to WB, 3 rd and 4 th generation immunoassays. Commercial panels (HIV-1 and HIV-2) Seroconversion Rapid antigen test: 7-15 days earlier than WB. Accuracy Rapid antigen test was reactive in 99.0% and 92.5% of 3rd and 4th generation Masciotra, S. et al., Journal of Clinical Virology 58S (2013) e54 e58

32 Laboratory Testing: Studies 4 th generation HIV Ab/Ag, WB and HIV-1 RNA HIV treatment center: Amsterdam, Netherlands ~5,000/years over 10 years: 75 cases Ab/Ag positive, WB negative, HIV-1 RNA not detected 7 cases Ab/Ag positive, WB negative, HIV-1 RNA detected 6 cases Ab/Ag negative, WB negative, HIV-1 RNA detected Sensitivity/specificity: 99.6%/99.8%. Jurriaans, S. et al., Journal of Clinical Virology 52S (2011), S67-S69

33 Laboratory Testing: Studies Comparison of 3 rd and 4 th generation IA Multi sites study: CA and WA Seroconversion 4 th generation vs 3 rd generation: 7-11 days earlier Specificity 4 th generation vs 3 rd generation: % Mitchell, E.O. et al., Journal of Clinical Virology 58S1 (2013), e79-e84

34 Laboratory Testing: Studies Comparison of WB to RAT and NAAT. 11 U.S. public health laboratories 570 remnant 3 rd gen IA reactive, HIV WB neg or indeterminate. ~90% confirmed HIV negative ~10% HIV positive (~3% acute HIV and 0.2% HIV-2). Linley, L. et al., Journal of Clinical Virology 58S1 (2013), e108-e112

35 Laboratory testing: Genotyping Utility: Monitoring HIV-1 resistance Baseline before start of antiretroviral therapy (ART) Guide alternative ART selection in cases of drug therapy failure

36 Laboratory Testing: Genotyping Examples of Genotyping assays Manufacturer Test Methods LOD Target Abbott Molecular Siemens Molecular Diagnostics Viro Seq HIV-1 sequencing system TRUGENE HIV -1 Genotyping Assay Sequencing 2000 copies /ml Protease RT Sequencing 1000 copies/ml

37 U.S. Preventive Services Task Force

38 CDC HIV Testing Guidelines 1989: Testing for HIV-1 immunoassays and confirmation with HIV-1 WB or IFA (MMWR v38, 1989) 1992: Testing for HIV-2 antibodies (MMWR v41, 1992) 2004: Use of HIV-1 WB/IFA to confirm reactive rapid test results (MMWR v53, 2004)

39 CDC HIV Screening Guidelines 2014: Recommended algorithm Initial testing for HIV should be done using 4 th generation immunoassay Reactive screen should be confirmed with an Ab test that differentiates HIV-1 and HIV-2 If reactive samples do not confirm, additional testing with a HIV-1 NAT Algorithm should be applied to reactive rapid tests CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, Accessed [04/2015]

40 CDC HIV Screening Guidelines 2014: Alternative algorithms Reactive 3 rd generation immunoassay followed by recommended algorithm Reactive WB/IFA followed by HIV-1 NAT and HIV-2 ab test if necessary Reactive 4 th generation IA followed by HIV-1 NAT Nonreactive 3 rd and 4 th generation IA followed by HIV-1 NAT CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, Accessed [04/2015]

41 New Testing algorithm CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, Accessed [04/2015]

42 Reporting results CDC guidelines, 2014

43 Conclusions Several, new, sensitive and user-friendly assays are now available for accurate diagnosis of HIV Recent CDC/APHL recommendations use combinations of these new methods to identify more cases of acute HIV infections

44 Self-Assessment Questions 1. The new CDC/APHL recommends that a reactive 4 th generation immunoassay be followed by testing with which of the following tests? A. HIV-1/HIV-2 ab C. Western Blot B. HIV-1 RNA D. HIV-2 RNA 2. Which of the following HIV antigens is detected by antibody/antigen combo assays? A. p24 C. gp120 B. p55 D. gp160

45 Self-Assessment Questions 3. Use of the new CDC/APHL HIV testing algorithm improves detection of acute HIV? A. True B. False 4. Which of the following test is reactive during the eclipse period of HIV infection? A. qualitative HIV-1 RNA C. Both of the above B. 4 th generation IA D. None of the above

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