List the steps in the fourth generation HIV screening algorithm Describe the relationship between rapid HIV antibody tests and the fourth generation

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1

2 Alere (honoraria)

3 List the steps in the fourth generation HIV screening algorithm Describe the relationship between rapid HIV antibody tests and the fourth generation testing algorithm Interpret challenging HIV screening results Recall the role of molecular testing in the diagnosis and management of HIV

4 30 year old pregnant female admitted to Labor and Delivery Found to be in early stages of labor Upon questioning, it was revealed that the patient did not receive routine prenatal care Several laboratory tests were ordered, including a rapid HIV antibody test Rapid HIV test was found to be positive

5 Follow-up fourth generation HIV antigen/antibody test performed Negative Does this patient have HIV? What follow-up testing would you recommend? Would you have tested this patient differently?

6 Enveloped single stranded RNA retrovirus HIV-1 Responsible for AIDS worldwide pandemic Groups M (major), O (outlier), and N (non- M, non-o) Derived from chimpanzees HIV-2 Limited geographic distribution (Africa and parts of Europe) Subgroups A-G Derived from sooty mangabeys

7 Commonly transmitted sexually through direct contact with body fluids including blood, semen, and vaginal secretions Tropism for CD4+ T cells and macrophages Three clinical stages of HIV infection Acute HIV infection- associated with non-specific symptoms (fever, rash, malaise, lymphadenopathy, and pharyngitis) Clinical latency- symptom free period with low level viral replication AIDS- marked by increased viral load, decrease in CD4 positive T lymphocyte, and resultant opportunistic infections

8 RNA P24 Antigen Diagnosis may be missed if inappropriate test is used Antibody Infection Days Eclipse Period Acute Retroviral Syndrome (symptomatic and very contagious) Adapted from Laboratory Testing Recommendations for the Diagnosis of HIV, Updated Recommendations, Centers of Disease Control and prevention, June 2014 Clinical Latency (asymptomatic)

9 First Generation Third Generation Viral lysate antigen target Detect IgG only Only ~95% specific 8-10 week window Recombinant antigen target Detect IgG and IgM 99.5% specific 2-3 week window Second Generation Fourth Generation Recombinant antigen target Detect IgG only 99% specific 4-6 week window Recombinant antigen target Detect p24 antigen Detect IgG and IgM 99.5% specific 2 week window Adapted from: Chappel R J, Wilson K M, Dax E M, Immunoassays for the Diagnosis of HIV: Meeting Future Needs by Enhancing the Quality of Testing, Future Microbiol. 2009;4(8):

10 HIV RNA Detectable ~10 Days Following Infection HIV Antibodies Detectable ~20 Days Following Infection HIV Antigen Detectable ~15 Days Following Infection HIV Antibody Response Expands Nucleic Acid Detection Test Positive Fourth Generation Antibody/Antigen Assay Positive Third Generation Antibody Assay Positive Western Blot Positive

11 Traditional screening performed using a third generation enzyme immunoassay (EIA) Traditional confirmation performed by Western blot immunoassay Discerns antibody specificity to immobilized HIV proteins Must have antibodies to multiple key proteins to be interpreted as positive Novel fourth generation assays detect both antibody and p24 antigen Allow for earlier diagnosis than serology alone Currently recommended by CDC for routine screening Still require confirmatory testing (Western blot no longer good option)

12 Laboratory Testing Recommendations for the Diagnosis of HIV, Updated Recommendations, Centers of Disease Control and prevention, June 2014

13 Detects all immunoglobulin classes to HIV-1 and HIV-2 Detects p24 expressed by HIV-1 and HIV-2 Increased sensitivity and specificity compared to many third generation assays Most are performed on large chemistry lab analyzers ADVIA Centaur <1 hour run time Abbott Architect <30min run time Bioplex 45 min run time Capable of differentiation between p24 and HIV1/2 antibodies Positive results require further confirmation

14 Laboratory Testing Recommendations for the Diagnosis of HIV, Updated Recommendations, Centers of Disease Control and prevention, June 2014

15 The Multispot Performed following positive antigen/antibody screen Second generation assay Detects only IgG antibody Steps Immobilized HIV-1 and HIV-2 antigens treated with patient serum After washing alkaline phosphatase labeled goat antihuman IgG is added Developer is added and positive test spot turn purple Control HIV-2 Peptide Recombinant HIV-1 HIV-1 Peptide = Nonreactive = HIV-1 Positive = HIV-1 Indeterminate = HIV-2 Positive

16 FDA approved supplemental HIV test Successor to the Multispot Multispot no longer in production by manufacturer Immunochromatographic assay Tests for antibodies against 4 HIV-1 proteins 2 HIV-2 proteins Results interpreted by an automated reader Helps prevent user error Sample Application HIV2 targets HIV1 targets Control

17 Laboratory Testing Recommendations for the Diagnosis of HIV, Updated Recommendations, Centers of Disease Control and prevention, June 2014

18 HIV RNA Detectable ~10 Days Following Infection NAAT is performed to detect acute cases HIV Antibodies Detectable ~20 Days Following Infection HIV Antigen Detectable ~15 Days Following Infection HIV Antibody Response Expands Nucleic Acid Detection Test Positive Patient may have acute HIV if fourth gen positive, differentiation assay negative Fourth Generation Antibody/Antigen Assay Positive Third Generation Antibody Assay Positive Western Blot Positive

19 Currently only one FDA approved molecular test is available for the diagnosis of HIV-1 Aptima HIV-1 RNA Detects viral RNA using transcription mediated amplification Quantitative tests are FDA approved for monitoring only But more readily available than qualitative tests Can be used as qualitative diagnostic tests following appropriate validation Important caveat: HIV-2 is not detected by these assays!

20 How Does This Algorithm Function? Laboratory Testing Recommendations for the Diagnosis of HIV, Updated Recommendations, Centers of Disease Control and prevention, June 2014

21 4 th Generation HIV-1/2 Antigen(AG)/Antibody(Ab)Combo, blood (n=10,536) 1% 99% HIV-1 Ab reactive: confirmed Reactive for HIV-1 (n=62) Reflexed to HIV-1 Viral Load 76% Reactive (n=82) Multispot for HIV- 1/HIV-2 Ab Differentiation (n=82) 22% Non-reactive for HIV-1/HIV-2 Ab (n=18) 0% Non-reactive (n=10,454) HIV-2 Ab reactive: confirmed Reactive for HIV-2 (n=0) 2% Reflexed HIV-1 Viral Load (n=17) 94% HIV-1 viral load Not Detected: Possible false positive screen (n=16) 6% No further testing. The final result is Nonreactive Undifferentiated recommend retesting or HIV-1 viral load (n=2) HIV-1 viral load Detected: consistent with acute or early HIV-1 infection (n=1)

22 Approximately 25% of antigen/antibody screens were false positives Is this too high??? A study of 10,014 life insurance applicants of low seroprevalence were tested by this algorithm 13 patients were positive on initial testing (85% false positives) A study of 51,935 Florida patients in a high seroprevalence setting were tested by the algorithm 1089 patients were positive on initial testing (7.2% false positives) Take home- Population sero-prevalence affects positive predictive value!

23 Conditions implicated with false positives Rheumatoid arthritis, lupus, Sjogren's and other autoimmune conditions Cross reacting viruses Pregnancy Chart review of patients with false positive screens (n=14) 7/14 patients were either pregnant (n=3) or had a documented autoimmune disorder (n=4) 2/14 had identified risk factors (IVDU) Both positive for HCV, though ultimately HIV negative Remaining five patients included Patient with alcoholic pancreatitis Patient with sepsis Patient with FUO that spontaneously resolved Patient with cystic fibrosis s/p lung transplant Patient with unknown medical history

24 Positive rapid antibody test, negative antigen/antibody screen Pregnant (associated with false positives) Can we say her rapid antibody test is a false positive?

25 Variety of different formats Some detect IgG only (second generation) Some detect IgG/IgM (third generation) Some detect IgG/IgM and p24 antigen (fourth generation) Advantages Easy to perform Results often in under 30 min Many are CLIA waived, so can be used POC Can use a variety of specimens (ie. saliva, blood, etc) Allows for immediate patient counseling

26 Test Chembio DPP HIV-1/2 Clearview COMPLETE HIV1/2 Clearview HIV1/2 STAT-PAK OraQuick ADVANCE Rapid HIV1/2 Antibody Test INSTI HIV-1/HIV-2 Antibody Test Uni-Gold Recombigen HIV1/2 Determine HIV1/2 AgAb Combo Test Detects HIV IgG antibody (second generation) HIV IgG/IgM antibody (third generation) HIV IgG/IgM antibody and antigen (fourth generation)

27 n= 7/32 n= 7/31 n= 8/33 n= 8/27 n= 11/33 n= 19/33 n= 25/33 n= 29/33 Sensitivity (%) Sensitivity in patients with positive nucleic acid amplification test and negative/indeterminate Western Blot Patel P, Bennett B, Sullivan T, Parker M M, Heffelfinger J D, Sullivan P S, and CDC AHI Study Group, Rapid HIV screening: Missed opportunites for HIV diagnosis and prevention. Journal of Clinical Virology 54(2012) 42-47

28 Western blot or IFA was previously recommended to confirm rapid tests This was because certain rapid tests were actually more sensitive than in-lab immunoassays This has changed with the fourth generation testing Fourth generation in-lab tests are more sensitive and specific than currently available rapid tests (even rapid fourth generation tests)

29 Fourth gen antigen/antibody tests have much greater sensitivity than third gen tests (should ALWAYS be positive if third gen test is true positive) HIV RNA Detectable ~10 Days Following Infection HIV Antibodies Detectable ~20 Days Following Infection HIV Antigen Detectable ~15 Days Following Infection HIV Antibody Response Expands Fourth Generation Antibody/Antigen Assay Positive Third Generation Antibody Assay Positive

30 Any reactive rapid antigen test should be tested by the fourth generation algorithm starting at the beginning Supplemental testing is NOT required for any patients positive by rapid antigen, and negative by fourth generation The role of the rapid test is to screen for those who should get fourth generation testing

31 Advantages Allow for bedside diagnosis Allow for same visit counseling Allow for taking the testing to the patient Is rapid HIV testing appropriate in the hospital? It depends! If your hospital is able to provide same visit in-lab testing probably not

32 Adapted from: Reactive rapid HIV antibody test (3 rd or 4 th generation) HIV-1/2 antigen/antibody combination immunoassay (preferred screen) (+) HIV-1/HIV-2 antibody differentiation immunoassay (-) Negative for HIV-1 and HIV-2 antibodies and p24 antigen HIV-1 (+) HIV-2 (-) HIV-1 antibodies detected HIV-1 (-) HIV-2 (+) HIV-2 antibodies detected HIV-1 (+) HIV-2 (+) HIV antibodies detected HIV-1 (-) or indeterminate HIV-2 (-) HIV-1 RNA* HIV-1 infection HIV-2 infection Cannot differentiate HIV-1 and HIV-2. Consider molecular testing or testing serology at later date HIV-1 RNA (+) Acute HIV-1 infection HIV-1 RNA (-) Negative for HIV-1

33 Proper HIV testing stewardship is a laboratory responsibility Challenges to consider Result reporting/communication Specimen requirements Assuring follow-up testing is performed Algorithmic testing should be developed in collaboration with the entire patient care team!!!

34 OB patient with positive rapid, negative in-lab fourth gen assay No further testing was performed Patient was told she did not have HIV In retrospect, physician did not know which HIV test to order It was discussed with OB providers that the inlab fourth gen is nearly as fast though more accurate Rapid HIV testing is no longer performed in this area

35

36 Treatment requires combination of highly active antiretroviral drugs (HAART) Nucleotide reverse transcriptase inhibitors (NRTIs) Non-nucleotide reverse transcriptase inhibitors (NNRTIs) Protease inhibitors Fusion inhibitors CCR5 entry inhibitors Integrase inhibitors Nature Reviews Drug Discovery 6, (Dec 2012)

37 Reverse transcriptase is prone to errors HIV lacks a proofreading enzyme 1 random mutation occurs per site per day Patients must be on multidrug therapy Patients on ineffective combination therapy will rapidly develop drug resistance Zidovudine studies show nearly 100% resistance in 6 months Patients need to be carefully monitored for resistance

38 Derived from Health and Human services working group for HIV guidelines CD4 Useful measurement of immune status at diagnosis Conveys urgency of starting treatment for HIV and opportunistic infections Viral load Useful predictor of disease progression at diagnosis Useful marker of disease progression after the start of therapy

39 Measure immediately before treatment and 2-8 weeks later 2 log copies/ml decrease in viral load after first 2-3 months indicates treatment response Monitor every 3-4 months to ensure response Treatment goal: undetectable by week log copies/ml difference is clinically significant (threefold change) Biologic variation 0.3 log Assay variation 0.2 log

40 Only detect HIV-1 Earlier developed assays used a variety of technologies Conventional reverse transcription PCR Branched DNA signal amplification Nucleic Acid Sequence Based Amplification More recent viral load assays use real time PCR Signal generated during amplification process by fluorescent probes

41 Less manipulation of specimens so less chance for contamination Highly automated for high throughput Higher sensitivity for recombinant forms and less common genotypes Lower levels of detection (40 and 20 copies/ml)

42 March 2008 Clinical Infectious Disease publication 1387 patients visited clinic March thru June 876 had been followed frequently (ie seen every 1-3 months) and had previously negative viral loads by Amplicor Monitor system (LOD 50 copies/ml) Switched to CAPTAQ (RealTime PCR) HIV viral load was >50 copies/ml in 253 patients (28.9%) Gatanaga et al, Detection of HIV Type 1 Load by the Roche Cobas Tapman Assay in Patients with Viral Loads Previously Undetectable by the Roche Cobas Amplicor Monitor, CID, Vol48, pp , 2009

43 Is next generation viral load testing too sensitive? Added cost Over-interpretation of drug failure Psychological distress Are all positive results significant?

44 Defined by the 2014 Panel on Antiretroviral Guidelines for Adults and Adolescents (Department of Health and Human Services) Viral loads transiently detectable at low levels (typically <400 copies/ml) May be seen in patients with transient infections Recommended NOT to check viral loads when patients are acutely ill May be seen transiently after vaccination for influenza, tetanus, or pneumococcus

45 All patients at initiation of treatment Patients with true virologic failure and HIV RNA levels >1,000 copies/ml If viral load >500 but <1,000 testing may be unsuccessful, but should be considered Patients with suboptimal initial response to therapy Failure to completely suppress viral load initially

46 Genotypic methods Two FDA approved assays for the sequencing of reverse transcriptase and protease genes Lab developed assays for the sequencing of other genes of concern (ie. integrase) PR RT

47 Phenotypic methods Able to test more drugs than those tested by commercial genotype assays Only performed at commercial laboratories The amount of drug necessary to inhibit 50% and 90% is reported

48 Tropism assays HIV binds either CCR5 or CXCR-4 as a co-receptor for cell entry Miravaroc is a CCR5 entry inhibitor and only effective when virus is CCR5 tropic The ability of cloned vector to infect CCR5 positive cell lines and CXCR-4 positive cell lines is measured CCR5 Positive CXCR-4 Positive Growth???

49 Patient Isolate Amplify and Sequence Compare to database of known mutations Limitations of all Methods Genotyping Phenotyping Tropism Assays Must have adequate viral load ( cp/ml) Resistant clone must comprise 20-30% of the viral population Patient Isolate Amplify and clone viral nucleic acid into vector Grow new virus in presence of different antiviral concentrations Patient Isolate Amplify and clone viral nucleic acid into vector Grow new virus in cell lines expressing either CCR5 or CXCR-4 Pro: Easy to perform Con: Limited amount of drugs detected Pro: Able to test many drugs Con: Laborious and expensive

50 HIV molecular testing plays an important role in the diagnosis of acute HIV HIV viral load should be monitored during treatment to assess for drug resistance Sustained or large increases in HIV viral load should be followed up with resistance testing While genotyping is most common, several different resistance detection assays are available All forms of HIV resistance testing have limitations which should be considered prior to ordering

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52 Rapid qualitative molecular testing Currently only one FDAapproved molecular test is available for diagnosis Cepheid Xpert HIV-1 Qualitative test has been CE marked and marketed abroad 90 minute run time and amenable to near-poc

53 Bioplex (5 th generation HIV testing) Tests separately and differentiates HIV 1 ab, HIV 2 ab, and p24 Acceptable 4 th gen screening assay though technically also an HIV1/2 differentiation assay Could change testing algorithm dramatically

54 THEORETICAL!!!! HIV-1 AB -, p24 - Negative HIV-1 AB +, p24 - HIV-1 AB -, p24 + Additional testing required What should be used for confirmatory testing? 5 th Generation Assay Is this specific HIV-1 AB +, p24 + Positive enough to establish a diagnosis? More data is needed regarding the performance of the Bioplex

55 Measuring drug resistance in archived viral DNA integrated into CD4 T cells In theory this should allow resistance testing on patients with suppressed viral loads Data is currently conflicting regarding utility Free virus CD4+ Lymphocyte

56 Used to detect small resistance populations amongst circulating virus Limit of detection approximately 1% Many mutations are indeed low abundance Simon et al. Low-Abundance Drug-Resistant Viral Variants in Chronically HIV- Infected, Antiretroviral Treatment-Naïve Patients Significantly Impact Treatment Outcomes, JID 2009:199 (1 March)

57 Minority Variant % Virologic Failure Total Hazard Ratio Minority Variant No Minority Variant Minority Variant No Minority Variant (95% CI) < ( ) < ( ) Li et al. Low-Frequency HIV-1 Drug Resistance Mutations and Risk of NNRTI- Based Antiretroviral Treatment Failure. JAMA, April 6, 2011 V Systematic review published in JAMA -Compiled pooled outcome data from 10 different studies -Examining outcomes in patients with minority variants receiving treatment Resistant minority variants likely are clinically significant and their detection may allow for the better management of patients with HIV

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59 25 year old male with fever, malaise, and history of IVD HIV serology screen (4 th generation ELISA) Fourth generation ELISA positive, multispot test negative HIV RNA PCR (quant or qual) HIV qualitative PCR positive HIV quantitative PCR and resistance testing

60 Patient has no detectable baseline drug resistance. Initial viral load is 100,000 copies per ml, repeat viral load two days later is 200,000 copies/ml No testing at this time, wait 2 weeks/months Patient viral load undetectable within two years. Five years later patient found to have viral load of 100 copies/ml Recheck one month later (could be blip) One month later viral load at 1000 copies/ml Resistance testing ordered

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